Talk:American Journal of Anatomy 28 (1920-21)

From Embryology

THE AMERICAN JOURNAL


OF


ANATOMY


EDITORIAL BOARD


Charles R. Bardeen

University of Wisconsin

Henry H. Donaldson

The Wistar Institute

Simon H. Gage

Cornell University


G. Carl Huber

University of Michigan

George S. Huntington

Columbia University


J. Playfair McMurrich

University of Toronto

George A. Piersol

University of Pennsylvania

Henry McE. Knower, Secretary

University of Cincinnati


VOLUME 28 NOVEMBER, 1920— MARCH, 1921


THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA.


V'-'S^


CONTENTS


No. 1. NOVEMBER, 1920

S. Saguchi. Cytological studies of Langerhans's islets, with special reference to the problem of ^ their relation to the pancreatic acinus tissue. Six plates (sixty-one figures) and four text figures 1

Hayato;Arai. On the cause of the hypertrophy of the surviving ovary after semispay ingj(albino rat) and on the number of ova in it 59

JosfilF. NoNiDEz. Studies on the gonads of the fowl. I. Hematopoietic processes in the gonads of embryos and mature birds. Three plates (twenty-nine figures) 81

No. 2. JANUARY, 1921

Charles R. Stockard. Developmental rate and structural expression: An experimental study of twins, 'double monsters' and single deformities, and the interaction among embryonic organs during their origin and development. Thirty-two text figures and six plates ' 115

Geo. a. Thiel and Hal Downey. The development of the mammalian spleen, with special reference to its hematopoietic activity. Three text figures and three plates (eight figures) 279

Chester H. Hetjser. The early establishment of the intestinal nutrition in the opossum — The digestive system just before and soon after birth. Six plates (twenty figures) 341

John Lewis Bremer. Recurrent branches of the abducens nerve in human embryos. Diagram A and four figures 371

ToKUYASU Kudo. Studies on the effects of thirst. I. Effects of thirst on the weights of the various organs and systems of adult albino rats 399

No. 3. MARCH, 1921

Warren H. Lewis. The effect of potassium permanganate on the mesenchyme cells of tissue cultures. Sixteen figures (one plate) 431

George A. Baitsell. A study of the development of connective tissue in the Amphibia. Six figures (four plates) 447


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THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 1, NOVEMBER, 1920


Resumen por el autor, S. Saguchi. Escuela Medica, Kanazawa, Japon.

Estudios citol6gicos sobre los islotes de Langerhans, con especial

menci6n del problema de su relaci6n con el

tejido acinoso pancreatico.

Para resolver el problema de la dependehcia o independencia de los islotes, el autor ha llevado a cabo un cuidadoso estudio citologico de este tejido. Las celulas de los islotes pancreaticos contienen granulos especificos, mitocondrias, corpiisculos lipoides, el aparatourano-argentofilo, granulos argentofilos, el cordon intracelular y aparato reticular, y granulos de pigmento.

Teniendo en cuenta la cantidad relativa de estas estructuras intracitoplasmicas y la forma, posicion y reacciones colorantes de los nucleos, las celulas de los islotes pueden clasificarse en los tipos a, b, c, d, y, e. El hecho de que estos tipos esten relacionados entre si, asi como con las celulas de los acini pancreaticos, mediante formas de transicion, demuestra que el islote no es un 6rgano independiente, sino que las celulas que le constituyen son elementos de los acini, transformados, mientras que algunas de aquellas pueden transformarse en estos ultimos.

El autor acepta la idea que supone al islote como un organo de secreccion interna y cree que la secrecci6n debe derivar de los corpusculos lipoides y del aparato urano-argentofilo. Estas celulas de secrecci6n interna form an, no solo los islotes tipicos, sino tambien otros no tipicos, y a veces, hasta celulas solitarias pueden encontrarse entre las celulas de los acini.

Translation by Jose F. Nonidez Cornell Medical College, New York


author's abstract of this paper issued by the bibliographic service, july 26


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS, WITH SPECIAL REFERENCE TO THE PROBLEM OF THEIR RELATION TO THE PANCREATIC ACINUS TISSUE

S. SAGUCHI Anatomical Laboratory, Medical School, Kanazawa, Japan

SIX PLATES (sixty-one FIGURES) AND FOUR TEXT FIGURES

_ CONTENTS

Introduction 1

Minute structures of islet cells 5

Historical 5

Observations 7

Specific granules : cell types : the nucleus 7

Mitochondria 11

Lipoid corpuscles 13

The urano-argentophile apparatus 15

Argentophile granules 17

The intercellular cord- and net-apparatus 18

Pigment granules 20

Mitosis and amitosis 20

The relation between the different types of islet cells 21

Distribution of islet cells in the pancreas 24

Relation between islet cells and acinus cells 28

Postembryonic development of islet cells 28

Ultimate fate of islet cells 34

Formation and fate of the typical islet 35

Functional significance of islet cells 38

Bibliography 41

INTRODUCTION

As is well known, the pancreas of vertebrate animals is studded with groups of peculiar cells which are characterized by a transparent appearance due to the lack of zymogen granules, and which accordingly can be distinguished with ease from the neighboring acinus cells. Since Langerhans's ('69) discovery of these


Z S. SAGUCHI

groups in the pancreas of the rabbit, many investigators who have devoted themselves to the study of the pancreas have concentrated their attention on these structures — designated later as Langerhans's cell-groups or islets — and there has been much discussion as to their morphologj^, development, and significance. This led to the formulation of various hypotheses, at present far from being in agreement. The most complex and difficult problem is that of the origin and ultimate fate of the islet of Langerhans, especially its genetic relation to the acinus tissue. Some believe that the islet is formed in an early embryonic stage by budding off and separating from the solid primitive cell-cord, and that once formed, it may exist as a permanent organ thi'oughout life. Others, while not denjdng its embryonic development, hold that the islet may originate from the acinus tissue of the adult pancreas. Laguesse and his supporters claim that islet cells may be formed, on the one hand, by the transformation of acinus cells and, on the other hand, by a reversion to the latter, the two tissues being thus well balanced (balancement theory). There are other observers who incline to the belief that islet cells are derived from the duct epithelium.

Decisive points in these discussions, especially of the problem of the genetic relation between the islet and the acinus tissue, have been the determination as to whether the islet is sharply marked off from the acinus tissue by a connective-tissue capsule or is in immediate contact with it, and whether or not the two behave independently under experimental and pathological conditions. Manj^ investigators seem to attach considerable importance to the question of the existence or non-existence of a capsule around the islet. We find it impossible to accept as adequate the postulation that the existence of a capsule is a sign of the independence of the islet, since it is difficult to decide whether the capsule is perfect or not. Even when the islet appears to be sharply separated from the- acinus tissue, it can hardlj" be said that the two have no genetic connection. On the other hand, it would be rash to assume a transition between the islet and the acinus tissue merely because the two are sometimes found in immediate contact, for there are many instances in which cells


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 3

belonging to different tissues are in close apposition, although a transition between them is not conceivable.

Those who have tried to solve this problem experimentally have hgatured the pancreatic duct, allowed the animal to fast, or injected pilocarpi!! or secretin, and studied the changes thus produced in the size and number of the islets. Their observations have not, however, been in accord. Some (Fischer, Dale, and Vincent and Thompson) believed that they found an increase in the number of islets in starvation, while others noticed neither an increase nor a decrease of the islet tissue. On the other hand, ligature of the pancreatic duct caused, in some instances, the disappearance of only the acinus tissue, the islet remaining intact; in others, the disintegration of both tissues. That lack of uniformity in the results of these experimental investigations is perhaps due primarily to an inequality in the experimental methods employed; secondly, to the fact that the islets vary greatly in size and number not only in the different animals, but also in different portions of the same pancreas, as has been pointed out by various investigators. Under these circumstances there is always a danger of misinterpretation when one attempts to draw any general conclusion from the changes in size and number of islets in any circumscribed area of the pancreas.

It will be seen from the foregoing that the evidence brought forward to show a transition between the islet and the acinus tissue is by no means positive. The points which morphologists must solve first of all are: What are islet cells? What is their internal structure or specific property? The characteristics of islet cells pointed out by various investigators are not an accurate index. It has been claimed that these cells are distinguished from acinus cells in the following details: 1) they contain no zymogen granules and therefore appear more transparent; 2) they form groups showing a definite disposition; 3) a lumen in continuity with the pancreatic duct cannot be discovered within the islet; 4) the form, structure, and staining reactions of the nuclei of islet cells are different from those of acinus cells; 5) some islet cells contain a certain type of granules. Of the above, 1 and 3 are not characteristic of islet cells alone; there are, on the


4 S. SAGUCHI

one hand, acinus cells from which almost all of the zymogen granules have been extruded; on the other, acini in which no lumen is evident. The specific disposition of cells is better seen in the larger islets. The character of the cytoplasm is of greater importance in distinguishing islet cells from acinus cells. Not all of the islet cells contain the minute granules which have been regarded as specific of them. From these considerations it is evident that the large islet is easily defined by the above characteristics, but the smaller one, consisting of only a few cells or even of a solitary cell, can be identified only with the greatest difficulty if it contains no specific granules. In other words, it is a difficult problem to decide whether the cells which are interspersed among acinus cells and which are devoid of zymogen granules belong to the acinus cells proper or not. The specific granules mentioned above are the only bodies of which we should take account in characterizing any islet cell, other properties being of a rather negative nature, as Benslej^ ('11-' 12) pointed out. We must have more positive identifying characteristics in order to define the islet cell accurately ; the more numerous these are, the more accurately can the cell be defined, so that even a solitary islet cell among acinous cells may be detected with ease. It is then only that the solution of the problem as to a possible transition between the acinus and islet tissue will become an easy one; for the process in question, if there be such, must be accompanied not only by definite changes in the shape, position, and arrangement of the cells, but also by changes in their internal structure, by which we should be able to trace the gradual disappearance of intracellular structures in the acinus cell and the appearance of new cell-constituents which are characteristic of the islet cell, or vice versa. This is the reason why a cytological study of the islet is of great importance in the definition of the islet cell and, consequently, to the question of a transition between the islet and acinus tissue.

The pancreas of Rana temporaria was selected as the material for this study as well as for the cytological study of the acinus in a preceding paper (Saguchi, '20). The latter investigation has greatly facilitated not only the study of the minute


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 5

structure of the islet cells, but also the solution of the question of a transition. The methods employed for fixing and staining were the same for both, and this has enabled me to compare the structures thereby brought to light, both in acinus cells and in islet cells, in one and the same preparation.

MINUTE STRUCTURES OF ISLET CELLS

Historical

The islet cells, though they have long been a problem for observers, have not yet been satisfactorily investigated in regard to their minute structures, at least not by the newer cytological methods. In looking over the hterature, one finds several references to the cytoplasmic structure. These briefly may be summarized as follows:

Since Langerhans's time, it has been frequently stated that the islet, either in whole or in part, consists of clear, transparent cells which are devoid of zj^mogen granules. Lewaschew ('86) describes two kinds of islet cells, both of which have the transparent cytoplasm, but which can be distinguished from each other by the shape and mode of delimitation of the latter.

Massari ('98), Diamare ('99), Schulze ('00), Levi ('04), Vincent and Thompson ('07), and others distinguish between two kinds of islets cells, one of which stains more heavily than the other. Vincent and Thompson, among others, pointed out that this difference is observed in the pancreas of various vertebrates; they designated the faintly staining islet as the leptochrome tissue and the heavier staining one as the bathychrome. According to these authors, the former constitutes the well-known islet of Langerhans; the latter, in mammals, consists of small groups of cells or even of solitary elements scattered throughout the secreting alveoli, while in birds, reptiles, and amphibia, it forms a solid mass of cells. The bathychrome tissue shows a remarkable tendency to form syncytium and stains deeply with ordinary dyes; this is especially true of the Flemming fixation.

Some investigators, on the other hand, have found special granules in some of the islet cells. Laguesse ('93) stated that


6 S. SAGUCHI

there are present in the developing islet cells of the sheep embryo minute safranophile granules. These have since been observed by Laguesse ('95-96;' '01, '09-10) and by many other investigators (Carlier, '96; Gianneh and Giacomini, '96; Diamare, '99; Mankowski, '02; Ssobolew, '02; Pearce, '02-'03; Levi, '04; Marchioni, '04; Tschassownikow, '06; Lane, '07-'08; Gelle, '11; Piazza, '11; Bensley, '11-12, etc.). According to Laguesse, these granules correspond in a general way to zymogen granules in their behavior toward fixing and staining, differing from the latter, however, in their smaller size and greater resistance to acetic acid. Mankowski, Ssobolew, and Gelle call special attention to their affinity for safranin, while Carlier and Pearce designate them as eosinophile. Mankowski and Piazza have found that the granules in question have the property of reducing silver nitrate, so that in silver preparations they take a brown or black color.

According to the above authors, the granules are not evenly distributed throughout the islet cells, being abundant in some, while other cells are almost devoid of them. On the contrary, Lane and Bensley hold that there are two kinds of granules which are present in different kinds of cells, both of which stain with neutral gentian, although they differ from each other in their behavior toward certain fixing reagents.

That the protoplasm of islet cells assumes an alveolar appearance in fixed preparations, caused by the presence of small vacuoles, has been shown by Laguesse ('9o-'96, '01, '09-'10), Diamare ('99), Bensley ('11-' 12), Retterer and Lelievre ('13), and others. Laguesse is of the opinion that these vacuoles are filled with clear liquid which, when stained, presents under certain conditions a gummy aspect.

Fat droplets may also occur in islet cells. Dogiel ('93) noticed certain corpuscles which stained black in osmic-acid solution; he claimed that they were fat globules produced in the protoplasm in consequence of the regressive metamorphosis of the cell. Laguesse ('09-'10), Stangl ('01), and Saleni ('05) have confirmed the presence of fat corpuscles in islet cells, without, however, attributing any special importance to it.


CYTOLOGICAL STUDIES OF LANGERHANS S ISLETS 7

Trophospongia are also found in islet cells. Holmgren ('04) was the first to describe this network as being connected with the processes of interstitial cells and which can often be canalized. Tschassownikow ('06) stated that the trophospongium of the islet cell was not constant, while Bensley ('11-12) claimed to have observed it in both fresh and fixed preparations.

The occm-rence of mitochondria in the islet cells was described by Bensley ('11-'12), Herwerden ('12), and Arnold ('12). According to these authors, the mitochondrial substance is present in the form of either minute granules or delicate filaments, the thick, coarse chondrioconts, such as are seen in the acinus cell, being entirely lacking.

It will be seen from these observations that the protoplasm of the islet cell presents either a homogeneous or a reticular appearance, and that the islet cells are of two kinds, distinguishable from each other by the tingibility of the plasma and by difference in granular content. It was also reported that fat droplets, mitochondria, and the intracellular network may occur in the islet cell, to which, however, no special importance has yet been ascribed.

Concerning the nuclei of these cells, the observations of vari.ous investigators are not in accord. The nuclei are described as being either smaller or larger than those of acinus cells, and the occasional occurrence of even giant-nuclei has been pointed out. They may be spherical, elliptical, or even rod-shaped. Many investigators believe that the nucleus has a dense reticulum which is rich in chromatin corpuscles, while others say that it is poor in these granules. Such diversity of opinion may be due to the technical methods employed. The only point of coincidence is that the nucleoli are either very small or altogether absent from the nucleus.

Observations

Specific granules: Cell types: the nucleus. We designate as specific granules of the islet cell those minute granules which are seen in the preparations fixed in sublimate-osmic-chromic acid, formalin, Maximow's, Regaud's, Meves's or Zenker's fluid, and


8 S. SAGUCHI

stained with acid fuchsin, iron-hematoxylin, etc. These elements are best brought into view by fixation with Zenker's fluid and staining with Heidenhaiii's iron-hematoxylin. From these reactions, it is obvious that osmic acid is not essential to the preservation of the granules. Furthermore, acetic acid does not dissolve them, but appears rather to favor their fixation. From these properties, it is most probable that the granules are similar with those which have been described by Laguesse and many others; contrary to the view of Laguesse, Mankowski ('02), and Tachassownikow ('06), who maintain that they are stained with safranin, as is the case with chromatin, I have found that these granules do not stain with alum-hematoxylin, but are, rather, acidophilic and show a tingibility analogous to that of the nucleolar and mitochondrial substance, differing only from the latter in that the specific granules offer greater resistance to the action of acetic acid. These granules, in part at least, seem to have a certain affinity for silver salts, for in Cajal's preparations they are stained a pale brown color, as has been pointed out by Mankowski ('02) and Piazza ('11).

The specific granules are of very minute size (figs. 1 to 3, b; 14 to 17) and they are so closely packed that it is almost impossible to distinguish the individual granules; thus the protoplasm exhibits, at first glance, a granular or powdery appearance, as was first pointed out by Laguesse. Under these circumstances, it is exceedingly difficult to decide whether or not this granulation may be due to the interlacement of fine, tortuous filaments. The granules, in the majority of cases, are distributed throughout the whole cell-body, although not infrequently they are accumulated in large numbers at one end of the cell.

Most of the islets consist of cells which contain the specific granules, and others which are either entirely devoid of them or seem to contain only a very small number so that the protoplasm is characterized by a hyaline, transparent appearance. These two types of cells may be designated, respectively, as granular and non-granular islet cells. The granular cells can further be classified under three types according to the tingibility of the granules, the shape and structure of the cell-body and of the


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS 9

nuclei, etc. These types are designated as a, b, and c. The descriptions are based mainly upon preparations fixed in osmic acid mixtures and stained with acid fuchsin or iron-hematoxylin.

The specific granules of the a cells (fig. 14) stain heavily, thus giving to the protoplasm a distinctly granular appearance. The cells of this type are of various shapes: cylindrical, pyramidal, polygonal, or even, as often occurs, more or less irregularly shaped, due apparently to pressure from neighboring cells. The nucleus, spherical or polygonal in shape, is provided, in the majority of cases with a thick nuclear membrane which is thrown into folds; the nuclear network is well marked and its meshes are closer than those of the nuclei of acinus cells.

The cells of b type (figs. 1 to 4, b; 16, 17) are characterized first of all by the fact that their specific granules stain more faintly with iron-hematoxylin than those of the a cells. Like the latter, the b cells are, for the most part, cylindrical or pyramidal in shape, but narrow, elongated cells are not infrequently met with. The shape and structure of the nucleus constitute the second criterion by which these cells may be distinguished from a cells. The nucleus is large and oval in shape, with a smooth and indistinct contour ; the nuclear network, as seen in iron-hematoxylin preparations, is represented by indistinct, faintly staining cords or threads on which one or more small nucleoli may be suspended.

The c cells (figs. 52, 53) contain specific granules which stain even more faintly than those of b cells, and which, in most cases, are not evenly distributed throughout the cell-body, being interrupted here and there by transparent, non-granular protoplasm, especially at one end of the cell. The c cells are usually smaller than the preceding two types of cells and are either long and narrow or short-cylindrical, pyramidal or even polygonal in shape. The nucleus, irregular in form, is enclosed by a less distinct membrane which may be thrown into folds. The nuclear network is as indistinct as that of the b cell, and the mainnucleolus is likewise small.

Non-granular cells (figs. 1 to 5, e, 18 to 21), which compose the greater part of the islet elements, contain no specific granules


10 S. SAGUCHI

or at most, very few. Most of these cells are cylindrical or pyramidal in shape, either straight or curved, the short, cylindrical or polygonal cells being few in number. Usually the nucleus is oval, though more or less spherical nuclei are never entirely absent. Ordinaril}^ its position is in the middle of the cell, but cells in which the nucleus is situated near one end are also often met with. According to the behavior of the- protoplasm and the nucleus toward iron-hematoxyhn or acid fuchsin, the non-granular cells can be divided into two groups which will be designated as d and e cells. The d cells (figs. 1, d, 20) are characterized by the fact that their protoplasm has a greater affinity for the abovementioned dyes; either the whole or a part of the cell-body may be heavily stained. The nuclear membrane is less smooth. The intranuclear network is very apparent and the meshes are closer, as is the case with the a cells. The nucleolus is very small.

The d cells are few in number as compared with the other type of non-granular cells which constitute a large part of the islet elements. The protoplasm of the e cells (figs. 1, 3, 4, e; 18, 19, 21) is transparent when stained with iron-hematoxyhn or acid fuchsin, which, in fact, is responsible for the clear appearance of the islet in general. The nucleus has an even contour and contains one or two small nucleoli and several nucleolar corpuscles suspended in the indistinct nuclear net. Large nucleoli such as are seen in acinus cells were never observed here. The nuclei of the cells are similar to those of b cells in form, contour, and character of the intranuclear network, differing from them only in their heavily staining nucleolar corpuscles.

Although, on the whole, the elements constituting the islets of the pancreas fall into five groups mentioned above, there are some cells which do not belong to any of these types, but which possess the combined characteristics of any two of them and must therefore be regarded as intermediate or transitional forms. These forms will be described when relation between the types of islet cells comes under consideration.

As noted above, the nuclei of the islet cells are, in general, of oval form, though the more spherical type is always represented.


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 11

That the nuclei of the islet cells exceed more or less those of acinus cells in bulk is evident from the figures 1 to 5. The nucleus is mostly situated in the middle of the cell-body, but there are cases in which it is placed near one end of the cell, which seems to be dependent upon the arrangement of the cells in the islet. The nuclei of b and e cells have an even contour, while those of a and c and sometimes d cells are surrounded by a membrane which is thrown into folds. The nuclear network, as seen in the preparations fixed in osmic-acid mixtures and stained with iron-hematoxylin or acid fuchsin, is most apparent in the nuclei of a and d cells; that of b, c, and e cells stains more or less faintly. In alum-hematoxylin preparations (fig. 23) it is seen that the chromatin granules are smaller and more numerous and that the network is closer in the nuclei of islet cells than in those of acinus cells. Another and more important difference is that the main-nucleolus of the former is always smaller than that of the latter, and contains one or two nucleolini which are situated at the periphery. The nucleolini are stained black by the Cajal method (figs. 48 to 50), while in ordinary preparations they are dissolved out so that there appear one or two small, clear vacuoles (fig. 51). In addition to the main-nucleolus, there are several side-nucleoli. In contradistinction to the preparations stained with iron hematoxylin, in the aium-hematoxylin preparation no structural differences of the nuclei between the various types of islet cells can be observed. This is due to the fact that the two staining methods exhibit two essentially different constituents of the nucleus: alum-hematoxylin stains mainly the chromatin, iron-hematoxylin the nucleolar substance. The structural differences in the nuclei of the various types of islet cells are therefore observed with respect, not to the chromatin,, but to the nucleolar substance. The chromatin content of the nuclei is fairly constant. Mobile the amount of the nucleolar substance undergoes a considerable change; this seems to point to the important significance of the latter in the metabohsm of the cell (Saguchi, '20).

Mitochondria. In the study of mitochondria, the following methods were applied : fixation in Meves's, Benda's, or Altmann's


12 S. SAGUCHI

fluids, sublimate-osmic-chromic acid, sublimate-osmic acid, or Zenker's fluid, and staining with iron-hematoxylin or acid fuchsin.

The specific granules present in the a, b, and c cells bear, in their staining reactions, a striking resemblance to mitochondria, except that they offer greater resistance to the action of acetic acid. Except for these granules, there are no mitochondrial filaments in these cells. If we assume that mitochondria are a constant constituent of all cells', then they should also be found in these three types. I am of the opinion that the specific granules in these cells represent mitochondria. Evidence in favor of this view is: 1) the resemblance of the staining reactions of the two as described above; 2) the development of the specific granules takes place in a manner identical to that of the mitochondria in the acinus cell (Saguchi, '20); and 3) it is probable that the ultimate fate of the specific granules is the same as that of the mitochondria in the other types of islet cells, a question which will be taken up when the lipoid corpuscles are considered. It is necessary to say here a few words with regard to certain granules of mitochondrial nature. These are scattered sparsely throughout the cytoplasm of the a and b cells (fig. l,b); they are larger than the ordinary specific granules. Some of them are certainly of nuclear origin, since it can often be clearly seen that the nucleolus constricts off pieces which pass out of the nucleus into the cytoplasm (fig. 15); some of them, however, especially the smaller ones, may be formed by the growth and eventual fusion of the specific granules.

The non-granular cells, i.e., d and e cells, contain typical mitochondria in two forms — fiJamentous and granular (figs. 1, 2, 3, 7, e; 18 to 21) Chondrioconts in these types of islet cells are tor. tuous, very delicate filaments which can be made out only with diflficulty in the stained preparations. In the preparations treated with the method of Meves or of Altmann they stain very faintly, whereas in those which were fixed in osmic-sublimate mixture and stained with iron-hematoxylin, they appear as relativelj^ thick, more heavily staining filaments (fig. 19). They are distributed evenly throughout the cytoplasm, without any local accumulation. They seem to be present more abun


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 13

dantly in the d cells, especially in the neighborhood of the nucleus, than in the e cells (fig. 1). The mitochondrial granules are of various sizes: from very small to several times the diameter of a filament. They are commonly scattered throughout the cytoplasm, but frequently they are gathered together in greater or less amount near one end of the cell. It is my belief that a certain proportion of these granules are produced by the disintegration of chondrioconts; in fact, it is not infrequently observed that the latter carry bulbous enlargements which may become separated from them and increase in size (fig. 20). Some of the granules are most probably of nuclear origin, as is the case with a and b cells.

Lipoid corpuscles. Many of the islet cells contain some spherical corpuscles of a fatty or lipoid nature, which may be designated as lipoid corpuscles. These stain gray with osmic-acid solution or in mixtures containing this reagent. If the piece fixed in osmic acid is treated with any reducing reagent, for example, by the Faure-Fremiet method, they appear as deeply stained corpuscles. The corpuscles are also found in the Weigl (figs. 38, 41) and Golgi preparations, although in the latter they present a shrunken, thorn-apple appearance. In the first method of Ciaccio ('11) they are stained a reddish-yellow color with sudan III (fig. 5), while in the second method, they take a gray color with a reddish tinge. If Ciaccio's assumption be true, these corpuscles must contain neutral fat in addition to the lipoid substance. They are likewise easily stained with sudan III and scarlet red as applied to frozen sections. They cannot be seen in the preparations fixed in non-osmic mixtures and stained with alum- or iron-hematoxylin, but are represented by clear vacuoles (figs. 22, 23) which are nothing more or less than the lipoid corpuscles emptied of their contents by the dissolving effect of reagents, and which, when present in abundance, give a clear, reticular, or alveolar appearance to the protoplasm. Even in osmic preparations, it often occurs that the corpuscles are dissolved out by dehydrating alcohol, and xylol, or chloroform (figs. 18, 20, 21).

The lipoid corpuscles are characteristically spherical or droplike in shape; they vary in size, showing all grades of transition


14 S. SAGUCHI

between the smallest and the largest granules which often reach the diameter of a nucleus. In most cases they are evenly distributed throughout the protoplasm, but it not infrequently occurs that they are accumulated near one end of the cell where the latter is in contact with a blood-vessel.

These lipoid corpuscles are present in b, d, and e cells, the largest and greatest numbers being found in the last. They are usually lacking or, if present, are few in number and of very small size in a and c cells; the smallest can be manifested only by the Golgi method.

It should be mentioned in this connection that the lipoid corpuscles, in their physical and chemical properties, bear a strong resemblance to the lipoid granules found in the basal portion of the acinus cell, for the details of which the reader is referred to my previous paper (Saguchi, '20). It may be that the two structures have the same functional significance, although in acinus cells the production of the substance which constitutes them does not take place to such a marked extent as in the islet cells.

As regards their genesis, no definite conclusion can be reached at present. It seems probable, however, taking into account my previous observation that the lipoid granules in the acinus cell are derived from mitochondrial filaments, that this is also the case with the lipoid corpuscles in the islet cell. We have seen that the mitochondrial granules in the e cells are formed by the disintegration of the chondrioconts; it is possible to assume that these granules, after having increased in size to a certain extent, may change their physical and chemical properties and become transformed into the smallest Upoid corpuscles, which gradually increase in size, either by growth or by fusion. In the same manner, the lipoid corpuscles of the h cells may be regarded as derived from the specific granules or the granules of mitochondrial nature which are contained in this type of cell.

The problem as to the fate of the lipoid corpuscles is not a simple one. The view of Dogiel ('93) that the islet must be regarded as a dead spot, since it contains fat droplets which are produced in consequence of the metamorphosis of the cell, is not the correct one, for no degenerative process can be seen either in


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 15

the cells or in the nuclei. This and the fact that the islet is composed mainly of cells of this type, rather strongly suggest that the lipoid corpuscles are a cell constituent which plays an important part in the function of the islet. It is presumable that this lipoid substance is to be extruded, either as such or after having undergone certain changes, into the blood-vessel with which the cells in question are intimately connected.

The urano-argentophile apparatus. By the term urano-argentophile apparatus, I understand those filamentous or granular corpuscles which can be made manifest by the Cajaluranic nitratesilver method. Since this method is one which is employed for exhibiting the Golgi intracellular network apparatus, the question arises whether the urano-argentophile and the Golgi apparatus are to be identified or not. In fact, I have found that the same intracellular network can be brought into view in pancreatic acinus cells not only by the Kopsch, Sjovall, and Weigi method, but also by the Cajal method. This is not, however, the case with islet cells; the Cajal method does not exhibit the net, but a different apparatus which can easily be distinguished from it by both its morphological and chemical characteristics, and which therefore must be treated of separately.

The urano-argentophile apparatus is stained brown or black with the Cajal method, as mentioned above, so that it stands out clearly from the faintly staining ground substance of the protoplasm (figs. 6, 28 to 31). These corpuscles are filamentous or granular in shape. The filaments are of varying thickness and length and are more or less tortuous in their course. They rarely ramify or anastomose with one another, and are usually distributed evenly throughout the cytoplasm, although it often occurs that they are gathered together in a given region of the cell-body. The thinner filaments have an even contour, while the thicker ones often have spherical or fusiform enlargements along their course, which may be set free and may give rise to urano-argentophile granules. The latter, spherical or oval in shape, are also of various sizes, and stain more heavily than the filaments. They are either evenly scattered throughout the cytoplasm or accumulated at one or both ends of the cell. Neither

THE AMERICAN JOURXAL OF ANATOMY, VOL. 28, NO. 1


16 S. SAGUCHI

granules nor filaments pass into the vacuoles which correspond to the dissolved out lipoid corpuscles.

As regards the distribution of urano-argentophile granules and filaments in islet cells, it can be said that cells containing granules in association with filaments are rarely seen; consequently, cells containing granules are usually devoid of filaments, or at the most, contain short, thick rods. By far the largest number of islet cells are of the latter type; these cells are cylindrical in shape and are in contact with blood-vessels at one or both ends. They also contain a great number of spherical vacuoles corresponding to Hpoid corpuscles (figs. 6, e, 29, 30). On the other hand, there are cells which are provided wdth granular uranoargentophile corpuscles, but in these the vacuoles are not evident; the cells of this type usually occur in small groups between acinus cells.

The cells containing urano-argentophile filaments can be subdivided into three kinds according to size, shape, and position. In the first place, there are some cylindrical cells which occur in the peripheral part of the islet or are interspersed between acinus cells, and which are laden with a large number of either thin or thick filaments. They have, in most cases, spherical nuclei which are usually situated near one end of the cell (figs. 25 to 27) . The second type of cell is small, and often long, narrow, and cylindrical in form. It is always situated at the periphery of the islet and contains a small number of filaments (fig. 32). The third tA^pe is situated between the cells containing urano-argentophile granules; its protoplasm stains more or less deeply and possesses a few tortuous filaments. It is characteristic of this kind of cell that the vacuoles corresponding to the lipoid corpuscles appear to be lacking, at least no larger ones are visible (fig. 33).

The question now arises, how can we identify these five types of cells with the a, b, c, d, and e cells. This is an exceedingly difficult problem, since we possess at present no technical methods by which to bring into view both specific granules and uranoargentophile corpuscles in one and the same preparation. The presence of large numbers of both lipoid corpuscles and urano


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS 17

argentophile granules in the cells forming the chief constituent of the islet seems to point to that fact that these cells correspond to the e type. Of the cells in which vacuoles are not evident, those which are laden with delicate urano-argentophile filaments may be identified with the a cells, as the form and position of the cell-bodies and the nuclei of the two are in accord. Secondly, the non-vacuolated, granule-containing cells, which form small groups scattered throughout the acinus tissue, may be regarded as corresponding to the h cells; in fact, the lipoid corpuscles of the latter are small in size and few in number, so that it is difficult to make them out in the Cajal preparations. Whether the narrow cells (figs. 31, 32) in the periphery of the islet belong to the h type or c type is difficult to decide, although the cells which contain the deeply staining protoplasm, and which are interposed between the e cells, correspond in all likelihood to d cells, for other types of cells than they are not usually found between the e cells.

That the urano-argentophile granules are formed by the disintegration of filaments has been mentioned above; but the development of the filaments themselves and the ultimate fate of the granules are points which have not been definitely determined. It not infrequently occurs that the disintegration of the filaments into granules takes place near one end of the cell or that the granules are especially abundant at one or both ends of the cell. From these observations, it would seem that there is some intimate relationship between them and blood-vessels. I am of the opinion that these granules, after having undergone certain modifications, pass into the blood-stream.

Argentophile granules. In preparations fixed in formalin and treated with silver nitrate, according to Cajal, there can be seen in the islet, or scattered through the pancreas cells which contain brown-stained granules (figs. 46, 47). Although these may be designated argentophile granules, they can also be made manifest by fixation in formalin and by staining with iron-hematoxylin. These granules are of small size, although larger than the specific granules previously spoken of. In some cases they are so numerous as to fill up the cell-body, in others they are few in number or located only near one end of the cell.


18 S. SAGUCHI

The cells containing argentophile granules contain few or no vacuoles corresponding to the dissolved out lipoid corpuscles; from this fact and from their position in the periphery of the islet, it is conceivable that these cells belong to the type which contains specific granules. I am of the opinion that the argentophile granules are present in both a and h cells; in fact, some of the nuclei of the cells containing argentophile granules are spherical in shape and situated near one end of the cell. Unlike these granules, the specific granules are stained only a faintly brown color in the Cajal preparation. The e cell contains no argentophile granules (figs. 48, 49, 50).

The genesis of argentophile granules may easily be followed in certain cells which are met with in the periphery of the islet or between acinus cells. In shape these cells (figs. 45, 46) are similar to acinus cells and contain spherical nuclei in which numerous argentophile granules of various sizes are visible. In some cases these granules are found in the nucleus; in others, they are also met with in the cell-body, while in the nucleus they are reduced in number. From these observations, the inference that the argentophile granules produced within the nucleus pass into the cytoplasm would appear justifiable. In my preceding work on the acinus cells of the pancreas I have referred to the fact that the nucleolini are stained a brown color by the Cajal method, and that they often pass out of the nucleolus, even out of the nucleus into the cytoplasm. In a similar manner the argentophile granules, I think, must be derived from the nucleolini, the production being very active in this case. As regards the ultimate fate of these granules, no definite conclusion can be reached at present. It is, however, well within the bounds of possibility that, after having undergone certain changes, they are eliminated from the cell-body or transformed into other cell constituents.

Intracellular cord or network apparatus. A structure comparable to the Golgi network is also found in islet cells. Although it is manifested with ease by the Weigl method, it can also be seen in preparations fixed in sublimate, sublimate-osmic-chromic acid, or Rabl's mixture, and stained with iron-hematoxylin. The structure presents four principal forms (figs. 34 to 40, 42 to 44) :


CYTO LOGICAL STUDIES OF LANGERHANS's ISLETS 19

1) There are found irregularly spherical or elliptical corpuscles of various sizes, which consist of a deeply staining cortex and a pale main mass (figs. 35, 36). These corpuscles are often connected with one another by threads of varying thickness which show the same staining reaction as the corpuscles (fig. 37). From the shape and position on the one hand and from the absence of lipoid corpuscles on the other, it is probable that the cells which contain these bodies belong not to the d or e type, but to the granular type of cell, especially to the a and h types. 2) There are typical nets which are formed by the anastomosis of thin or thick, often double-contoured threads (figs. 38, 39, 42). These are situated either between the nucleus and an extremity of the cell or along the side of the nucleus; in the latter case, the net is flattened and elongated (fig. 42). 3) Another form of Golgi's apparatus is represented by rings or loops (figs. 40, 43, 44). The ring (usually there is only one) is generally situated near the nucleus. There are also cases in which the ring or loop emits various prolongations which either anastomose with one another, end freely in the surrounding protoplasm, or run along the side of the nucleus toward the other end of the cell. 4) The apparatus sometimes appears as threads (fig. 21), which may be straight or curved, situated near one end of the cell, or alongside of the nucleus. The latter three forms of the Golgi's apparatus are found in cells with lipoid corpuscles. The lipoid content of these cells, however, is small; therefore they must be looked upon as belonging not to the e type, but to the h type. Most of the e cells, which contain a large amount of lipoid substance, are devoid of the apparatus in question (fig. 41). There are also other forms which may be regarded as transitional between the types mentioned above. I am of the opinion that these gradations point to a course of development and of involution of the structure. In fact, it is not difficult to see how the corpuscles become elongated and connected with one another (fig. 37) in order to form the typical net. On the other hand, the net undergoes regressive metamorphosis by a gradual thinning and adherence of the trabeculae or threads, so that, at last, it leads to the formation of a single ring or loop, or even a delicate filament w^hich gradually disappears (fig. 21).


20 S. SAGUCHI

It will be seen from the above account that the b cells contain the Golgi apparatus in the height of its development; in the a cells it is in the earlier stage of its existence, while in the e cells it is in the terminal stage.

Pigment granules. Some of the islet cells contain yellowishbrown pigment granules which are well preserved in sublimate or in Rabl's mixture (fig. 22). They stain black by the Cajal method (fig. 49), as is the case with the pigment in nerve cells. They are present in two forms — small granules and relatively large, somewhat spherical corpuscles, which when viewed in sublimate or Rabl preparations, consist of a cortex of pigment granules, and of a more faintly staining internal substance. In the Cajal preparations, on the contrary, the whole of the corpuscle takes a deeply brown color and its surface is furnished with short, thorn-like prolongations which correspond to the row of pigment granules found in ordinar}^ preparation. Although varying in number and intracj^toplasmic position, they are present in the e cells, that is to say, in those which are laden with lipoid corpuscles. In addition, the pigment content of the cells is subjected to much variation according to the islet examined.

MITOSIS AND AMITOSIS

Bizzozero and Vassale ('87), Laguesse ('95-'96), Ssobolew ('02), Lane ('07-'08), and others record that they have found mitotic figures in islet cells. Laguesse ('09-'10), in addition, describes that in man mitosis is rarely found in the islet, whereas amitosis is not infrequently met with. In the cytological study of the islet m}^ attention was repeatedly attracted to the presence of karyokinetic figures in both b and e cells (figs. 56 to 58). Cells with two nuclei are also found in the islet, the nuclei being situated either one upon another or side by side according to the shape of the cell. It is my belief that they are produced, not by mitosis, but by an amitotic fragmentation of the nucleus. Cases are occasionally observed where a connection exists between the two nuclei which may be regarded as being in the nature of a constriction (fig. 59). On the other hand, the amitosis accom


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS 21

panies no degenerative process of the islet cell. So far as I have been able to ascertain, the islet cell undergoes no degeneration, at least there are visible no changes in the nucleus which maj^ be looked upon as degenerative. In a word, the islet cells multiply either by mitosis or amitosis, without decreasing in number by degeneration.

THE RELATIONS BETWEEN THE DIFFERENT TYPES OF ISLET CELLS

x4.s shown in the preceding pages, the islet cell contains several cytoplasmic structures, w^hich are not found in an equally developed state in one and the same cell, but rather in such a way that a given group of cells will be rich in one structure, while another group is poor in it. This makes possible several modes of classification of islet cells according to the character and amount of the structures concerned. These classifications have already been referred to. The next important step is to determine the types of islet cells by summarizing the results thus obtained. As I pointed out in the introduction, there is always danger of misinterpretation if one tries to classify the islet cells according to one or two in distinct* properties. We must therefore take into account as many positive structural characteristics as possible. The most practical and convenient plan is to sort out the cells according to a given structure, which serves as the fundament of classification, and then assign other structures to the types thus determined.

The following descriptions of the nuclei are based upon the preparations stained with iron hematoxylin or acid fuchsin, as the alum-hematoxylin preparation does not reveal any marked differences between the nuclei in the different types of cells.

1. a cells. This type of cell (fig. 14) is heavily laden with specific granules which are deeply stained with iron-hematoxylin or acid fuchsin. While mitochondria are lacking in these cells, it is highly probable that they are represented by the specific granules, the two structures bearing a striking resemblance to each other in their microchemical reactions. On the other hand, a cells contain few or no lipoid corpuscles, while the urano-argento


22 S. SAGUCHI

phile apparatus exists in the shape of dehcate tortuous filaments (fig. 25). The Golgi apparatus consists of spherical or elliptical corpuscles of irregular contour (figs. 34 to 36). Pigment granules are not seen. The cell-body of this type is cylindrical or conical in shape; its spherical nucleus is situated near the basis of the cell; it is surrounded by a thick nuclear membrane which is often thrown into folds and stains deeply with iron-hematoxyhn; the nuclear net is very distinct. The nucleolus is always smaller than that of the acinus cell.

2. b cells. The specific granules of b cells are faintly stained so that the granulation of the cytoplasm is indistinct (figs. 1 to 4, b, 16, 17). As is the case with a cells, mitochondrial filaments cannot be found. Lipoid corpuscles, however, are always present in b cells, though most of them are small in size (figs. 2 b, 16). The urano-argentophile apparatus is fairly well developed, and is either filamentous or granular in form (figs. 26 to 28). The Golgi apparatus occurs in the form of the typical network which may be regarded as being at the height of its development (figs. 38, 39, 40, 42). The 6-cell is cubical or cylindrical in form and contains an elliptical nucleus, the boundary of which, though indistinct, has generally an even contour; it is in the middle of the cell-body. The nuclear net stains very faintly. The nucleolus is small.

3. The c cells are the most ill- defined type of islet cells. The granulation of the cell-body is very indistinct and is replaced here and there, especially near one end of the cell, by a homogeneous protoplasm (figs. 52, 53). Although it is certain that these cells contain neither mitochondria nor lipoid corpuscles, it is difficult to decide in what form urano-argentophile and argentophile granules, as well as the intracellular apparatus of Golgi occur. It seems, however, that these structures are never found in a well-developed state (figs. 31, 32). The c cells are generally small in size and either cubical or narrow, long and cylindrical in shape. The nucleus is irregularly shaped and surrounded by a wrinkled boundary membrane; one or more small nucleoli, which are suspended in the ill-defined nuclear net, are visible in the nucleus.


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 23

4. The d cells are few in number and are interspersed among e cells (figs. 1, d, 20, 33). They differ from the latter in the following details: 1) the number of lipoid corpuscles is smaller than the e cells (figs. 1, d, 33); 2) the urano-argentophile apparatus seems to occur in the form of delicate filaments (fig. 33) ; 3) the protoplasm of the d cells stains more deeply and appears darker in iron-hematoxylin or uranic nitrate-silver preparations; 4) the mitochondria are more abundant than in e cells (fig. 1, d); 5) the nucleus exhibits a distinct network with closer meshes (fig. 1, d).

5. The e cells contain very few if any specific granules and thus the cell-body has a more transparent appearance. The most characteristic feature of the protoplasm of the e cells is the great abundance of mitochondrial filaments (figs. 1, e, 18, 19, 21), lipoid corpuscles (figs. 5, e, 41) and urano-argentophile granules (figs. 6, e, 29, 30), whereas argentophile granules appear to be entirely absent (fig. 48). The Golgi apparatus, in the form of either rings or threads (fig. 21), and pigment (figs. 22, 49) are also visible in them. The cells are the largest of the islet cells and are mostly of cylindrical form. The nucleus, as seen in iron-hematoxylin preparations, is similar to that of the b cell. It is oval in shape and usually situated at the middle of the cell-body, though not rarely it is placed near an end of the cell. The faintly staining nuclear network is provided with one or several small nucleoli.

It can scarcely be said that all the islet cells belong to one of the five types just spoken of, as there can be found intermediate forms. For example, the cell shown in figure 15 must be regarded as a transitional form between the a and 6 or a and e cell. The specific granules of this cell are of the same nature as those of the a cell, but the nucleus has an oval form with an even contour, and it has an indistinct nuclear network, just as is the case with the 6 or e cells. The b and e cells, on the other hand, have lipoid corpuscles in common. There are cells which, like the e cells, contain a large number of lipoid corpuscles, but in which specific granules, though few in number, are visible. These cells must be considered as intermediate between b and e cells. In fact,


24 S. SAGUCHI

there are found cells which contain both specific granules and mitochondrial filaments. The relation of c cells to other types of islet cells is a problem which is difficult to solve. From the granular appearance of their protoplasm and the absence of mitochondrial filaments, it seems not unlikely . that they bear some relation to b cells. But the possibilitj^ of a transition between e and c cells is by no means to be excluded.

Summarizing the above observations upon the cytological character of the islet cells, we come to the conclusion that the five types of islet cells are connected with one another by all grades of transition: a cells pass into b cells and the latter into e cells ; on the other hand, e cells pass into b cells and perhaps into c cells. The d cells and e cells have many characteristics in common, so that a transition between them is quite conceivable. I am of the opinion that the d cell is a resting phase of the e cell; the nucleolar hyperchromasy of the nucleus and cytoplasm with abundant production of mitochondrial filaments strongly suggests this view. From these considerations it must be admitted that a cells and c cells, though connected with 6 or e cells, have no relation to any other types of islet cells; in other words, they cannot be regarded as intermediate between any two types. Are they to be regarded as a resting phase of other types of islet cells, or as an intermediate condition between the islet cell and some other cell which forms a constituent of the pancreas? To solve this complex and difficult question it is not sufficient to investigate the islet tissue only, but other elements of the pancreas, especially the acinus cells, must be subjected to a careful examination. If, in this way, a transition between the a cells or c cells and the acinus cells can be traced, there can be no doubt that the islet is not an independent organ. A discussion of this matter wdll be taken up in connection with the relation between islet and acinus cells.

DISTRIBUTION OF ISLET CELLS IN THE PANCREAS

Since the pancreas of the frog is small, it is an easy matter to cut cross-sections through it and to examine the distribution of the islet cells. If search is made in such sections for cells which


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 25

show the characteristics mentioned above and the cells sketched, it will be found that islet cells (for the sake of simplicity, this designation is given not only to cells which constitute the typical islet, but also to those which occur either in small groups or individually, and are to be identified with the former) are scattered throughout the pancreas either individually or in smaller or larger groups, as shown in figures 60 and 61. Whether the predominant occurrence of the islet cells is single or in groups seems to depend upon the region from which the section is taken.

That islet cells occur alone or in small groups among acinus cells has been noted and described b^^ Laguesse ('01), TscKassownikow ('00), Vincent and Thompson ('07), Bensley ('11-' 12), and others. According to Laguesse, these cells contain safranophile granules which are either scattered throughout the cellbody or confined to the basal portion of it. Tschassownikow believes these granules to be chromatophile, while Vincent and Thompson say that the protoplasm of these cells gives a bathychrome reaction. Piazza ('11), on the contrary, is of the opinion that these solitary cells belong to islets, the main part of which is not contained in the section under examination, for they are laden with argentophile granules as is the case with cells of the typical islet. On the other hand, the cells, containing minute granules, which Arnold ('12) regards as a sort of acinus cell, are to be identified with solitary islet cells. At least, from the microchemical reactions of the granules found by him, it is probable that they correspond to the a or h type above referred to.

In the pancreas of the frog the solitary islet cells are met with everjrwhere, not only in the center, but also near or even at the periphery immediately beneath the capsule. Most of them are of the h type, while cells belonging to the a type are few in number. I was unable, however, to determine whether or not other types of solitary islet cells occur. The solitary cell (fig. 2, h) is situated between the acinus cells ; at one extremity it is in contact with the basal membrane, while the other extremity ends either between the neighboring acinus cells or reaches the lumen. Under these circumstances, it often occurs that the solitary cells, especially those belonging to the a type, extend along the basal


26 S. SAGUCHI

membrane, thus assuming a polj^gonal or stellate form. The fact worthy of note is that the solitary islet cells are in close relation to the blood-capillaries, just as the case with the typical islet. In fact, the blood-capillary with which they are in contact is in a distended condition.

Besides the solitary islet cells, there are found scattered through the pancreas small groups of cells. These behave, in relation to the neighboring acinus cells as well as to the basal membrane, like the solitary cells. These groups of cells may be designated as non-tj'pical islets. The cells composing them belong either to one or to several acini (figs. 3, b, e; 7). In the latter instance the islet cells belonging to different acini are usually separated by a blood-capillary to which they converge. In either case, the islet in question is composed, in most cases, of two kinds of cells — b and e. It is extremely rare to see an islet composed of e cells only. The b cells are situated at the periphery of this group, while the e cells are in the center; in other words, the e cells are separated from the neighboring acinus cells by b cells. Islets of this type are distributed throughout the pancreas; in the superficial portion of the organ, however, they are rarely met with.

The most characteristic feature of the solitary islet cells and of the cells composing the non-typical islet above referred to is that they are situated in the acinus and are arranged in the same row as the neighboring acinus cells (fig. 7) . In the third form of cell groups, which may be designated as typical islets, the cells are disposed in an entirely different manner. Figures 1, 4, 5, and 6 show parts of such a typical islet. It consists of cell cords which anastomose with one another and form a sort of network, the meshes of which are occupied by blood-capillaries. The ends of the cord may pass gradually into the pancreatic acini or may be separated from the latter by the basal membrane. In the former case islet cells are in immediate contact with acinus cells; in other words, the islet cells are enclosed by a connective-tissue membrane which is in direct continuity with the basal membrane of the acini. The cells composing the cord are mostly of elongated cylindrical form, and are arranged in a row in side-by-side


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 27

apposition, the both ends of the cell being in contact with the membrane which encloses the cord. There are, however, cells which end freely before reaching the membrane of the opposite side; this is dependent, in part at least, upon the direction in which the section is carried through the cords. Cases in which the cells of the islet show no regular arrangement may also be due to the same cause. It sometimes happens that two cords run side by side, separated only by the connective-tissue membrane (fig. 6). My observations show that all typical islets, however diversified they may appear, consist of a system of cellcords which intertwine with blood-capillaries.

The typical islets usually consist of cells already mentioned. In most of the islets, especially in the larger ones, the e cells are the most abundant, although the h cells may sometimes exceed them in number, especially in the smaller islets, while a, c, and d cells are few in number or entirely lacking (fig. 1). As regards the position of these different types, it can generally be said that the h cells are situated at the periphery of the islet so as to form a sort of cortical layer. As is the case with the non-typical islet, the e cells are here, too, separated from the neighboring acinus cells by h cells. In addition, the h cells not infrequently are of such a form and arrangement as to suggest compression from the acinus tissue, d cells, if present at all, are almost always interposed between the e cells, while a and c cells are located at the periphery of the islet. The e cells lie in contact v/ith the bloodcapillaries, which is not always the case with h cells.

The typical islets are, in the majority of cases, larger than the non-typical ones, and may be found everywhere in the pancreas except in the peripheral layer, the largest ones being located generally near the center of cross-sections of the pancreas.

It follows from the above observations that the pancreas contains either solitary islet cells or groups comprised of few or many cells. These cell groups may be subdivided into nontypical and typical islets. Most of the islet cells belong to the e and h type, while a, c, and d cells are few in number. The e cells form an essential constituent of the typical islet, whereas h cells are usually isolated or occur in small groups. Solitary cells


28 S. SAGUCHI

and cells in small groups are enclosed by the basal membrane, thus forming a part of the acinus. The cell cord of the typical islet is enclosed by a delicate membrane which is either in direct continuity with the basal membrane of the acinus or separates the islet cells from the neighboring acinus cells; a special boundary membrane or capsule around the islet does not exist.

RELATION BETWEEN ISLET CELLS AND ACINUS CELLS

Postembryonic development of islet cells

Concerning the development of the islet, there is wide divergence of opinion among different investigators. Gianneh ('98), Diamare ('99), Opie ('00), Pearce ('02-'03), Kuster ('04), Helly ('06), and others beheve that the islet, although derived from the primitive undifferentiated pancreatic cell cords of the acinus remains independent through life. There are many others who assume that not only primitive cell cords, but also specific differentiated pancreatic cells (i.e., acinus cells containing zymogen granules) may give rise to islet cells. Lewaschew ('86), Pischinger ('95), Laguesse ('95-'96, '01, '05,'09-'10),Mankowski ('02), Tschassownikow ('06), Dale ('04), Vincent and Thompson ('07), and Fischer ('12) are supporters of this hypothesis. The arguments on which it is based are: 1) the absence of a boundary membrane between the islet and acinus tissue and the immediate contact or connection between the two; 2) the presence of transitional forms between them. It has been assumed by some that acinus cells transform into islet cells by the gradual disappearance of zymogen granules and by the change in shape, size, and staining reactions of the cell-body and the nucleus, while the lumen is lost to view and the cell boundaries become indistinct. Laguesse ('01), among others, states that in the transformation of acinus cells into islet cells the zymogen granules diminish in number and come to stain more faintly, while the minute granules characteristic of the islet cells appear in the base of the cell. These granules gradually increase in number so that the cell in question finally assumes the appearance of a typical islet cell. Tschassownikow ('06) regards the cells which contain chroma


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS 29

tophile granules, and which are scattered through the pancreas individually or in small groups, as transitional between the acinus and islet cells.

Other investigators have opposed the above view, and they attach considerable importance to the presence of the connectivetissue membrane which forms a sort of capsule around the islet, sharply marking it off from the acinus tissue. Piazza, who takes into consideration the fact that the behavior of the blood and nerve supply and of the connective tissue of the islet is quite different from that of the acinus tissue, and especially the fact that the chemical characters of cells of the two tissues differ widely from each other, has come to the conclusion that a transition between the two is impossible.

Experimental studies also have been made to solve this complex and difficult problem. Some investigators have found that the ligature of the pancreatic duct results in the disappearance of the acinus tissue, while the islets remain intact. Schulze ('00), Ssobolew ('02), and others have, from these observations, been led to the conclusion that the islet is an independent organ, whereas Laguesse ('05) claims that these findings are not contrary to his view that the islet is derived from the acinus tissue. Mankowski found that both tissues disappeared in the same experiment. On the other hand, there are many investigators who have observed, after the injection of pilocarpin or secretin or after a period of fasting, that the islets increased in number; according to Gelle ('11), Fischer ('12), and Retterer and Lelievre ('13), this is due to the increased transformation of acinus cells into islet ceUs.

Kyrle and Weichselbaum (Kyrle, '08; Weichselbaum and Kyrle, '09) believe that the islet cells are derived from small pancreatic ducts, while Hansemann ('02) attributes a mesenchymal origin to the islet. Their views, however, seem to have gained few adherents among histologists.

The behavior of the capsule or the size and number of the islets, in either normal or experimental conditions, is not sufficient to solve the problem of the genetic connection between the islet and acinus tissue. Transparent cells containing no zymogen


30 S. SAGUCHI

granules cannot always be regarded as transitions, for the resting acinus cells and centroacinus cells may have the same appearance; a distinction between the two is often made with difficulty in cases where the islet cells are interspersed among acinus cells. Cells containing, on the one hand, minute granules which are assumed to be a characteristic constituent of the islet cell, and, on the other, zymogen granules, as has been described by Laguesse, can certainly be considered to be transitional between the acinus and islet tissue. It must be borne in mind, however, that there are islet cells in which the minute granules cannot be manifested and the protoplasm of which, in ordinary preparations exhibits a clear, transparent appearance.

That the islet cells can be characterized by the presence not only of minute granules, but also of several other protoplasmic structures, has been mentioned above, and if there be a transformation of acinus cells into islet cells, the formation of the specific cytoplasmic constituents in the latter cells and the disappearance of those in the former must take place in a visible manner; thus the general process of transformation can be followed more accurately than with any other methods. For this reason as many recent cytological methods as possible must be employed in the investigation of the islet, and, at the same time, the minute structures of the acinus cells must be taken into account.

From a careful study I have come to the conclusion that there is a transformation of the acinus cell into the islet cell, whereby not only the cell-body and the nucleus change shape and position, but the intracytoplasmic structures also undergo a series of definite alterations. This process of transformation may be divided into two stages:

First stage. The acinus cells which are about to transform into islet cells gradually decrease in volume, and some of them take a rather elongated form as if from compression between the neighboring cells (figs. 3, A', 8, 9). Concomitant with this change, a gradual reduction in the number of zymogen granules takes place. The nucleus seems to undergo no remarkable change in size, although it takes the form best adapted to that


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 31

of the cell-body. The striking changes are those of the intranuclear network. The nucleolus or nucleoli, as well as nucleolar corpuscles, increase in volume so that the network becomes more pronounced; the nuclear membrane, in addition, increases in thickness, stains more deeply than before, and is often thrown into folds. In other words, the nucleus exhibits a marked nucleolar hyperchromasy (Saguchi, '15). The process is not confined to the nucleus; it extends at successive periods, to the cytoplasm so that the latter stains more or less deeply with iron-hemotoxylin or acid fuchsin.

The most striking change in the cytoplasm is that of the mitochondria. In my previous work I mentioned that the acinus cell contains thick and coarse mitochondrial filaments. In the first stage of transformation (fig. 8) there appear along the course or at the ends of these filaments, which have meanwhile become thickened and more or less shortened, spherical or oval enlargements in which one can distinguish very soon after their formation a clear inner part and a heavily staining cortical layer, due perhaps to the production of liquid in the accumulated mass of mitochondrial substance. This process of liquefaction extends over the whole length of the thickened mitochondrial filaments, while the heavily staining cortical layer disappears; at last there remain only canaliculi or spaces filled with the clear liquid (figs. 9, 10).

In preparations treated by the uranic nitrate-silver methods of Cajal, one often sees acinus cells containing 'ong or short, brown or black, spherical or oval, rod-, club-, or dumb-bell-shaped corpuscles which are scattered through the cytoplasm, often reaching the distal end of the cell (fig. 24). Concerning the significance of these corpuscles no definite conclusion could be drawn from my observations since I could not follow out the process of their formation. One thing, however, is certain — they have no definite genetic connection with the Golgi apparatus, which shows the same staining reaction, for there are found cells in which these structures are visible, independent of the network apparatus near the base of the cell. I am of the opinion that the formation of these corpuscles and the liquefaction of mitochondrial filaments are one and the same process; the mitochon THE AMERICAN JOUKNAL OF ANATOMY, VOL. 28, NO. 1


32 S. SAGUCHI

dria undergo a definite change and there is formed a substance which no longer exhibits the specific mitochondrial reaction, but becomes impregnated by the Cajal method. It will be seen from a comparison of the preparations that the above-mentioned bodies are in full accord not only in position, but also in shape, with the clear spaces or vacuoles produced by the liquefaction of mitochondria, so that it is conceivable that the former is only the positive figure of the latter. The corpuscles gradually disappear from the cell. From the fact that they show a tendency to proceed from the basal part of the cell toward the lumen, and that they are often gathered near the latter, it is presumable that they become in part, at least, extruded into the lumen.

Second stage. A remarkable phenomenon of this stage is the passing out of a certain intranuclear constituent. In the preparations fixed in osmic mixtures, especially in sublimate-osmicchromic acid, or in Zenker's mixture, and stained with ironhematoxyhn or acid fuchsin, there can easily be found nuclei from which a deeply stained substance passes out in the form of delicate, tortuous filaments or of minute granules (figs. 10, 11). These at first are accumulated around the nucleus, but gradually extend over the whole cytoplasm, and are finally so densely packed together that the individual filaments are no longer visible, the cytoplasm exhibiting rather a granular appearance (fig. 13). These granules or filaments are nothing else than what we have designated specific granules and the cell in question now becomes a cell (fig. 14).

The nucleus maintains its original spherical or polygonal form and the meshes of the nuclear net become closer. The most striking feature in this stage consists in the decrease in size and volume of the nucleolus and nucleolar corpuscles, which may perhaps be correlated with the passing out of the filaments or granules. I am of the opinion that the specific granules of the islet cell are derived from the nucleolar substance. The nucleolus passes out of the nucleus not only in the mode described above, but also in the form of larger spherical droplets or corpuscles formed by its constriction as is seen in figure 15.


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 33

Argentophile granules and urano-argentophile filaments seem to make their appearance in this stage; their development, so far as can be seen from my material, has already been mentioned (figs. 25, 45, 46). Certainly, the cells with nuclei containing many argentophile granules, as well as those with delicate, tortuous urano-argentophile filaments, judging from their shape and position, are nothing but acinus cells which are destined to transform into islet cells.

The Golgi apparatus of the acinus cell decreases in volume in the first stage (fig. 24), and seems to disappear finally. The irregularly spherical or oval corpuscles described above, which show the same staining reaction as the apparatus and which form a network by fusion, in all probability appear in the second stage (figs. 34 to 36). How they are formed and what relation they bear to other cell constituents it is impossible to determine definitely, although it is certain that they are neither the remains of, nor are they derived from, the Golgi apparatus of the acinus cell from which the islet cell has taken origin.

It will be seen from the foregoing that the most striking features in the transformation of acinus cells into islet cells are, on the one hand, the disappearance of zymogen granules, mitochondria, and the Golgi apparatus characteristic of the former cells and, on the other, the increase in the amount of nucleolar substance and of nucleolini, followed by their passing out of the nucleus. This nucleolar substance, after passing out, forms the specific granules of the a and h type of cell, while the nucleolini give rise to the argentophile granules. The urano-argentophile apparatus and the intracellular network of the islet cell are also newly formed cell-structures. The passing out of the nucleolar substance and nucleolini leads to the decrease in volume of the main nucleolus — a condition which is regarded as characteristic of the islet cell. It must be mentioned, however, that this process is not limited to the above case; I have also found (Saguchi, '20) that the mitochondrial filaments of the acinus cell are derived from the nucleolar substance, and that nucleolini can pass out of the nucleolus and eventually out of the nucleus. In the case of the islet cells, the process must be regarded as being accelerated to a considerable degree.


34 S. SAGUCHI

Ultimate fate of islet cells

Most of the investigators who beheve that islet cells are derived from acinus cells seem to admit that there is also a reversal of this transition. According to Lewaschew ('86), the transformation begins with the accumulation of the cytoplasm around the nucleus and ends with the appearance of zymogen granules, while the cell boundaries become gradually more pronounced and the cell-body larger. Laguesse ('95-'96, '01) likewise describes that, in this transformation, the cell-body becomes pyramidal; the nucleus, in which a large nucleolus now appears, passes to the base of the cell and the minute granules of the cj^toplasm disappear, while zymogen granules are produced. In addition, this author pointed out that it is more difficult to follow 'the period of involution' (that is to say, the stage in which the islet returns to the acinus) than 'the period of deconstitution of the acinus,' as the process of involution rapidly spreads over the islet. Laguesse ('01) regards, as transitional, areas that are occupied by somewhat larger, indistinctly bounded cells, and by more numerous centroacinus cells, and through which solitary islet cells or small groups of them are scattered.

It is, as pointed out by Laguesse, a difficult matter to follow the transformation of islet cells into acinus cells; this is perhaps due to the very indistinct nature of change which takes place in the process. Nevertheless, I have been able to detect what are to be regarded as transitional forms between the two tissues. While c cells (figs. 52, 53), as shown under the previous heading, can be considered as derived from 6 or e cells, there exists, on the other hand, no connection between these and any other type of islet cells. They seem rather to be in close relationship with the acinus cells. In the peripheral part of the islet we often find cells with a non-granular, rather transparent cytoplasm, and a more round, vesicular nuclei (figs. 54, 55). These cells may be regarded as transitional between c cells and acinus cells; in common with the former, they contain no zymogen granules and the nucleolus is small, while they have the non-granular cytoplasm with a small number of mitochondrial filaments charac


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 35

t eristic of acinus cells. They cannot be regarded as acinus cells in the resting state, since the resting cell, besides still containing some zymogen granules, has a large nucleolus and a great number of thick mitochondrial filaments.

From these observations I have been led to the conclusion that the transformation of islet cells into acinus cells takes place in the following manner: First, the cytoplasm of the c cell becomes transparent by losing its granular contents; the nucleus then rounds off and comes to stain a darker color; finall}^, there appear mitochondrial filaments around the nucleus. The cells thus formed, yet containing neither zymogen granules nor large nucleoli, remain for some time in the peripheral part of the islet, during which period they store up, on the one hand, the nucleolar substance so that the nucleolus increases in volume, and produce, on the other, mitochondrial filaments which afterward can take part in the formation of zymogen granules, as do the typical acinus cells.

Formation and fate of the typical islet

From the above observations it follows that a cells are derived from granular cells and that c cells are transformed into acinus cells. On the other hand, there exist transitions between the various types of islet cells; that is to say, between a cells and h or e cells and between h and c cells. Considering the existence of these transitions, it is conceivable that the cells constituting the typical islet are supplied from acinus cells on the one hand, and transformed into them on the other. The formation of the typical islet (see text-figures) must be regarded as being inaugurated with the development of solitary a cells interspersed among acinus cells from which they are derived (fig. 2 and text fig. a). These solitary islet cells with the addition of a few newly formed ones constitute small islets. They consist, for the most part, of h cells, situated at the periphery, and of e cells, placed in the center, the cells belonging either to an acinus or to two or three neighboring acini (fig. 3). The latter is especially the case if the islet shows a tendency to enlarge. But it cannot be said that all of the small islets pass into the large typical ones. Most


36


S. SAGUCHI


of them return, I think, after a relatively long duration, to the acinus tissue through the stage of the c cell; this seems particularly to be the case in the peripheral part of the pancreas, where large islets usually are not found. Some of them, however, especially those which are situated in the center of the pancreas,




y^y


j-^-z^-f-^J



c a

Figs, a, b, c, d Schematic figures representing the development of islet cells and the change of their arrangement for the formation of the typical islet. Islet cells are shaded; 6c, blood-capillary.


may enlarge at the expense of the whole acinus, even of the several neighboring acini. At the beginning of this transformation the initial islet cells maintain the same arrangement as is seen in an acinus; at one end, they are in contact with the basal membrane; at the other, with the cells of the opposite side so that the contact line is in continuity with that of the neighboring


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 37

acinus cells, as seen in figure 7 (also text fig. b) . In the course of time, this arrangement changes; the cells become elongated so that there is interdigitation between the opposite cells (text fig. c) ; the process continues until the upper ends of the cells touch the basal membrane of the opposite side. Thus the typical cellcord of the islet is formed (text fig. d, and fig. 1). From this mode of formation, it is evident that the diameter of the cord is nearly the same as that of the acinus, most of the islet cell being double the length of the acinus cell. The typical islet thus formed seems to be in existence for a relatively long period, during which the islet cells are formed by the conversion of acinus cells, while some of them revert to the latter. This influences not only the form, but also the size of the islet, and conditions the inconstancy of the contour. From these circumstances, it is conceivable that, if the newly formed islet cells exceed in number those cells which are destined to return to the acinus tissue, the islet would increase in size; and that, if the reverse is the case, it would be reduced. In addition, the increase in volume of the islet seems, in part at least, to be effected by the mitosis and amitosis taking place within it; the decrease in volume, on the contrary, is due entirely to the return of the islet cells to the acinus tissue. Notwithstanding close inspection, I have not been able to detect any degenerative process in islet cells, the amitotic figure often found in the islet being no sign of cell degeneration as shown in the previous chapter,

I cannot say how long the typical islet exists as such. The process of increase and decrease in bulk must take place very slowly. It is also an extremely difficult matter to determine whether an islet is in the course of development or of involution. Laguesse ('01, '09-' 10) distinguishes between three periods in his evolutionary cycle of the islet: the deconstitution period of the acinus, the resting period, and the involution period of the islet. I am of a different opinion, so far as I have been able to ascertain, no such distinction can be made in the structure of various islets, but one islet may contain cells in all three periods; in other words, it may have not only b and e cells, but also a and c cells. The ratio of these types of cells in the islet is not in every


38 S. SAGUCHI

case the same, which fact may be taken advantage of in determining whether an islet is in the evolutive or in the involutive period. Some difficulty is met with, however, by this determination; for example, in a case where the islet is rich in b cells, it is hard to decide whether they are derived from a cells or from e cells. On the other hand, I have not been able to perceive a figure corresponding to the involutive change mentioned by Laguesse which occurs so abruptly that it spreads rapidly over the whole islet. In fact, solitary islet cells and small groups can be found scattered throughout the pancreas of the frog, and it would be obviously absurd to connect these with the involutive change.

FUNCTIONAL SIGNIFICANCE OF ISLET CELLS

As to the functional significance of the islet there is considerable difference of opinion, which may be briefly summarized as follows: 1) The islets have no function worth mentioning ( Nagay o) . 2) They have something to do with the nervous system (Langerhans, '69). 3) They belong to the lymphatic structure (Kiihne and Lea, Sokoloff, Renaut, Mouret, Pischinger, Pugnamt, Schlesinger, Katz and Winkler) . 4) They are either embryonic remains or incompletely developed acini (Gibbes, Piersol, Harris and Gow, Gianelli '00). 5) They are either exhausted or temporarily modified parts of the pancreatic acini (Lewashew, '86; Mankowski,'02; Vincent and Thompson, '06). 6) The islet cells are real glandular elements and yield the secreted matter to the pancreatic duct (a view advanced by Gianelli in 1898).

7) The islets are either the parenchyma changed pathologically or a stage of its regressive metamorphosis (Kasahara, Grineff, '11; Fischer, '12); Dogiel ('93) believes them to be dead spots.

8) The islets belong to that group of glands which are assumed to take part in the so-called internal secretion. This is the view of many investigators, such as Laguesse ('93, '95-'96, '09-' 10), Diamare ('99), Ebner ('99), Hansemann ('02), Pearce ('02-'03), Heiberg ('09), Piazza ('11), and others.

It is evident, from these various views advanced by different investigators, that the islet is a tissue, the functional significance


CYTOLOGICAL STUDIES OF LANGERHANS's ISLETS 39

of which is extremely difficult to determine. From the topographical and cytological behavior of the islet, it is impossible to conceive that it has no function, or that there exists any connecJ:ion between the islet and the nervous or lymphatic system. The protoplasm of the cell elements composing the islet is too well differentiated to be regarded as embryonic remains. In their fully developed condition they have no connection with the lumen, which excludes the possibility that they participate in the pancreatic secretion. The islet cells show no sign of regressive metamorphosis. There can be found no degenerative change of the nucleus; the latter rather multiplies by mitosis and, in part at least, by amitosis which never leads to the death of the cell. Most of the islet cells contain fatlike corpuscles; this accumulation of fat, however, is not associated in any way with the degeneration of the cell as mentioned by Dogiel, but seems rather to have an important share in the function of the cell. Finally, islet cells cannot be identified with a resting stage of acinus cells, for they differ from the exhausted cells in their cytoplasmic structure.

The various views mentioned above being excluded as improbable, we are left with a conception that the islet is an organ for internal secretion, giving certain substances to the bloodstream. I, for my part, do not hesitate to admit this hypothesis as the most probable one. A strong argument for it is that most of the islet cells are, at one or both ends, in contact with distended capillaries. Even when there is a solitary cell or a few cells forming a group, the blood-capillary to which the islet cells are attached has a wide lumen. It is also seen that the bloodcapillary widens with the development of the islet. These facts, together with the fact that the islets have no lumina which are continuous with the pancreatic duct, strongly suggest that the cells bear a close relation to the blood-vessels. Import and export of substances are faculties with which living cells are endowed; the gland cells, for example, take on materials from the blood-stream in order to give the secreted fluid to the secretory duct. In such a tissue as the islet, where most of the cells are not in contact with any other tissue than a, blood-vessel, the elaborated products can be given only to the blood-stream.


40 S. SAGUCHI

Since the substances imported from the blood usually do not appear as formed elements, it is safe to conclude that the various cytoplasmic constituents of the islet cell are morphological expressions of substances formed by the elaboration of the imported material and are to pass into the blood-stream.

Now the question arises, which of the cytoplasmic constituents must be regarded as the specific secretion of the islet cell? As for the specific granules, they are contained in the a and h cells which are situated in the peripheral part of the typical islet and which are often of such a form and an arrangement to suggest compression against the neighboring acinus tissue, so that they do not always come into intimate relationship with the blood-capillary. These facts demonstrate sufficiently that the specific granules take no important part in the secretion of the islet cell. Like the mitochondrial substance to which they are similar in chemical and staining reactions, they are to be regarded rather as the mother-substance of secretion.

The e cells, on the contrary, form the principal elements of the islet, being in close relation to the blood-vesselsi The lipoid corpuscles and urano-argentophile apparatus, which are present in a fully developed state in the e cells, must therefore be looked upon as specific, secreted matter of the islet. In a previous paper ('20) I have shown that the acinus cell produces two sorts of secretions: one derived from zymogen granules and the other collected in the form of the Golgi intracellular apparatus. In a similar manner, the islet cell produces the lipoid and urano-argentophile substance; the lipoid corpuscles differ in some respects from zymogen granules, although the two are in accord in that they both offer little resistance to the action of acetic acid; they bear rather a strong resemblance in its chemical character to the lipoid granules found in the basal portion of the acinus cell; at least, the result of fixing and staining of the two is nearly the same. The urano-argentophile apparatus, on the other hand, corresponds to the Golgi intracellular apparatus of the acinus cell in that they can both be brought into view by the Cajal uranic nitrate-silver method. But that there is some difference between them is obvious from the fact that the Weigl and Kopsch


CYTO LOGICAL STUDIES OF LANGERHANS's ISLETS 41

methods do not exhibit the same thing, but another network of a different nature in a and h cells. However that may be, I am of the opinion that the above two cell constituents, after having undergone a certain chemical alteration, pass into the bloodcapillary.

I have mentioned in my previous paper on the pancreas of the frog that the acinus cell contains lipoid granules which are collected in the basal portion, and that it is highly probable that these pass into the blood-vessel and therefore may be looked upon as internal secretion matter. From this it is conceivable with some reason that the internal secretion of the pancreas is derived from islet cells, as well as from acinus cells, though to a less extent, a view held by Lepine ('05), Sirtori, ('07), and Grineff ('11). But the same idea may also be formed from another point of view; that is to say, from the fact that the cells which are considered to yield internal secretions not only form larger and smaller groups but are also scattered throughout the pancreas.

Kanazawa, Japan

January 5, 1920


42 S. SAGUCHI

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mikros. Anat., Bd. 67. Vincent, S., and Thompson, F. D. 1907 On the relation between the 'islets of

Langerhans' and the zymogenous tubules of the pancreas. Internat.

Monatschr. Anat. u. Physiol., Bd. 24. Weichselbaum, A., und Kyrle, J. 1909 Ueber das Verhalten der Langerhans'schen Inseln des menschlichen Pankreas im foetalen und post foetalen Leben. Arch, mikros. Anat., Bd. 74.


PLATES


45


PLATE 1


EXPLAXATION OF FIGURES


1 A part of the typical islet, b, 6-type of cell; d, d-type; e, e-type; A, acinus cell; be, blood-capillary. Fixation: sublimate-osmic-chromic acid (saturated solution of sublimate, 4 parts; 2 per cent osmic acid, 2 parts; 1 per cent chromic acid, 4 parts); staining: iron-hematolxyin. XllOO.

2 Cross-section of an acinus containing a solitary islet cell, b. be, bloodcapillary; A, acinus cell. The same fixing as figure 1, and staining according to Altmann's acid fuchsin method. XllOO.

3 A non-typical islet consisting of a few numbers of islet cells; the latter belonging to three adjoining acini, b, 6-type; e, e-type; C, centroacinus cell; A, acinus cell; A', acinus cell showing the nucleolar hyperchromasy of the nucleus and the cytoplasm, and preparing for transformation into the islet cell. The same fixing and staining as figure 1. XllOO.


46


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS

8. SAGUCHI


PLATE I


« 4





l^




t;:;*"




■-^>i




'■9>J "r'-iS




' 1 ^ i:


be


V


2




?^— tA!


G


,^bG



47


THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 1


PLATE 2


EXPLANATION OF FIGURES


4 A part of the typical islet, b, b-type of cell; c, c-type; e, e-type; be, bloodcapillary; A, acinus cell. Fixed in 4 per cent formaldehyde and stained with iron-hematoxylin. X 1 100.

5 A part of the typical islet, e, c-type of cell; be, blood-capillary; A, acinus cell. Fixed and stained according to Ciaccio's method for lipoid proper ('11). XllOO.

6 A part of tj'pical islet, e, c-type of cell; be, blood-capillary; A, acinus cell; Treated according to Cajal's uranic nitrate-silver method for the Golgi's net. XllOO.

7 A non-typical islet; a group of islet cells at the end of an acinus, where they have not yet their definitive shape and arrangement, the contact line of the opposite acinus cells being in direct continuity with that of the opposite acinus cells. 7, islet cell (e-type); A, acinus cell. Fixation: sublimate-osmic acid; staining: iron-hematoxylin. XllOO.


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS

S. SAGUCHI


PLATE 2






bc


'r.




, > .


^' .*'.'>


e


be b


be


..V




e


• '•: -r•.•,■,•w-■-^c^.■iC^•"■


A




v}-vi:."p:y--:^


-"-■X<- ' r'





A 6


A ! •


e— -•• .-.•'••« be •-'




be ^ *••:•?;


« •,


— ^ .«k;.* • '


49


PLATE 3


EXPLANATION OF FIGURES


Figures 9, 10, 14, 15, 17, 20, and 21 are of sections of the pancreas fixed and stained as in figure 1, plate 1. Figures 8, 11, 12, and 16 are of preparations treated as in figure 2, plate 1. Figure 13 is of a section fixed in Zenker's fluid and stained with iron-hematoxylin. Figure 18: fixing according to Meves and staining with iron-hematoxylin. Figure 19: the same fixing and staining as figure 7. Figure 22: fixing according to Rabl (sublimate-picric acid) and staining with iron-hematoxylin. Figure 23: fixing in trichloracetic acid according to Holmgren and staining with Mayer's hemalum. X2400.

In figures 8 to 59, all cells are so delineated that the cell-ends where they are in contact with blood-capillaries are always directed toward the lower side of the plate, except the case in which the islet cell comes in contact with the blood-vessel at both ends. X2400.

8 to 11 Various stages of transformation of acinus cells into islet cells. In figures 8 and 9 the nucleus and the cytoplasm show the nucleolar hyperchromasy; mitochondrial filaments being in the process of liquefaction. Figures 10 and 11 show the passing out of an intranuclear constituent in the form of filaments or of granules; diminution of the nucleolus. X2400.

12 and 13 Accumulation of the granules and filaments passed out. X2400.

14 a-type of cell. X2400.

15 A transitional form between the a-type and b- or e-types of cell. X2400.

16 /^-type of cell, containing specific granules and some lipoid corpuscles. X2400.

17 6-type of cell. X2400.

18 e-type of cell, containing delicate mitochondrial filaments, and vacuoles corresponding to the lipoid corpuscles dissolved out. X2400.

19 e-type of cell, containing mitochondrial filaments, which are somewhat thickened in consequence of the action of osmic acid. X2400.

20 d-type of cell. X2400.

21 e-type of cell. X2400.

22 e-type of cell, containing pigment corpuscles and granules. X2400.

23 e-type of cell; the nuclear network is distinctly stained with hemahmi. X2400.


5:)


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS

8. SAGUCHI


PLATE 3



V





11



13


&.;




L **


R^X


15


< ' .

\ 'i -'s^'^


/..y"; ■:' v^ *1^^.




V ".^ .^:


16


i'i


I '


— -~^^v


.^/


I


V


mi

iiU 19


If-:


20





.:; n


i


22



51


PLATE 4

EXPLAXATIOX OF FIGURES

Figures 24 to 33 are treated according to the Cajal's uranic nitrate-silver method for the Golgi's network. Figures 34 to 41: according t ) Weigl's osmic acid method for the Golgi's net. Figures 42 to 44: fixing in saturated solution of sublimate, and staining with iron-hematoxylin. X2400.

24 Acinus cell preparing for transformation into the islet cell, the product of liquefaction of mitochondria being seen as droplets stained a darkly brown color. X2400.

25 to 28 Various stages of develojiment of the urano-argentophile apparatus. The cells belong to the a- and ?;-typ'3s. X2400.

29 and 30 Cells (e-type) containing the fully developed urano-argentophile apparatus, and vacuoles corresponding to the lipoid corpuscles dissolved out. X2400.

31 and 32 Cells belonging to the c-type. X2400.

33 A cell with the darkly staining protoplasm and the filamentous uranoargentophile apparatus, perhaps corresponding to the d-type. X2400.

34 to 37 Acinus cells preparing to transform into islet cells, round or oval irregularly shaped corpuscles appearing in the cytoplasm. X2400.

38 to 40, 42 to 44 Various forms of the Golgi's network. X2400.

41 A fully developed e-cell, which contains no Golgi apparatus. X2400.


52


CYTOLOGICAL STUDIES OP LANGERHANS'S ISLETS

S. SAGUCHI


PLATE 4




^^^l/:^


53


PLATE 5


EXPLANATION OF FIGURES


Figures 45 to 50 are of sections fixed in 10 per cent formalin and afterw^ard treated with silver nitrate according to Cajal. Figure 51: fixing in 10 per cent formalin, and staining with Altmann's acid fuchsin. Figures 52 to 55, 58 and 59: from sections fixed in sublimate-osmic-chromic acid, and stained in iron-hematoxylin except the figures 55 and 58, which are stained according to Altmann. Figures 56 and 57: Zenker-fixing and iron-hematoxylin-staining. X2400.

45 to 47 Transitional forms of acinus cells into islet cells, the passing out of argentophile granules from the nucleus. X2400.

48 and 50 e-cells, containing no argentophile granules, but clear vacuoles corresponding to the lipoid corpuscles dissolved out. X2400.

49 Pigment corpuscles in the cytoplasm. X2400.

51 Showing a nucleus, the nucleolus of which contains a small clear vacuole corresponding to the nucleolinus. X2400.

52 and 53 c-cells, in which the transparent protoplasm has appeared at one end of the cell. X2400.

54 and 55 Transitional forms between the islet and acinus cell, the reappearing of mitochondrial filaments around the nucleus. X2400. 56 to 58 Various phases of mitosis. X2400. 59 Amitotic cell-division. X2400.


54


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS

S. SAGUCHI


PLATE 5





45


.yf>'


46


J: Ml


^'


■<41


'§W4\


49


■ 50


51



53


y;


54


/,


^7r


"^ /"


'-' t



ci vi -..i


f>



55


|fr


K


W


iS^:^.


56




V


58


K U /^ ;^'


59


55


PLATE 6


EXPLANATION OF FIGURES


60 and 61 Sections of the pancreas, showing the distribution of internal secreting cells which either are scattered solitarily or form smaller or larger groups. The smallest black spot shows a solitary islet cell; cell groups or islets are delineated in various sizes according to the number of cells constituting them. Figure 60, X180; figure 61, XIOO.


56


CYTOLOGICAL STUDIES OF LANGERHANS'S ISLETS

S. SAGUCHI


PLATE 6




K



~7


>


60


4;


</


61


57


Resumen por el autor, Hayato Aral.

Institute Wistar de Anatomia y Biologia.

Sobre la causa de la hipertrofia del ovario superviviente despues de la ablacion del otro ovario (rata albina) , y sobre el numero de 6vulos en el contenidos.

En la rata albina sin uno de los ovarios, el ovario superviviente es mayor que el normal en todos los estados. En los que preceden a la puber tad, excede en tamano al ovario del animal que sirve como tipo de comparacion (control), en peso, en un 70 por ciento, pero despues de la pubertad le excede en un 100 por ciento 6 mas. El exceso de peso antes de la pubertad se debe al mayor numero de foliculos grandes, y despues de aquella, a un exceso de cuerpos amarillos.

No hay pruebas de que el tejido del estroma sea responsable de este aumento, como ha supuesto Bond. La ablacion de un ovario no produce mas modificacion en el numero de 6vulos del ovario superviviente que el apresurar ligeramente la reduccion de este numero con la edad mas avanzada. El numero de individuos en las crias de ratas desprovistas de un ovario es casi tan grande como el de las crias de ratas normales con ambos ovarios intactos, y el numero relativo de cuerpos amarillos coincide con este resultado. La mera presencia de machos en la misma jaula parece apresurar ligeramente los procesos del crecimiento del ovario, pero la pubertad coincide casi constantemente con un cierto tamano del cuerpo y tiene lugar al mismo tiempo en las ratas normales y en las desprovistas de un ovario. A causa de su mayor actividad, el ovario superviviente regula el crecimiento del cuerpo en la rata desprovista de un ovario, del mismo modo que en la rata intacta esta regulado por ambos ovarios.

Translation by Jos6 F. Nonidez Cornell Medical College, New York


AUTHOR S ABSTRACT OP THIS PAPER ISSUED BT THE BIBLIOGRAPHIC SERVICE, JULY 26


ON THE CAUSE OF THE HYPERTROPHY OF THE SURVIVING OVARY AFTER SEMISPAYING (ALBINO RAT) AND ON THE NUMBER OF OVA IN IT

HAYATO ARAI The Wistar Institute of Anatomy and Biology

INTRODUCTION

Bond ('06) has shown that in the rabbit a compensatory hypertrophy of one ovary may occur when the other has been removed. He states that this process takes place only if the operated animal is permitted to become pregnant or at least to have sexual intercourse. Moreover, he believes this compensatory hypertrophy of the surviving ovary to be caused by the overgrowth of stroma tissue. Carmichael and Marshall ('08) also noted in adult rabbits the compensatory hypertrophy of the surviving ovary at two to five months after semispaying, but contrary to Bond, hold that this process is independent of pregnancy or sexual intercourse. Furthermore, they found by removing portions of the surviving ovary that the power of compensatory growth is relatively greater the larger the amount of ovarian tissue removed. Doncaster and Marshall ('10) also reported hypertrophy of the surviving ovary in rabbits.

Hatai ('13) found that in albino rats after semispaying the compensatory growth of the surviving ovary is almost perfect, and the surviving ovary has therefore nearly twice its normal weight. Hatai operated on rats between sixteen and twenty-two days old and killed them at 326 days. In these same rats Stotsenburg ('13) followed the growth of the body up to 275 days and concluded that semispaying does not modify either the body weight or the body length, though total spaying produces a definite increase in weight, in a measure due to an accumulation of fat.

59

THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 1


60 HAYATO ARAI

In all the foregoing cases semispaying was practiced before puberty, but the final examination was made on the adult. Consequently, these studies do not tell us what influence semispaying has on the surviving ovary before puberty.

As already mentioned, Bond holds that this compensatory hypertrophy is probably due to an excessive growth of the ovarian stroma. In my previous paper ('20) I stated that before ovulation the weight of the ovary of the albino rat is most dependent on the number of the large degenerated follicles and of those nearly or completely mature, while after puberty the weight is mainly influenced by the number of corpora lutea present. It seemed desirable, therefore, to determine whether these facts observed on the growth of the intact ovaries were applicable to the hypertrophy of the surviving ovary, or whether, as Bond holds, the excessive growth of the ovarian stroma is mainly responsible.

At the suggestion of Professor Donaldson, I undertook, therefore, the problem of determining the influence of semispaying on the total number of ova in the surviving ovary, together with a study of the cause of the compensatory hypertrophy.

MATERIAL AND TECHNIQUE

Albino rats twenty days old were chosen for operation, because in my previous study ('20) it had been found that from twenty until sixty or seventy days of age the number of ova is nearly constant. If, therefore, the compensatory growth of the surviving ovary occurs during this period, we may be able to determine whether the number of ova is responsible for it. For this special point examination of the operated rat after puberty is not so instructive, owing to the presence of the corpora lutea in the ovary, which complicates the interpretation.

For this study I used eight litters of albino rats. Each of these contained at least four females. Three females in each htter were operated on at twenty days of age, and the remaining females used as the controls. These eight litters were divided into two series. The rats belonging to series I were returned to


CAUSE OF HYPERTROPHY OF SURVIVING OVARY 61

the mother, together with the control females, and one or two males of the same litter.

On the other hand, the rats belonging to series II were also returned to the mother after operation, but were kept with the control females only, and thus were absolutely separated from the influence of the males.

In order to avoid a possible seasonal effect on growth, all the eight litters were chosen from those born in the spring, from February 15th up to April 8th, and the members of the litters had approximately the same body weight, about 20 grams, at the time of operation.

I followed the technique employed by Stotsenburg ('13) for removing the ovary; that is, while the rats were under ether the hairs were clipped, then an incision was made along the edge of the lumbar muscles, a little caudal to the last rib. The incision need not be longer than 1 cm. and should not cause bleeding, A small blunt hook is inserted through the opening and the right ovary pulled out with the adherent structures and cut from its attachment to the fallopian tube. The whole operation takes but a few minutes.

The edges of the wound are brought together and covered with thin celloidin, and the animal is placed in warm cotton until consciousness has fully returned, when it is replaced in the cage. Beyond boiling the instruments, no other aseptic precautions were taken, yet in no case did ill effects follow the operation. I removed always the right ovary.

Within one or two hours after the operation, the rats were returned to the mother, and usually a week later the mother was removed from the cage. The excised ovary was carefully prepared and weighed in a small weighing bottle. After the weight had been determined, the ovary was fixed in Bouin's solution, embedded in paraffin, completely sectioned at 10 n, and finally stained with hematoxylin and eosin.

The operated rats with their controls were killed at the third, fifth, sixth, and seventh weeks after operation; i.e., at 41, 55, 62, and 69 days of age.


62 HAYATO ARAI

After recording the body weight and body length, the abdomen was opened and the left surviving ovary was carefully removed from the surrounding tissues. The examination of the uterus showed no abnormality except in some cases in which the stump of the right cornu was adherent to the intestine or peritoneum, though in the majority of cases it was free and mobile. So far as gross appearance was concerned, the right cornu appeared about the same as the left. No sign of atrophy could be found on either side, and when the left was hyperemic, the right was hyperemic also. The surviving left ovary was weighed and the sections of it made in the same way as that just described for the right ovary. The viscera of the control rats were also examined, but no abnormahties were found in them. The left ovary of the control rats was also prepared at the same time. The procedure just described gave for histological study in each group the right ovary of the operated rat at the age of operation — twenty days — and the left, surviving ovary of the operated rat at the time of killing — together with the left ovary of the control rat taken also at the time when the operated rat was killed.

The method of counting the ova has been fully described in my earher paper ('20). Briefly it is as follows: The number of ova was counted from all sections, but in order to avoid the counting of one ovum more than once only those which possess a distinctly outlined and deeply stained nucleus were recorded. I have divided the larger degenerated folhcles into medium-sized and large degenerated follicles. The medium-sized are represented by those follicles which may in some instances, though not always, show quite a small cavity, and in which the ovum has either degenerated or disappeared, leaving at times the mere contour of the ovum without a nucleus. The large degenerate follicles are represented by those which possess a completely formed cavity, as well as a cumulus ovigerus, and in all cases the ovum has either disappeared or lost its nucleus.

The Ust of rats used is as follows :


CAUSE OF HYPERTROPHY OF SURVIVING OVARY


63


TABLE 1


Killed at 3rd week

5th week

6th week

7th week after operation


days

41 55 62 69


SERIES I. WITH MALES


Semispayed


Control


SERIES II. WITHOUT MALES


Semispayed


Control


OBSERVATIONS

As is shown in table 2, there are individual variations in body weight as well as in body length within each Utter, though all the members were kept in one cage and thus raised under identical conditions. Moreover, the growth rate of the litters is not the same, although each litter had nearly the same body weight at the age of twenty days. The real influence of semispaying on the growth of the body in weight and in length, as well as on the weight of ovaries, is best seen in table 3, in which all the characters are given their average values.

Examination of table 3 shows a better growth in both the body weight and the body length in the operated rats in five out of eight litters. However, the difference is slight, as can be seen in table 4, and I do not think it significant. In table 4 we find the average ratio in body weight is 1.06 in series I and 0.98, in series II.

I therefore conclude that so far as the body weight is concerned, semispaying has no influence on the growth.

Similarly, we find the average ratio for body length 1.00 in series I and 0.98 in series II — a result which leads to a like conclusion.

Thus, my own observations agree with those of Stotsenburg ('13), who also studied the effect of semispaying on the body growth of the albino rat, and who concluded that the absence of one ovary does not modify the general body growth of the operated animal.


64


HAYATO ARAI


In the weight of the left surviving ovary, however, we do find a significant difference between semispayed and control rats. With the exception of the one litter (killed at 62 days)


TABLE 2


The body weight and other data for both operated and control rats at the time of

killing


APPLYING TO BOTH SERIES


SERIES I. WITH MALES


SERIES II. WITHOUT MALES



Weeks after

operation


Age


Body weight


Body length


Weight of surviving ovary


Corpora lutea


Body weight


Body

length


Weight of surviving ovary


Corpora lutea




days


grams


mm.


m^m.



grams


rmn.


mgm.



Test


3


41


36.3


108


5.0



46.0


117


6.0



Test


3


41


36.4


108


5.0



48.2


112


5.8



Test


3


41


33.1


102


'5.1



47.5


118


5.7



Control



41


40.2


110


3.5



46.1


116


3.6


_


Control



41


31.8


105


2.5



37.3


110


4.2



Test


5


55


63.8


136


7.7



64.5


131


7.0


_


Test


5


55


71.2


141


9.2



59.5


132


7.1



Test


5


55


57.4


140


7.0



68.5


131


7.2



Control



55


55.3


129


6.1


_


85.0


148


5.8


_


Control



55


50.1


128


5.5







Test


6


62


52.4


134


5.1



119.4


167


34.6


+


Test


6


62


58.8


134


7.8



126.4


172


33.2


+


Test


6


62


81.9


150


10.3



121.3


170


40.2


+


Control



62


84.0


152


15.3


+


120.9


169


13.6


+


Test


7


69


84.4


150


24.9


+


96.8


153


42.3


+


Test


7


69


92.1


157


33.2


+


87.8


148


20.5


+


Test


7


69


102.3


161


42.9


+


77.3


139


6.6



Control



69


78.5


148


21.3


+


90.0


154


13.6


+


Control



69


88.8


154


17.2


+


88.6


148


13.7


+


in series I, the weight of the left surviving ovary (as well as the relative size of the ovary on body weight) is greater than in the controls (table 3). The litter killed at 62 days in series I is exceptional, owing to the appearance of corpora lutea in the


CAUSE OF HYPERTROPHY OF SURVIVING OVARY


65


ovary of the control rat, while they were still absent in the semispayed members of this group.

TABLE 3 The average values for the data in table 1


APPLYING TO BOTH 8EBIES


SERIES


. WITH MALES



SERIES II.


■WITHOUT MALES



u





>•


-iif >.^





>


i£,>.« 




O




to a ,2


>

1>


ve percen size of le iving ova ody weigh


a


'3


J3

a


It


ve percen size of le iving ova ody weigh




^.2


V


^


>>


"mm


J2 M3


0.


T3


•a


MM


.2 M3


Q.




tc



o











&


<


pq


« 


^


Ph



pq


pq


&:

« 





days


grams


mm.


mgm.


per cent



grams


W,7Jt,


Tngin.


per cent



Test


3


41


35.3


106


5.0


0.0141



47.2


116


5.8


0.0123



Control



41


36.0


108


3.0


0.0093



41.7


113


3.9


0.0093



Test


5


55


67.5


139


8.0


0.0118



64.2


131


7.1


0.0111



Control



55


52.7


129


5.8


0.0110



85.0


148


5.8


0.0068



Test


6


62


64.4


140


7.7


0.0120



122.4


169


36.0


0.0294


+


Control



62


84.0


152


15.31


0.0183


+


120.9


169


13.6


0.0112


+


Test


7


69


93.7


156


33.7


0.0359


+


92.3


151


31.4


0.0340


+


Control



69


83.7


151


19.3


0.0242


+


89.3


151


13.7


0.0154


+


TABLE 4


The ratios of the body weights, body lengths, and ovary weights of the operated rats to those of the control rats. The control values were taken as standards


APPLYING TO BOTH SERIES


SERIES I. WITH MALES


SERIES ll. WITHOUT MALES


Weeks after operation


Age


Ratios

body

weight


Ratios body length


Ratios of ovary


Ratios body weight


Ratios body length


Ratios of ovary


3 5 6

7


days

41 55 62 69


grams

0.98 1.28 0.77 1.12


mm.

0.98 1.08 0.92 1.03


mgm.

1.66 1.38 0.501 1.74


grams

1.13 0.76 1.01 1.03


mm.

1.03 0.88 1.00 1.00


mgm.

1.50 1.22 2.66 2.30


Average


1.06


1.00


1.32 1.59


0.98


0.98


1.92


Average whe days omitt


tt group at 62 ed





1 Corpora lutea in control.


66 HAYATO ARAI

It is interesting to note (table 4) that before puberty tne surviving ovary is about 40 per cent heavier than that of the controls, while after the appearance of corpora lutea it becomes more than 100 per cent heavier.

As has been stated, Bond ('06) found in semispayed adult rabbits the compensatory hypertrophy of the surviving ovary. The hypertrophied ovary had twice the weight of the ovary previously removed. This growth he attributes to the increase in the ovarian stroma. Thus Bond compared the weight of the surviving ovary with the weight of the ovary of the same animal at the time of its removal. Bond simply stated, from counting the new large corpora lutea, that there are approximately the same number of these in the surviving and in the removed ovaries. However, since a new formation of corpora lutea usually occurs in association with sexual activity, there must remain a greater number of old corpora lutea in the surviving mature ovary, even granting that some of these were either degenerated or otherwise changed.

If such were the case, the presence of more corpora lutea in the surviving ovary would make it heavier and larger and so produce the compensatory hypertrophy.

Carmichael and Marshall ('08) semispayed three non-pregnant rabbits and found a definite compensatory hypertrophy in the surviving ovary after two, four, and five months; and in one rabbit in which pregnancy occurred subsequent to the removal of the ovary, the surviving ovary, when examined four and a half months later, weighed twice as much as the ovary previously removed. These authors concluded that sexual intercourse is not necessary to induce a compensatory hypertrophy in the ovary.

My own observations lead to the same conclusion as that reached by Carmichael and Marshall. In tables 3 and 4 are shown the two litters in series II, which were separated absolutely from the males, and in which after puberty the weight of the surviving ovary is twice or more than twice that of the corresponding ovary of the control at hke age.


CAUSE OF HYPERTROPHY OF SURVIVING OVARY 67

Hatai ('13) also found in the adult semispayed rats that the weight of the surviving ovary is almost twice the weight of the normal single ovary in the control of like age.

It is clear from the foregoing that both in albino rats and in rabbits the surviving ovary hypertrophies at least within one to five months after operation, and weighs usually twice or more than twice the normal ovary, and it seems also certain that the compensatory hypertrophy is not necessarily dependent on coitus or pregnancy. These statements are based, however, on observations made on adult animals, that is, after puberty, and so far as I am aware, there are no previous observations on the changes in the surviving ovary before puberty. My own observation shows, however, that even before puberty the surviving ovary exhibits a definite amount of hypertrophy within a short time after semispaying, namely, three to five weeks, though its increase is not so large as that found in the surviving ovary of adult animals, since in the prepubertal period, the surviving ovary weighs only about 40 per cent more than the normal ovary of control rats.

With the exception of the suggestion of Bond, the real cause or causes of the compensatory hypertrophy have not been considered. With the purpose of getting an explanation, therefore, I have compared the total number of ova and of corpora lutea in the normal ovary with those found in the surviving ovary in these eight litters of rats.

Total number of ova in the surviving ovary compared with total number of ova in the normal control ovary of like age

In each litter I have counted the number of ova in the left surviving ovary of two semispayed rats and in the corresponding left ovary of one control rat belonging in the same litter and of like age. I have also counted the number of ova in the ovaries removed from the semispayed rats at the age of twenty days, the time of operation. There are altogether eight litters, and thus results have been obtained for the number of ova in sixteen semispayed and eight control rats, as shown in table 5. For


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-Sm^,^ Hi 9 \4 10 11 vii/ (7 ^^'^■ ^ S) ^1 w / 14 d 111 PLATE 3 EXPLANATION OF FIGURES 16 and 17 Granulocytoblasts with crescent-shaped nuclei, representing the stage which precedes to the granulocytes represented in figures 7, 15, and 18. 18 Myeloid metaplasis in the connective tissue surrounding an egg-follicle in a pullet, g, degenerated granulocytes. Sections of a vein and an artery (b) are seen in the figure. Slide by Dr. H. D. Goodale. Hemat. eos. 19 Phagocytic cell found in a granulopoietic focus of the testis of the eighteenth-day embryo. The cytoplasm shows a faint network, but lacks a conspicuous sphere and centrosomes. Hem. eosin. 20 and 21 Phagocytic cells with engulfed granulocytoblasts in their cytoplasm; the cell in figure 20 is binucleate. Same material as in preceding figure. 22 Lymphoid metaplasis in the connective tissue of the regenerating testis of a mature Campine cock. Hem. eos. 23 to 28 Stages in the transformation of a small lymphocyte (first cell to the left in figure 23) into wandering cells. Figures 27 and 28 represent the large wandering cells thus produced, described by several authors under the names of Leydig cells and interstitial cells. Testis of a mature hen-feathered Campine cock, over one year old. Hem. eos. 29 Small lymphocyte with minute acidophile granules in the cytoplasm. From the mj^eloid metaplasis drawn in figure 18. 112 THE GONADS OF THE FOWL JOSE F. NONIDEZ PLATE 3 ^3 24 16 17 ...--7^ ^^.^' 25 ^6 as >^ rf 29 19 ^^1®® i/ •^'/ 22 A 113 THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2, JANUARY, 1921 Resumen por el autor, Charles R. Stockard. Cornell University Medical College, New York. La niarcha del desarrollo y la expresion estructural. Un estudio experimental de los gemelos, "monstruos dobles" y deformidades aisladas, y la interaccion entre los 6rganos embrionarios durante su origen y desarrollo. En una larga serie de experimentos el autor ha interrumpido de diversos modos el desarrollo normalmente continuo de un pez, para determinar los efectos de tales modificaciones sobre el desarrollo de las diversas estructuras. Los momentos criticos y pasivos del desarrollo han podido localizarse. La interrupci6n del desarrollo en un momento critico frecuentemente es causa de marcadas desviaciones estructurales, cuyo tipo varia segun el momento afectado. El autor ha localizado varios momentos definidos durante los cuales las interrupciones provocan un tipo particular de anomalia. Existe un momento temprano durante el cual la interrupci6n tiende a producir embriones dobles y gemelos; una interferencia un poco mas tardia induce la aparicion de varios defectos oftalmicos, y existen otros momentos en los cuales la interrupci6n origina deformidades en la boca y branquias, anomalias oticas, la supresion de las vesiculas cerebrales primarias etc. Todas las reacciones estructurales, incluso los gemelos y embriones dobles son "cesaciones de desarrollo" tipicas. L^n estudio de los componentes de monstruos dobles ha susministrado ayuda valiosa en la comprensi6n de la competencia del crecimiento que existe entre las partes que estan creciendo y los 6rganos del embri6n. El autor discute la importancia de estos experimentos sobre la poliembrionia en las formas inferiores y la formaci6n de gemelos en el hombre. Tambien analiza el factor tiempo en el desarrollo del individuo y sus diversos 6rganos. Translation by Jose F. Nonidez Cornell Medical College, New York AUTHOR 8 ABSTRACT OF THIS PAPER ISSUED BT THE BIBLIOGRAPHIC SERVICE, DECEMBER 6 DEVELOPMENTAL RATE AND STRUCTURAL EXPRESSION: AN EXPERIMENTAL STUDY OF TWINS, 'DOUBLE MONSTERS' AND SINGLE DEFORMITIES, AND THE INTERACTION AMONG EMBRYONIC ORGANS DURING THEIR ORIGIN AND DEVELOPMENT! CHARLES R. STOCKARD Cornell University Medical College, New York City THIRTY-TWO TEXT FIGURES AND SIX PLATES CONTENTS 1. Introduction 116 2. The specific rate of development in a given species 118 3. Continuous and discontinuous modes of development 120 4. Experimentally changing a continuous into a discontinuous mode of development 122 a. The method of experiment 122 h. Stopping or retarding the progress of development at stages of apparent indifference to such interruption 125 c. Stopping or retarding the progress of development at stages of critical susceptibility to such interruption 138 d. Differences in effect between greatly reducing the developmental rate and actually stopping the process temporarily 154 e. The types of arrests or deformities following a stop in development or a slowing of the rate 160 5. Experimental production of twins and 'double monsters' by an earlj^ arrest of the developmental rate 162 a. Arresting development by low temperature and the production of double embryos and twins in Fundulus 166 h. Arresting development by reducing the oxygen supply and the occurrence of double individuals in the trout and Fundulus 173 1. Results with Fundulus 173 2. Double embryos in trout eggs 177 c. An explanation of the frequent occurrence of twin and double chick embryos 186 1 This investigation has been aided by the Memorial Foundation of St. Bartholomew's Hospital for Diseases of the Alimentary Canal, of New York City. The author expresses his appreciation for this valuable support. 115 116 CHARLES R. STOCKARD d. An explanation of polyembryony in the armadillo 190 e. 'Alternation of generations' and twins in vertebrates 197 6. The structural differences between the two components in connected twins and double individuals '. 198 a. Double individuals with identical or equal-size components 198 b. Double individuals with unequal components 201 1. Condition of the larger component 202 2. Condition of the smaller component 202 3. The small component and the actual frequency of double or twin individuals 207 4. The small component and certain theories of teratoma 209 5. Types of defects exhibited by the smaller component 211 c. The components in double human specimens 212 7. The double individual with unequal components and an understanding of the cause of all monstrous development 218 8. The double individual with unequal components and an analysis of the development of organs in the single individual 228 a. The growth influence of the apical or primary bud over the secondary and potential buds in plants 229 b. The initial linear growths, subsequent lateral buds, and the inter action among the organs of the vertebrate embryo 231 c. Developmental rate and post-natal changes 245 9. Continuity of the series from monstra in defectu through the single normal individual to monstra in excessu and finally identical twins. 247 10. The necessity of a controlled or regulated environment in which to develop highly complex individuals 248 11. Growth competition between the two components in double individuals and the time of occurrence of teratoma in man 251 12. Cancerous growths and the general cessation of all normal growth in the old individuals 252 13. General summary 255 14. Literature cited 263 1. INTRODUCTION In the present contribution an endeavor will be made to analyze the causes and conditions which determine the usual type of structural expression or form. The ordinary progress of embryonic development gives rise to individuals of rather uniform structure, yet there may be numerous slight variations and defects in the structural composition of various organs and parts. Minor defects in structure are found in almost every individual of a group, but rarely do two individuals present exactly the same kind or degree of defects. These facts are readily recognized in a group of human beings where small differences are easily appreciated, STRUCTURE AND DEVELOPMENTAL RATE 117 but no doubt the same conditions obtain among vertebrate animals in general, although in lower forms such differences in developmental results are more difficult to detect. The end products of development differ from one another to varying degrees, slight differences are of little concern and are classed as ordinary variations, but when the same deviations become exaggerated they may be ranked as serious deformities or, anomalies. This fact renders the analysis of normal developmental processes and the experimental study of monstrous development one and the same problem. It should be understood that the present study is not intended solely as a contribution to socalled 'teratology,' but is an experimental analysis and consideration of the processes involved in all normal embryonic development and growth. The experimental treatments have in many of the cases caused the formation of well-known monstrous structures, but the point of importance is not the production of the monster, but the simple alterations in the usual course of events which have induced the modified structural expression. For the past ten years I have claimed that all types of mon-/ sters not of hereditary origin are to be interpreted simply as deJ velopmental arrests. Such a position has been taken by otherst (Dareste, '91). However, I propose at this time to present evi-\ dence which clearly demonstrates the truth of the claim. By ^ arresting development in very simple ways all types of monsters may be obtained. The experiments have now reached such a degree of exactness that the following propositions may be stated as true, the evidence for which is recorded in the body of this paper. First, all types of monster, double as well as single, may be caused by one and the same experimental treatment ; second, any one type of monster, such as cyclopia, may be produced by a great number of different experimental treatments; third, all effective treatments tend primarily to lower the rate of development, and, fourth, the type of monster induced depends upon j the particular developmental moment or moments during which the developmental rate was reduced. Slowing the rate at one 118 CHARLES R. STOCKARD moment will produce a double monster or identical twins and at another moment slowing by the same method will give rise to the Cyclopean defect. In fact, the same thing which causes the double monster may later in development induce one of its heads to be cyclopean. Thus there is no longer any ground for considering certain defects as specific responses to particular treatments. And there is as little reason for further descriptions of individual monsters, since all belong to the same class and the individual differences simply result from the different moments during which the developmental interruptions have acted. The important consideration then arises as to what internal and external factors may tend to introduce the developmental arrests. Does one growing part in any way inhibit the activity of other developing organs? We shall devote a section to a consideration of the interaction among the developing and growing organs within the embryo. The study of the growth influences of one embryonic . organ on another is one of the most important problems in the analysis of structure. Finally, the interaction among growing parts and the inhibiting effects of one rapidly proliferating region over other regions will be very briefly considered in connection with abnormal and malignant growths. 2. THE specific' RATE OF DEVELOPMENT IN A GIVEN SPECIES It is a generally known fact that the eggs of different species do not progress at the same rate of development even during comparable stages. The lengths of time between fertilization and the first cleavage and the rates at which the early cleavages follow one another may differ decidedly among the eggs of even closely related forms. These differences in developmental rate are probably fundamentally connected with differences in chemical structure of the egg substances, and in particular with the different rates of oxidation of certain stuffs. It is a wellknown chemical fact that very slight differences in composition between substances may cause very great differences in their oxidation capacities. STEUCTTJRE AND DEVELOPMENTAL RATE 119 The efforts on the part of numerous embryologists to associate the differences in rate of cleavage and time required to attain certain stages of development with the size of the egg, the amount and position of the yolk substances, or even the types of cleavage have not been satisfactory. Certain meroblastic eggs develop much faster than certain holoblastic ones, while other holoblastic eggs have a rate of cleavage far more rapid than the meroblastic types. All of the so-called laws of cleavage rates based on morphological differences among egg types have been found to fail so decidedly when applied in general that one is forced to seek more deep-seated causes for the differences in developmental rate. At the present time we can only state that such causes probably , reside in the differences in chemical make-up of the several species of eggs. The rate of development certainly depends, particularly during later stages, on the amount of food available, but the supply of oxygen and the degree of temperature at \ which development is taking place have a far more striking influ- \ ence on the rate. Cessation of development also occurs much \ more promptly from absence of oxygen or sudden changes in temperature than from any other natural modifications which happen in the environment. These facts point decidedly to the rate of development as being dependent upon kind and rate of chemical change, most particularly upon rate of oxidation. The egg probably has a definite coefficient of metabohsm dependent upon the interaction of its specific cheiniCaT^tructure and the given environment in which it normally develops. The rate of development results from both the internal qualities of the egg and the nature of the surrounding environment. The present extremely crude state of our knowledge of the chemistry of development will permit of no more satisfactory statements of the principles underlying differences in developmental rate than those which have been attempted above. The inadequacy of such statements is as keenly appreciated by the writer as by the critical reader, but this inadequacy concerns chiefly, the absence of the details involved, while the statements in general I believe are correct. 120 CHARLES R. STOCKARD Although there is a definitely normal rate of development for a given egg, this rate is frequently subject to wide variations, usually as a result of variations in the surrounding conditions. The two chief, or most frequent, modifying causes are a change in oxygen supply or a change in temperature. An acceleration of the usual rate only takes place to a limited degree under natural conditions and but slight increases in developmental rate have been experimentally obtained. On the other hand, a very wide range of decrease in developmental rate is readily brought about. Slight changes in the surrounding temperature or reduction in the oxygen supply will readily tend to slow the rate of development to a marked degree. Finally, the entire progress of development is frequently stopped in nature by removing the supply of oxygen or by sufficiently lowering the surrounding temperature, as will be discussed in subsequent sections. 3. CONTINUOUS AND DISCONTINUOUS MODES OF DEVELOPMENT Although, as stated in the foregoing section, each egg has a more or less characteristic rate of development, this rate is not uniform throughout the different developmental stages. All eggs develop with rythmical changes in rate^ going alternately faster and slower from stage to stage. Certain stages are passed very ^'>| rapidly, almost suddenly, while others are slowly attained in a tedious manner, yet the process of development is as a whole continuous. That is, development begins with fertilization which is soon followed by cleavage, and then continues without interruption until a free living larva or young embryo is formed. This then proceeds to grow and change until the adult structure is attained. Such a continuous mode of development is most common, indeed so common, that it is often carelessly considered to be universal, while a discontmuous mode, is looked upon as something veiy strange or unusual and not as a phenomenon extremely important in an understanding of the more common continuous type of development. The continuous mode is found among the great majority of those animals in which the eggs develop in a uniform or homogeneous • environment, such as the sea-water. The general conditions of STRUCTURE AND DEVELOPMENTAL RATE 121 moisture, oxygen supply, and temperature are comparatively uniform, and although the eggs may develop faster or slower under slightly different conditions of temperature, etc., yet the variations in the medium are rarely sufficient to inhibit or stop development entirely, and when they are the eggs usually die. On leaving the sea the fresh-water and land-living invertebrates and vertebrates show most varied and complex methods and arrangements for insuring an environment of sufficient uniformity to permit an uninterrupted development. Many forms, as is also the case in certain sea-living animals, have evolved a method for the development of the embryo within the body of the mother. Such an internal environment tends to control very effectively the conditions of moisture and in mammals also the temperature, but at times, as we shall see beyond, the oxygen supply is not properly adjusted and the continuity of development may be interrupted or interfered with on this account. The land-living animals have not always succeeded in obtaining an ideal developmental environment, and there are many examples of a discontinuous mode of development as a result of environmental breaks in the strictest sense. That is, the egg begins to develop and attains a certain stage, when a more or less sudden change or break in the environment occurs and development stops completely and may remain at a standstill for various lengths of time — days or possibly weeks. Another alteration in the environment then occurs which again permits development to start and continue until the fully formed animal is obtained. Such a discontinuous mode of development is universal among one great class of vertebrates, the birds. Among the birds development, as far as studied, is invariably interrupted when about the stage' of gastrulation, at which time the egg is laid or passed out of the warm body of the mother. The fall in temperature causes development to stop and the egg remains in the gastrular stage until incubated by the heat of the parent's body or until artificially incubated at a similar temperature. The means of interrupting development seem to reside entirely outside the egg itself, they are properties of the environment. As far as is known, all eggs having once begun to develop will pro 122 CHARLES R. STOCKARD ceed in a continuous manner from stage to stage until the larva or free living embryo is formed, the environment permitting. Stops in development take place through lack of oxygen, unfavorable temperature, insufficient moisture, or shortage of available nutriment, but the egg itself is wound or set for development so as to continue through if possible. Thus experiments on discontinuous development must apply as methods various means for modifying the environment, and the results will depend upon the power of the egg to adjust itself to or withstand these changes. Being unable to meet the situation, abnormal or unusual developmental productions may arise. The question then presents itself as to whether the development of any egg may be interrupted for definite lengths of time and later be allowed to finish or proceed. What would be the consequences of such interruption in the case of a normally continuous mode of development? Would the effects of the manner of development be the same following interruptions at different stages, or would the effects vary depending upon the stage of development at which the interruption occurred? In other words, are there indifferent and critical moments of developmental interruption? Would a complete stop in development have an effect similar to a decided slowing of the rate, or would the one be more effective than the other? The experiments recorded in the following sections were devised in order to answer these and other queries. 4. EXPERIMENTALLY CHANGING A CONTINUOUS INTO A DISCONTINUOUS MODE OF DEVELOPMENT a. The method of experiment The continuous mode of embryonic development is the more common type in nature. We are, therefore, warranted to some extent in assuming that the discontinuous mode is nature's experimental modification of the continuous. What methods of modification has nature employed that may be artificially imitated? The simplest, commonest, and most evident natural method is change in temperature which causes the interruption of development in the eggs of all birds. STRUCTURE AND DEVELOPMENTAL RATE 123 Changing the temperature of the environment and, therefore, of the egg, is the method employed in most of the present experiments in order to interrupt or make discontinuous a normally continuous development. There are several definite natural cases of discontinuous development among mammals, the significance of which will be considered in another section of this paper. But in the present connection we may be certain that nature has here employed another method than temperature change in causing the interruption. The temperature of the maternal body in which the mammalian embryo is developing is sufficiently uniform never to interrupt the progress of the egg. For reasons to be more fully cited beyond, changes in the supply of oxygen would seem to be the most probable cause of interrupted development in the rare cases of this phenomenon among mammals. Lack of oxygen or excess of CO2 has also been resorted to in the present experiments as a means of interrupting or retarding the rate of a normally continuous development. Neither of the two methods is new. A number of experimenters have studied the influence of temperature changes on the manner of development of different eggs. The effects of abnormally high and low incubator temperature on the development of the hen's egg have been recorded by Dareste and many others, most recently by Miss Alsop ('19). The development of amphibian eggs under unusual temperature conditions has been considered by O. Hertwig ('96), King ('04), and others. The influences of low temperatures on the development of the fish's egg have been investigated by Loeb ('16) and Kellicott C16). These studies on temperature, however, are of interest in the present connection only in so far as they almost all show how readily abnormal development of the embryo may be induced by unfavorable temperature conditions. The attempted explanations of the deformities which were given in only a few cases, as by Kellicott, entirely disregard or dismiss the real point of fundamental importance; that is, the induced change in the rate of development resulting from the modified temperature. Kellicott 124 CHARLES R. STOCKARD attempted to refute the slow rate as a cause of structural modification in discussing my assumption of arrested development. The present experunents differ from the previous temperature experiments in that they were undertaken with an almost completely different problem in view. The former experiments will be considered only as they bear on the specific questions in the discussion to follow. Numerous studies on the behavior of eggs deprived of oxygen as well as in the presence of various reducing and anaesthetic substances have been conducted. All of these oxygen studies have little or no bearing on the immediate problems and are not treated in this connection. The material used in the present experiments were the eggs of the common minnow Fundulus heteroclitus. I have studied and experimented with these eggs for a number of years and am familiar with a great many common deformities which they may be induced to present. The exact method of experimentation with temperature change was as follows: the eggs were taken from the female and fertilized in a 'dry bowl.' About fifteen minutes later they were rinsed free of foreign material with seawater and left standing under water. The first cleavage takes place after about two hours, varying a little with the season and \ the temperature. The next cleavage follows after another hour, and development proceeds in a continuous fashion from then on until the fully formed fish hatches from the egg membrane and wims freely about within from eleven to eighteen or twenty ays, depending again upon the season and temperature. There s a wide variation in the rate of development of these eggs, yet nder all usual conditions after development once starts it is continuous. The eggs were placed during different stages of development in compartments of a refrigerator at temperatures of 5°, 7° and 9°C. and left for varying lengths of time, from one to five days. At the lowest temperature development was almost if not completely stopped, while in the other two compartments it was slowed idown to from one-twentieth to one-fiftieth of the normal rate. The responses shown in the manner of development are so differ STRUCTURE AND DEVELOPMENTAL RATE 125 ent in eggs stopped or slowed at different stages that the exact time of treatment will be considered in connection with the different effects obtained. The difference in effects between slowing and actually stopping development will also be considered. Other eggs were crowded close together in bunches and developed in bowls at room temperature. The eggs near the center of the masses or bunches obtained much less oxygen and were in a higher concentration of CO 2 than the more superficial ones. These were slowed in their rate of development. Sea-water was boiled so as to drive out most of the air and afterward kept stagnant. Egg masses were developed in this water and the inner eggs of the mass were almost completely stopped in many cases. In all such arrangements the rate of development was so retarded that many abnormal and deformed embryos resulted. These in general are the methods employed; the different times of application and the results will be discussed in the particular cases below. b. Stopping or retarding the progress of development at stages of apparent indifference to such interruption In order to successfully change a continuous into a discontinuous mode of development, without producing ill effects on the resulting embryos, it becomes necessary to locate certain indifferent periods during embryonic development at which the interruption may be induced. Certain of these indifferent periods are those moments at which the interruptions of development occur in nature. Should the stoppage naturally take place during a sensitive period, the species would readily be eliminated on account of the high proportion of abnormal embryos which would result. When the eggs of Fundulus are placed in low temperatures after having passed through the earliest active stages of development, cleavage, gastrulation, the formation of the germring and early appearance of the embryonic shield, they may be stopped for several days, or caused to develop at an extremely slow rate, without marked injury to the resulting embryos. In fact, when such eggs are returned to room temperature after 126 CHARLES R. STOCK ARD being in the refrigerator for three or four days, they may often resume development at such a fast rate, probably as a result of the stimulation of raising the temperature, that they may hatch only a day or so later than control embryos. The percentage of such eggs that do hatch may also be equally as high as that from the control. These statements may be illustrated best by a somewhat detailed consideration of the records from experiments. A large number of experiments have been performed and are recorded in my notes, but only a few of these may here be selected as typical examples of the series in general. Experiment 905. A group of eggs, 23 hours after fertilization, with high segmentation caps just beginning to flatten on the j'olk-sphere, were carefully selected, being certain that every one was developing, and arranged as follows. Lot Ci was placed in the refrigerator at 5°C., C2 at 6°C., C3 at 8°C., C4 at 9°C., and C5 was placed in the top compartment of the refrigerator which ranged from 9.5° to 10°C. When 27 hours old, the control group showed the germ-disc somewhat further flattened on the yolk-sphere, but there was no visible indication of a germ-ring and the disc had not begun to descend over the yolk. This expermient was being conducted during the early June season, and normal development at this time was unusually slow. At 27 hours old, three other lots were placed in the refrigerator as follows, Di at 5°C., D2 at 6°C., and D3 at 8°C. When 48 hours old, the control showed the germ-ring about one-fourth over the yolk-sphere with the embryonic shield clearly forming. The C and D series had become arrested and were still in much the same condition as when placed in the low temperatues on the previous daj'. The control at 3 days, or 72 hours old, showed the embryos well formed, though the germ-rings were not yet entirely over the j^olk-sphere (fig. 1). Lot Ci, having been 49 hours at 5°C., was still in high segmentation stages much the same condition as when placed in the refrigerator (fig. 2). These were now returned to room temperature. Lot C2 showed much the same condition as Ci and were also removed from the refrigerator. Lot C3 seemed as completely stopped as the other two and was returned to room temperature. The members of the C4 group were also in about the same stage as when placed in the refrigerator, though their temperature was 9°C. These remained in the refrigerator. The C5 lot in about 10°C. had developed slowly, the caps had flattened and the embrj^onic shield had just become visible, though the STRUCTURE AND DEVELOPMENTAL RATE 127 germ-ring had scarcely begun its descent over the yolk (fig. 3). These also remained in the refrigerator. Lot Di after 45 hours at 5°C., was still in about the same stage of development as when placed in the low temperature at 27 hours old. These are now placed at room temperature. Lot D2 was in a closely similar condition to D,, but remained at the reduced temperature. Lot D3 had also failed to make noticeable progress during the 45 hours at 8°C., but was allowed to remain at this temperature. Fig. 1 A control embryo 72 hours old, the body is well outlined and the germ-ring almost completely over the yolk. Fig. 2 An egg 72 hours old that had spent the last 49 hours at a temperature of 5°C. Development had been practically stopped in this high segmentation stage. Fig. 3 A specimen 72 hours old that had been during the last 49 hours at a temperature of 10°C. Development had progressed slowly, the germ-disc being flattened and the embryonic shield, indicated by stippling, has just become visible. 128 CHARLES R. STOCKARD When four days old, the control embryos were fully formed with prominent optic vesicles, hearts were formed, but not yet pulsating. Thus they were not more than up to a midsummer 72-hour stage, since the heart beat had generally begun about this time. However, all of these embryos were normal and well, as is shown by their later records, even though the cool season had thrown them about 24 hours behind within four days. Lot Ci, now having been at room temperature for 24 hours, were all going very well. The germ-rings varied in position from one-quarter to one-third over the yolk-spheres. Only a few had failed to resume development and the eggs in general were about up to the condition of the present control when they were 50 hours old. These Ci eggs had now actualh' developed at room temperature for about 47 hours, the first 23 hours after fertilization and the fourth day. Lot C2 was also after similar periods of experience in a uniformly good condition with the germ-rings all about one-third over the yolkspheres. Thus subjecting to low temperature after 23 hours of development is decidedly less injurious than similar treatment during the early cleavage stages, as will be seen from the records beyond. In lot C3 the germ-rings had all descended about half way over the yolk-sphere. The D series showed somewhat the same response. Lot Di, after 24 hours at room temperature, were developing normallj^ with the germrings from one-half to two-thirds over the j^olk-spheres and the embryos well formed. Thus stopping for 48 hours after 27 hours of development, when the segmentation caps were flattened over the top of the yolk, showed no ill effects on their present development except to render them almost exactty two days behind the developmental stage of the control. The control at 5 days old had a vigorous heart beat, but the circulation was just beginning to be well established. Lot Ci, almost all of the embryos were full length, the optic outpushings were just beginning, but not fully formed, thus about in the condition shown by the present control at 72 hours. These were still about two daj^s behind the control, or had practically lost the time spent in the refrigerator. There were a few with the germ-rings not entirely covering the yolk and with the body of the embryo short and poorly formed at the caudal end. Lot Co were about in the same condition as Ci. Lot C3 seemed on the average a little further along, though closely similar to the two foregoing lots. Lot C4, now four days in the refrigerator at 9°C., seemed in good condition, with the germ-rings well formed and descended about onehalf over the yolk. These specimens had thus continued their development at this temperature, although very slowly, and had advanced about 12 hours in development within the 4 days. They were now returned to room temperature. STEUCTURE AND DEVELOPMENTAL RATE 129 Lot C5 at aboift 10°C. for four days, were possibly a little further along then C4, though in general they showed a similar condition. These were also returned to room temperature. Lot Di contained full-length embryos, some with the optic processes already formed and others without. These specimens were about one and a half days behind the control or about in the stage of the two and a half day control. Lot D2, after four days at a temperature of 7°C., introduced after 27 hours of normal development, were still in about the same stage as when placed in the refrigerator. The segmentation caps were flat with early germ-rings forming and the embryonic shields just beginning. The descent of the germ-ring has been considerably prevented. All of the specimens were living and seemed well. They were now returned to room temperature. Lot D3, all seemed in good condition with germ-rings from onequarter to one-half over the yolk-sphere and with well-formed embryonic shields. Thus this slightly higher temperature of 8°C. had given the D3 group a considerable advantage in progress over the D2 lot. These were now also returned to room temperature. When six days old, the black and red chromatophores were fully expanded on the yolk and embryonic bodies of the control specimens. The embryos were now occasionally twitching and moving their bodies. Lot Ci, after being out of the refrigerator for 3 days, had embryos comparable to about a usual midsummer 70-hour stage, or about the condition of the present control when 4 days old. The heart beat had not begun. Lot C2, embryos were also in a stage just prior to the heart beat, and the C3 group was about the same. Lot C4 were now out of the refrigerator for one day after having been at a temperature of 9°C. for 4 days. The embryos were well formed and the blastopore was about closing, so they had made a considerable advance from the condition of the previous day when the germ-rings were only one-half way over the yolk. The C5 group are still further advanced with the optic outpushings prominently shown. Lot Di now showed chromatophores both on the yolks and on the embryos' bodies, yet no heart beat could be detected in any of those examined. Lot D2, when one day at room temperature after being at 7°C. for four days, showed the germ-rings two-thirds over the yolk-sphere, with the embryonic axis well formed in the shield. Lot D3 contained long embryos with the optic outpushings just beginning, so these were still ahead of D2. At seven days the control embryos were actively moving and the yolk vessels were now clearly mapped out by the pigmented arrangement. Lot Ci, now out of the refrigerator for 4 days, showed many embryos with good circulations and pigment migration, some had a 130 CHARLES R. STOCKARD heart beat, but had not estabhshed a circulation and others had not yet developed a heart beat. Lot C2 showed a good circulation in almost all. Lot C3 presented a majority with good circulation, there were, however, many with imperfect circulation or no circulation, although the heart was pulsating. When 9 days old, the control presented a perfectly normal condition. Lot Ci showed practicalh' every specimen normal and strong, apparently just as good as the control, though somewhat behind. Lot Co were in equally as good a condition. Lot C3 was much the same as the other two groups. Lot C4 also seemed to contain all normal embryos. Lot C5 were further advanced than C4, since they had continued to develop slowly while in the refrigerator at the higher temperature of about 10°C. They had, therefore, developed slowly for 4 days, and after having been out for 4 days were practically perfect in their development. Lot Di were all normal at 9 days old and as perfect as the control except for the fact of being behind in developmental time due to the few days stand-still spent in the refrigerator. Thus development can be discontinued for 3, 4, or 5 days at the stages used in this experiment (27 hours old, just after gastrulation has started) with no subsequent ill effects on the development and structure of the earlj^ embryos. Lot D2 contained specimens further behind in development than the Di group, since they remained in the cold longer, but all appeared perfectly normal at this tune. Lot D3 were all normal. At 12 days old, the control seemed about in the condition to hatch. The C series which had been subjected to developmental interruptions after being 23 hours old now presented perfectly normal conditions. In lot Ci three specimens had not developed and sixty were normal This is as good a record as is usually found under ordinary conditions. Lot Co contained about 100 specunens, which were all living and nornial. Lot C3 had about the same number in similar conditions. Lot C4 also contained about 100 normal specimens, so that the numbers examined were sufficienth' large to furnish a very reliable index of the reactions. Lot C5 contained a few more than 100 normal specimens and a single individual that was abnormally small, yet even this one was sufficientl}^ normal to have a free blood circulation. Lot Di, which was put in the refrigerator 27 hours after fertilization, contained six specimens that did not develop out of a total of seventy-five eggs. The other sixty-nine specimens were normal. The D2 lot were all normal, and so was the D3 group, yet all were behind the control in their developmental stage corresponding to about the length of time they had spent in the refrigerator. STRUCTURE AND DEVELOPMENTAL RATE 131 When 19 days old the control were almost all hatched actively free swimming young fish. The. few yet imhatched seemed normal and ready to hatch at any time. Lot Ci contained a majority hatched and all seemed normal. In lot Co there were not quite as many hatched, but all were in good condition. Lot C3 were about the same in hatching record, so there was little effect to be noticed at this time resulting from the two days spent in the refrigerator following their first 23 hours of development. Lot C4 had remained longer in the refrigerator, 4 days, and at this time none had hatched, though they seemed fully ready. In lot C5 also none had hatched. Lot Di contained a majority hatched, almost as large a proportion as the control. These had remained in the cold only two days. Lots D2 and D3 had remained in cold for 4 days, and only one specimen in the two groups had hatched. All appear normal and ready to hatch. When 20 days old, the first one in C4 had hatched. In lots D2 and D3 many had now hatched, so these are not very much later than the control in spite of their 4 days' arrest. In lot C5 none had yet hatched, although during the next 24 hours many of them did hatch. When 22 days old, a few of the control were still unhatched, though they were normal. Lot Ci had 12 unhatched and 50 hatched. Lot C2 contained 18 unhatched and about 80 hatched. I^ot C3 had 29 unhatched, one with a deformed body, and about 70 normal ones hatched. This record was about as good as a usual control. About half of the C4 lot had hatched, and all seemed normal, though they remained in the refrigerator twice as long as Ci, C2, and C3 had. Lot Co also showed about half of the specimens hatched. Lot Di had 7 unhatched and about 60 hatched, all of them seemed normal. Lot D2 contained 29 unhatched and about 40 hatched, all of which were normal. Lot D3 showed 20 unhatched and about 30 hatched. When 25 days old, every egg in the control had hatched. Lot Ci, only 4 were unhatched, one of these had abnormally small defective eyes and no blood circulation. So these are a little behind their particular control in quality at this stage, but very little, and probably their disadvantage is of no significance, since such a single specimen might occur in any group of eggs. Lot C2, every specimen hatched. In lot C3 only 3 failed to hatch. One of these was grossly deformed and the other two had slightly abnormal eyes. So this group is somewhat inferior when compared with the control record. Lot C4 contained 12 specimens still unhatched. One hatched specimen was bent and unable to swim. One of the 12 unhatched was abnormal, so this record also was a little worse than the perfect control. 132 CHARLES R. STOCKARD Lot Cs contained 10 unhatched, one of which was abnormal, the others were all normal. Lot Di, only one unhatched, all seem fine. Lot D2 contained 2 unhatched, and lot D3 had 3 unhatched, though all of these seemed normal. This experiment shows very clearly that stopping or arresting the development of Fundulus eggs after about twenty-four hours of development, when gastrulation has definitely begun, produces very slight or no ill effects on such specimens up to the time of hatching and becoming free swimming little fish. Whether during later stages of growth these fish might show some disadvantages following the developmental interruption we have not attempted to determine. It is probable, however, that these specimens were interrupted in their development during a particularly passive period and that no later disadvantages would accrue. This would seem further probable since it is at just such a stage in development that the eggs of birds are normally interrupted, and clearly wdthout ill effects on the group. These experiments not only show that stopping development at this stage, just after gastrulation has started, is not noticeably injurious in effect on the development of the young fish, but further, that after gastrulation has commenced the rate of development of the embryo may be slowed to a most extreme degree, as occurred in the upper temperatures of the refrigerator, without serious injury to the structure of the young fish. To further establish the correctness of the above results, we may record one other similar experiment in brief detail. Experiment 906. 64.1. A group of eggs when 24 hours old containing all normal fine specimens were placed at a temperature of 5°C., and later compared with a selected control from the same parents. At 46 hours old, the control were developing rapidly, with the germrings almost completely over the yolk and the embryos well formed. The B4.2 lot now in cold for 22 hours showed the same condition as when placed in the refrigerator except that the segmentation cavities were distended so that a vesicle appeared below each disc. These eggs were now moved to an upper compartment of the refrigerator to allow them to develop slowly at a temperature of about 9°C. When four days old in the 9°C. temperature they were developing slowly but normally, with the germ-rings about one-half over the yolk-spheres and with embryonic shields in which the axis of the embryo was beginning to form. STRUCTURE AND DEVELOPMENTAL RATE 133 At five days old these eggs were still developing remarkably well although very slowly. The germ-rings were a little further over the yolk. They were now returned to room temperature after having spent 4 days in the refrigerator, 24 hours at 5°C. and 3 days at 9°C. One day later, all of the eggs were developing and almost every one presented a well-formed embryo normal in appearance. When ten days old, all were living with a fine circulation of the blood and otherwise apparently normal. When 17 days old, 18 of these embryos had hatched and 24 were unhatched. After 24 days, 12 were still unhatched, one of these being very abnormal. All of the embryos had seemed normal when ten days old, but at this time it was readily seen that the 12 unhatched specimens were really far behind the control. While showing no gross deformities they were smaller and not so well developed as the control. Although these early arrests do not give marked effects on the very young fish, it is certainly possible that many later symptoms might develop if their existence was observed through longer periods of time. When 29 days old, 4 embryos were still unhatched, one had died and 3 seemed normal and ready to hatch. Thus the record of this group for the length of time it was followed does not compare unfavorably with the ordinary control records of Fundulus embryos up to a comparable period. As might be expected, however, eggs after being 24 hours old which were stopped or retarded in development for 4 days are not able to hatch on schedule time with the control; but are several days late in reaching the hatching stage. Such results will be found to differ entirely from those considered beyond as obtained when the eggs are stopped during more critical developmental stages or at times when rapid cell proliferation and developmental changes are occurring. Therefore, it may be stated in general that certain indifferent moments in development do exist during which time the rate of development may be slowed to almost stopping, or development may be actually stopped, and later resumed at a normal rate without causing structural anomalies or unusual conditions in the resulting young fish. It is also shown by the above experiment that development may be stopped at certain indifferent periods, in a temperature of 5°C. and then resumed at an extremely slow rate in 9°C. for several days, and later increased to a normally rapid rate at room temperature without injury. Thus it is not always necessary that development be promptly resumed at a normal rate in order to avoid structural defects. 134 CHARLES R. STOCKARD The next experiment is cited to show the behavior of eggs arrested in still later periods of general indifference. Experiment 907. — Eggs with germ-rings one-quarter to one-third over the yolk sphere and with embryonic shields well formed, a stage acquired after 48 hours of development during the early cool June season, were placed in the refrigerator in two groups, Eo at 6°C. and Eg at 8°C. After 24 hours in the refrigerator they had advanced only shghtly beyond the condition of the day before. The E3 group had advanced somewhat more than the E2 lot particularly in {he formation of the embryonic line, or axis, in the shield. When 5 days old, and after having been in the refrigerator for 3 days, the Eo group at 6°C. have advanced the germ-ring to about two-thirds over the yolk sphere. Thej^ were thus not as completely stopped by this temperature of 6°C. as were eggs placed in the same temperature during early cleavage stages, as will be seen be3'ond. These eggs were now, after 3 dscys of extremely slow development, returned to room temperature. The E3 lot at this time showed the germ-ring almost completely over the yolk-sphere, and the embryonic body was well formed in the majority of the eggs. These specimens at a shghtly higher temperature had developed somewhat further than those above. They were now also returned to room temperature. After being at room temperature for 24 hours, the rate of development had greatly increased in both lots. The E2 group now showed long embryos with the optic outpushings well begun in many. The E3 lot showed optic outgrowths well formed in all, and were thus a little ahead of the E2 ones in development. At 9 days old, the specimens in both lots seemed behind the control to the extent of their 3-day stay in the refrigerator. When 12 daj^s old, they were closely examined for slight anomalies. The E2 lot showed one abnormally small embryo with no blood circulation, 4 had stopped, and did not develop after removal from the refrigerator, and 45 specimens seemed to be in normal condition. The E3 lot all appeared to be normal except that the}^ were about 3 days behind the control in their development. Thus subjecting the embryos to a severe reduction in developmental rate after they were 48 hours old had only slight, if any, detrimental effect on their ability to resume a normal developmental rate and to form apparently normal young embryos. Very probably, however, minor effects are produced which would be indicated in the later structural or physiological history of the specimens could they be studied through a longer season of their existence. At 19 daj^s old, when a large majority of the control had hatched and were free swimming, none of the Eo or E3 lots had hatched. But when 21 da3's old, a number were hatched in both lots. STRUCTURE AND DEVELOPMENTAL RATE 135 When 22 days old, the Eo group contained 25 hatched and 20 unhatched. Three of the latter were abnormal with no blood circulation, two being small and inactive, and the third was grossly deformed. The E3 group had 25 hatched and 11 unhatched, all of which seemed normal in structure. At 25 days old, 4 of the Eo group were still unhatched, but all of the E3 lot had hatched. They were kept until 34 days old, at which time many had died on account of the difficulty in feeding them, but the 4 specimens in lot E2 never succeeded in hatching. When these records of late arrests are compared with those from eggs arrested during early cleavage stages, one will be struck with the low mortality following removal from the refrigerator in the case of the former. The complete absence of double monsters, ophthalmic deformities, etc., among the specimens arrested during late stages also contrasts with the common occurrence of such conditions among specimens arrested during cleavage stages. The general nature of the circulatory disturbances, etc., which do occur after late arrests is also characteristic. A contrast is further noted by considering this experiment in comparison with the specimens described above which were introduced into the cold after one day of development — there again the advantage in subsequent development is on the side of those specimens caused to develop very slowly during the later developmental stages. But of the specimens almost completely stopped in development, those stopped very soon after gastrulation seem to have an advantage over specimens stopped when one day older, or further advanced in development. The stage immediately following the first rapid changes of gastrulation would seem to be an extremely indifferent period. Two other sample experiments will be reviewed in brief to illustrate the gross reaction following still later developmental interruptions. It must be realized that in all of these experiments we are at present sunply recording the outward gross appearance and behavior of the specimens. A closer microscopic examination of the young fish in section might show a considerable depression in the development or expression of certain internal organs, for example, the conditions in the branchial regions, digestive glands, etc., while observation of the living specimen had given no indication of its inner defective condition. 136 CHARLES R. STOCKARD Experiment 908. Specimens 72 hours, or three days old, with the optic cups ah'eady invaginated and formed, but just before the beginning of a heart beat, were carefully selected, so that every individual was normal and good, and arranged in two groups. Group Fi, consisting of 62 vigorous specimens, were placed in the refrigerator at 5°C. and group F2, containing 36 normal embrj'^os, were subjected to a temperature of 8°C. When 6 days old and after being 3 days in the refrigerator the Fi lot were in much the same condition as when put in the cold, the hearts had not begun to beat and the general structural appearance had not changed. The F3 lot were a little further advanced, but there was still no heart-beat. The control embryos at this time have, of course, a vigorous circulation of the blood, they are well pigmented and the yolk vessels are mapped out by the chromatophores. At 8 days old, the Fi group were still in the same condition as when put in the 5°C. temperature 5 daj^s before. There was no heart beat and the embryos appeared as if about 3 days old. They were now returned to room temperature. The F3 lot, after 5 da3^s at 8°C., were further advanced, their hearts were pulsating feebly and very slowh^, blood-cells were formed on the yolk-sacs and masses of blood were frequently observed in the tail regions. These embryos were also now returned to room temperature. After being at room temperature for 3 days, with a total age of eleven days, the Fi lot seem recovered and are developing well, though about 4 or 5 days behind the control. All of this lot were living. The F3 lot were also all alive and in apparently perfect condition. When 18 days old, almost all of the control embryos had hatched. The Fi lot all seemed normal, but none had hatched, and the same was true of the F3 group. Two days later, however, many had hatched in both lots. Thus they were 3 or 4 days later than the control in hatching, which was a little less than the time they had spent at low temperature. Finally, when 27 days old, none of the embrj^os in the two lots had died, which indicates that they were all unusually good specimens. Every one of the 36 in the F3 group hatched, and but 2 in the Fi group failed to hatch, although these appeared normal in structure. A complete stop or an arrest in developmental rate of as much as five days after the optic cups are already formed and just before the beginning of a heart beat does not exert an injurious effect upon any organ that would prevent the normal development of the body form or the capacity to hatch and swim freely. Experiment 909. Embryos 6 daj'-s old, with fully vigorous blood circulation over the yolk-sac and within the embryonic body, with chromatophores fully migrated and expanded, and with their bodies moving and twitching, were placed in a temperature of 7°C. After STRUCTURE AND DEVELOPMENTAL RATE 137 24 hours the hearts were still beating, but much slower than the control, and they had fallen about 20 hours behind the control in development. After 3 days in the cold these embryos had fallen far behind the control in size and development. The heart was beating slowly and the blood was circulating in all. Two daj^s later, when the embryos were 11 days old, they were still in about the 6-day condition, although all were living at a slow rate during the 5 days in the refrigerator. When 13 days old, and after being 7 days in the low temperature, the embryos were all alive. They had a slow heart beat and a circulation which in many was so sluggish as to allow large sinuses in the 3^olk-sac to remain distended with blood, although the circulation within the embryonic body was complete. At this time they were returned to room temperature, and after 24 hours the heart beat had regained a normal rate and the blood was circulating freely and fast in each of the specimens. All seemed fully recovered from the depression caused by the low temperature. At 19 days old, almost all of the control embryos had hatched, but none of these that had spent 7 days at 7°C. were yet up to the point of hatching. At 22 days old, stijl none were hatched. But when 23 days old, 16 had hatched and 38 were unhatched. They were thus 5 days behind the control in beginning to hatch as a result of their 7 days of slow development at the low temperature. On the 25th day only 2 were still unhatched, and finally, on the 27th day, these two had not hatched, although they seem normal in structure. There is, therefore, no evidence that any harm was done by subjecting advanced embryos with blood freely circulating to low temperatures. Although under the cold conditions the heart rate was greatly reduced and the circulation rendered extremely sluggish for a period of seven days. On return to normal temperature recovery was rather prompt and seemed on superficial examination to be complete. A number of similar experiments to those reviewed above are recorded in my notes, and in all cases the results are in close accord. If we consider them entirely from a standpoint of the external evidence of injury produced, a fair comparison may be made with the results of further experiments in which the eggs were stopped and arrested at other developmental periods or moments. It will be readily shown that periods very close to some of those used above are decidedly dangerous moments at 138 CHARLES R. STOCKARD which to stop or interrupt the progress of development. From such experiments one seems justified in classing these moments in development as indifferent at which arrests may be induced without causing subsequent high mortality among the embryos and without a considerable percentage of gross structural deformities resulting. The eggs treated in the above experiments were all stopped at comparatively indifferent moments in the course of development so far as their gross structure and behavior up to the newly hatched free swimming stage of life would indicate. In the section following a review of experiments with decidedly different results will be considered. c. Stopping or retarding the progress of development at stages of critical susceptibility to developmental interruption From facts we know of development in nature, as well as, from the experiments discussed in the preceding section, it becomes evident that the course of embryonic development need not necessarily progress in a continuous manner, but may be stopped entirely for a considerable length of time or may be decidedly reduced in rate without necessarily injuring the end result. On the other hand, it is equally well known in a general way, and even more widely believed, that when a developing egg is injured in such a manner as to cause its development to stop, it is usually incapable of resuming development at all, or if it does start again to develop it will only continue for a short time and often in a very abnormal fashion. These two apparently contradictory statements are equally true. This is due to the fact that the way in which a developing egg responds after having had the progress of its development stopped or arrested by any unfavorable condition depends entirely upon the stage in development at which the interruption occurred. In the first case stated above, the interruption is introduced at a stage in development when no unusually rapid changes are taking place, a comparatively quiescent moment during which all parts are developing, but during which no particular or important part is going at an excessively high rate. Such a time we may term a 'moment of indifference.' STRUCTURE AND DEVELOPMENTAL RATE 139 In the second case, the interruption occurs at a time when certain important developmental steps are in rapid progress or are just ready to enter upon rapid changes, a moment when a particular part is developing at a rate much in excess of the rate of the other parts in general. Gastrulation is an important developmental step which apparently cannot be readily interrupted without serious effects on subsequent development. Many of the chief embryonic organs seem also to arise with initial moments of extremely high activity, processes of budding or rapid proliferation and growing out. During these moments a given organ may be thought of as developing at a rate entirely in excess of the general developmental rate of the embryo. Such moments of supremacy for the various organs occur at different times during development. As is well known, a certain organ arises much earlier or later in the embryo than certain others. When these primary developmental changes are on the verge of taking place or when an important organ is entering its initial stage of rapid proliferation or budding, a serious interruption of the developmental progress often causes decided injuries to this particular organ, while only slight or no ill effects may be suffered by the embryo in general. Such particularly sensitive periods during development I have termed the 'critical moments.' That we may analyze the responses of embryos in which developmental interruptions have been introduced during some of these critical moments, resource may again be had to the records of the experiments. Here also a large number of experiments have been performed, but we shall only attempt a review of certain typical examples from the entitle series. Experiment 901, B Series. Eggs were fertilized at 11 a.m., and three hours later, immediately before the first cleavage, they were divided into four lots, one for control and three others which were placed in a refrigerator at temperatures of 5°, 7°, and 9°C. When 24 hours old, the control had reached a high segmentation stage, the germ-discs in only a few had flattened down on the yolk sphere, but in none had the cap begun to descend over the yolk or to form the germ-ring. The night had been unusually cool and the control was thus developing far more slowly than the normal summer average rate. At 24 hours old, the germ-ring is usually well formed and has descended about one-third to one-half way over the 140 CHARLES R. STOCKARD yolk-sphere. The inhibition resulting from the cool nights of the early season very probably accounts for the almost uniform inferiority of emljrj^os developed at this time as compared with those developing during early July, the height of the spawning season for thus locality. Lots Bi and B2, in temperatures of 5° and 7°C., respectively, for 19 hours, were all in either 2- or 4-cell stages. They were thus almost completely stopped in development. The 2-cell stage was about reached when they were placed in the low temperatures, and probabl}' some were dividing the second time before the surrounding water had cooled to the temperature of the refrigerator (all dishes contained 60 cc. of sea-water). Lot B3 at 9°C. contained after 19 hours fairly regular 16- and 32-cell stages. At this temperature cell division had been able to continue, although at a greatly reduced rate, accompHshing only three or four divisions in the 19 hours. The control eggs 48 hours after fertilization showed the germ-ring onl}^ one-quarter over the 3^olk sphere, with the embryonic shield beginning to form (fig. 4), a stage that should be attained within 24 hours during the warmer part of the season. Lot Bi, after 45 hours at 5°C., was in first-, second-, or third-cleavage stages. The arrangement of the cell groups was often very irregular and many cells contained large vacuoles. There were a very few almost typical 2- and 4-cell groups. In some of the '2-cells' a large central vacuole seemed to ahnost divide each of the cells (fig. 5) . These eggs at 5°C. have thus only in rare cases divided more than once during 48 hours. This lot was now removed from the refrigerator and returned to the room temperature after being 45 hours in the cold. Lot B2, at 7°C., was in much the same condition as lot Bi, except that some eggs had undergone one or two further cleavages. There were many irregular cleavage patterns and a few almost regular 16or 32-cell stages. A number of the germ-discs consisted of irregular partly divided masses (fig. 6). Lot B3, at 9°C., had developed very slowly but fairly well, and now after 45 hours in the low temperature contained germ-discs composed of from 64 to about 128 cells. The cell arrangements and shapes of the discs were almost uniformly regular. Therefore, at this temperature development progresses, though very slowly, and none of the cell masses had yet begun to flatten down to cap the yolk-sphere. When 3 daj^s old, the control embryos were well formed, although the germ-ring was not yet entirely over the yolk-sphere, much the same stage as shown above in figure 1. Lot Bi, after being at room temperature for 24 hours, had passed from the 2-, 4-, and 8-celled conditions and had reached a high segmentation stage. The discs had not fully fiattened on the yolkspheres, but were beginning to descend. There was no gross indication of germ-ring or embryonic-shield formation. Many eggs had promptly recovered their ability to develop on return to higher temperature and had progressed during the 24 hours about as far as the control had gone during the first 24 hours of their development. STRUCTURE AND DEVELOPMENTAL RATE 141 Lot B2 had now been for 70 hours at 7°C. These showed r.iany irregular germ-discs, but some were fairly regular 16- and 32-cell stages. Their condition was thus much the same as on the day before and they had scarcely progressed at all during the 24 hours. These eggs were now returned to room temperature. Fig. 4 A control embryo 48 hours old, the germ-ring only one-quarter over the yolk, far behind the usual stage on account of the cool season. Fig. 5 A group of cleavage patterns 48 hours after fertilization and after 45 hours at a temperature of 5°C. Development is practically stopped. In many of the two-cell stages large vacuoles, V, occupy the entire center of the cells. Fig. 6 An irregular partly undivided protoplasmic mass with blastomeres at its ends, 48 hours old after 45 hours at 7°C. Lot B3 still had, after 70 hours at 9°C., high segmentation discs about the 128-cell stage. The discs were normal in general appearance. Thus at this temperature development continues, but at an extremely slow rate. This lot was now also returned to room temperature. When the eggs were 96 hours, four days old, the control embryos w^ere fully formed with prominent optic vesicles, the embryonic heart was not yet visible, and there was no pulsation. These embryos were thus scarcely up to the midsummer 72-hour stage, since the embryonic heart beat is often fully established before such a time. The cool 142 CHARLES R. STOCK ARD weather of early June had caused this control to fall about 24 hours behind in the four da3"s. Although such embryos appear to be normal, many of them are inferior in size and general appearance when compared with more rapidly developing specimens of the later warmer season. This advantage is no doubt due to the retarded development primarily resulting from the cooler tempei'ature, and not to a poorer quality of the eggs, since the midsummer eggs will fare in a similar fashion when caused to develop at the same temperature. Such a retardation, however, is too slight to produce gross defeats in any average lot of eggs, yet the embryos very probably are somewhat below par as their physiological responses would indicate. Lot Bi had now been for 2 days, 48 hours, at room temperature after having spent 45 hours at 5°C. The germ-caps were about one-half over the yolk-sphere, the germ-rings and embryonic shields were well formed in most of them. The}^ presented the condition of a midsmnmer 24-hour stage, or were about up to the condition of the present control at 48 or 50 hours. Thus during the 48 hours at room temperature these eggs had developed about as rapidh' as did the control during their first 48 hours of development. The embryonic shields with the embryo in outline appeared normal, although some were considerably behind others and a great many failed to resmiie development after being removed from the refrigerator. The lot B2, after 24 hours at room temperature following a stay of 70 hours at 7°C., showed disc-like caps flattened down, but no germrings were yet formed and the disc had not begun to descend over the yolk-sphere. Some caps were still high or mound-like and many were irregular, containing cells of different sizes (fig. 7). A large number of eggs failed to resume development and there were many discs with vacuoles in their centers, etc. The mortality resulting from this exposure was, therefore, high and many embryos were rendered abnormal during these early stages. The lot B3, after 24 hours at room temperature, were in an even worse condition than those in B2, although a single individual had a germ -ring one-fourth over the 3'olk-sphere and was thus the most advanced specimen of the two lots. The majority, however, presented high germ-discs with a peculiar vacuole occupying about half of the disc and distorting the position of the cells (fig. 8). Vacuoles similar in appearance are frequently present in eggs slowed by other methods, such as solutions of LiCl, etc. But in this case the vacuole differs somewhat in not being a simply distended segmentation cavity. It will be recalled that these eggs developed very slowly at 9°C. for 70 hours, so that they had progressed much beyond the lots Bi and B2 when removed from the cold. Yet after 24 hours at room temperature they were at a disadvantage rather than an advantage when compared with Bo at this moment. The extremely slow progress during the 70 hours would seem to be more detrimental at this stage than the almost complete cessation of development in lot B2. In later stages, however, STRUCTURE AND DEVELOPMENTAL RATE 143 those eggs which have been subjected to the higher temperature will gain a decided advantage as compared with the lower-temperature groups. At 5 days old, the control showed the heart beat just beginning, but no circulation. Lot Bi, after 3 days at room temperature, con 8 Fig. 7 Three specimens 4 days old, having been 24 hours at room temperature following a stay of 70 hours at 7°C. The upper outline shows a disc-like cap flattened down on the yolk-sphere; the middle one, a high segmentation cap; and the bottom specimen has a cell mass comparable to a normal 12-hour stage. Fig. 8 Top and lateral views of 4-day specimens, having been 24 hours at room temperature following 70 hours at 8°C. These segmentation masses are very abnormal and are distorted by the presence of a huge vacuole, V. tained short embryos on the surviving eggs, but the majority of eggs failed to develop at all after being removed from the cold. Lot B2 had germ-rings onh' about one-half, or a little more, over the yolksphere. Thus the one day longer in the refrigerator had caused these to be far behind B]. 144 CHARLES R. STOCKARD The Lot Bs had germ-rings also a little more than half over the yolk, though here again a great many were not developing at all. The 6-day-old control presented black and red chromatophores full}^ expanded on the yolk-sac and the embryo. The circulation was completely estabhshed both within the embryonic body and on the yolk-sac. The embryos had begun twitching and moving their bodies. Lot Bi had now been at room temperature for 4 days after having been arrested for 45 hours at a temperature of 5°C. The embryos were small with no circulation, almost all seemed abnormal at the head end and many were short; the tail region was not properly formed. They were thus far behind a usual 4-day embryo. Lot B2, after now developing at room temperature for 3 days, contained many small cyclopean and otherwise defective embryos, but the majority of eggs had stopped and did not develop beyond the condition shown by them after the 70-hour stay at 7°C. Lot B3 contained some fairly regular 3-day embryos, but with no circulation, and many of these were deformed. Seven days after fertilization the blood-vessels of the control embryos were well mapped out by the alignment of pigment and the embr3^os themselves were vigorously active. Lot Bi contained at this time many well-formed embryos with good circulation, pigment migration, etc. Others had a sluggish and poorly established circulation, some showed a heart beat, but no circulation, and many more had stopped in development and the cells had wandered apart to lie over the yolk surface. Some eggs presented simply yolk-sacs with blood-spots scattered over them, but without an embryo. A few of the apparently well-formed embryos were abnormal in various ways. Lot B2 showed no circulation, many eggs did not develop, and almost all were readily seen to be abnormal. The lot B3 also showed no circulation, but contained some well-formed embrj^os just about in condition for the heart beat to begin. When 9 days old, the control contained all fine vigorous embryos. Lot Bi still showed those with only blood and pigment on the yolk-sac, with no embryonic body present. Others still had the cell-mass confined to the upper yolk-pole and there were a few abnormal embryos, some with and others without a circulation. The majority of the living specimens were now normal in appearance with a vigorous circulation, as if some degree of regulation and recovery had taken place Lot B2 contained many apparently normal embryos with a good circulation, while some were small and some were abnormal without a circulation. Some eggs showed the old mass of early cleavage cells at the upper yolk-pole still alive after 9 days, though not developing; the cell-masses were irregular and the individual cells spherical in form. Several yolk-sacs also contained blood-cells and a few pigment cells, although no embryo was present. Lot B3 contained a few eggs with early cell-masses similar to those in lot B2. The large majority of the surviving individuals now seemed STEUCTURE AND DEVELOPMENTAL RATE 145 normal with a good circulation; very few were slightly deformed with poor or no circulation. The majority in all B lots were now normal in appearance with a good circulation. In the B3 lot 47 seemed normal out of 61, so that 14, or about 25 per cent, were abnormal, and of these 6 showed the early cell-mass condition or were not developing. Thus only 8 embryos were smaller or slower than normal. Yet it must be recalled that many dead eggs had been removed during the first few days following return to room temperature. In the control, however, there were no abnormal ones and there had been no unusual mortaUty. When 12 days old, the control were all normal and about in the condition to hatch. In lot Bi 6 showed that development had stopped during an early stage, 4 showed yolk-sacs with blood and pigment but no embryos, 10 were deformed embryos with no circulation, 4 were also deformed, 2 being eyeless, but with a circulation. Of all the survivors in this lot 24 were affected and 45 were apparently normal at this time, thus over 34 per cent were bad. In lot B2 14 failed to develop beyond the cell-mass stage, 4 presented only yolk-sacs with blood-spots and pigment cells, 8 were abnormal with no circulation, and 3 were abnormal with a circulation, while 31 appeared to be normal. Thus 15 of those that continued to develop, or about 33 per cent, were abnormal and 25 per cent of the total number that lived were unable to resume development after their stay at 7°C. In lot B3 6 stopped development early, though continuing to live, 3 were deformed and possessed a circulation, 5 were deformed without a circulation, and 47 individuals were apparently normal. Here, then, only 14 per cent were deformed of those that developed. Such a record is twice as good as that attained by either of the other groups. Thus the 9°C. temperature, at which an extremely slow rate of development is possible, is not so injurious to the later development of those individuals which survive it as are the more severe temperatures of 5° and 7°C., which practically stopped the progress of development entirely. The control when 15. days old had not yet begun to hatch, on account of the cool season. In lots Bi, B2, and B3 one or two more of the abnormal embryos in each had died and all of the individuals were behind the control in their developmental condition, though, as stated above, many in all groups now appeared normal. When 19 days old, a large majority of the control were hatched and swimming about in a typically active fashion. In lot Bi none had hatched and several more had died. In B-^ none had hatched and a few more also had died. In B3 none had hatched, many still seemed nonnal, and many were deformed, showing distinctly typical eye anomalies, cyclopia, etc., and there were many types of head and caudal end deformities. THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 146 CHARLES R. STOCKARD When 21 days old, in lot Bi 2 more had died and 3 had hatched, in B2 2 had hatched, and in B3 many had hatched. The control at 22 days old showed 47 hatched and 18 imhatched, although all were normal. In lot Bi 5 had hatched, and 52 were unhatched, the majority were normal in appearance, but 13 were grossly deformed in the head region and possessed small ill-formed bodies. In lot B2 4 had hatched and 36 were unhatched, of these 11 were grossly deformed and 25 seemed normal in structure. In lot B3 15 were hatched and 39 were not, of the latter 7 were grossly deformed, one a typical cyclops and one a monophthalmia. Four others had slightly underdeveloped eyes in addition to the 7 actually deformed. When 23 days old, only 2 of the control were still unhatched. In lot Bi there were 35 hatched and 20 unhatched. Lot Bo contained 27 hatched and 12 unhatched. In lot B3 over 40 had hatched and only 9 were unhatched. One had died and 2 of those that had hatched showed their bodies so badly twisted that they were unable to swim. One of these had a badly deformed body and one eye was abnormally small with the lens protruding. At 25 days old, every individual in the control lot had hatched. Lot Bi had 16 unhatched and 4 of those that had hatched showed deformed bodies and could not swim in a straightforward manner. Thirty-seven of those hatched were normal in appearance, 3 of the unhatched had died. The following deformed conditions existed: One was a double-headed specimen, man}^ had no eyes, monophthalmia, abnormally small eyes, short bodies, etc. Thus at this time after the great number of specimens had died there were still over 20 per cent deformed. In lot B2 11 were unhatched and 29 had hatched. Two of those hatched were so deformed and twisted as to be unable to swim. The 11 unhatched ones were all grossly deformed, so there were 13, or 33 per cent, of the total living specimens deformed at this time. Lot B3 showed 5 unhatched and about 40 hatched. Four of those hatched were so deformed as to be unable to swim in a normal fashion. One of these presents the peculiar condition of a heart beat, but no circulation in a hatched fish with a long normally shaped body. There was a large accumulation of blood-cells within the sinus venosus and the median vein in the region of the anus was filled with red corpuscles. This specimen could swim poorly from place to place, had fairly regular respiratory movements, and waved its fins without a circulation of its blood. When 34 days old, the Bi lot finally had 9 specimens which were unable to hatch, all of them were deformed. Lot Bo showed 6 unable to hatch, all deformed and without a blood circulation. In lot B3 4 failed to hatch. It must be recognized that a great many specimens in each of the lots Bi, Bo, and B3 had died during the preceding 20 days. The weaker and actually most defective individuals are eliminated as shown b}^ the early mortality records. STRUCTURE AND DEVELOPMENTAL RATE 147 The above 6 unhatched embryos in lot B2 were kept in order to determine how long such specimens might be able to survive. When 52 days old, these specimens were still alive, although the yolk-sphere had become very small, being almost absorbed. The small monsters were practically at a stand-still as to their life processes and were not kept after this time. These experiments are here considered in a general way with-] out going into the details of the deformities concerned. Theyl demonstrate the fact that a normally continuous development' may be modified into a discontinuous one by stopping its course during a very early cleavage stage. The fact is also shown that this stoppage is followed by a too slow resumption of the developmental rate and results in about 33 per cent of gross anomalies among those specimens able to survive the treatment. The mortality induced by stopping at such periods is high, the majority of eggs in all cases dying after return to normal temperature. Great variation in ability to withstand such treatment is shown by these hardy Fundulus eggs. The weakest ones succumb without resuming development on removal from the cold. Stronger specimens may undergo a few^ further divisions and live for some time in a high segmentation stage without being able to continue or progress further in their development. Other eggs continue development, but in such extremely abnormal fashion as to fail completely to form the embryonic body and only differentiate certain tissues scattered irregularly over the yolk-sac. Still more hardy specimens succeed in forming the embryonic body, but many organs requiring a high degree of cell proliferation and growth for their development, such as the eyes, other brain diverticula, mandibular, hyoid, and branchial pouches, etc., are unable to form in a normal fashion, and numerous defects in these parts are to be found. Finally, the most resistant or hardiest eggs withstand the stoppage due to the low temperature and are able to resume development at an almost normal, though slightly retarded rate. These individuals may seem typically normal in structure, and often develop into hatched free-swimming fish, yet even these not infrequently show some indication of a subnormal condition in having their bodies slightly twisted or bent, and in being unable 148 CHARLES R. STOCKARD to swim in a perfect fashion. Veiy probably the best of these specimens would present various ill effects from their early arrest could they be kept and observed throughout a longer life period. There are only a few simple performances to be observed in the actions of a newly hatched fish. Whether they are later capable of feeding and digesting food, reproducing, and performing other functions in a normal fashion is unknown for such individuals. The probable later effects as well as the classification of the deformities following stoppage at various developmental moments will be more fully considered in the subsequent sections. One other similar series of experiments may be briefly recorded to further make clear the results which follow various degrees of interference with the rate of development during its early stages. A careful consideration of these records also brings out some of the differences between the effects of completely stopping and of slowing to a decided degree. The significance of the very varied types of deformities which result from early interruptions will be considered in connection with the records in the following sections of the discussion. Experiment 902, B, C series. Three hours after fertilization, when in the 2-cell stage, eggs were placed in the refrigerator in the following arrangement: Bi and Ci at 5°C., B2 at 7°C., and B3 at 9°C., with a control from the same groups of eggs kept at room temperature. When 24 hours old, the control were all developing in a perfect manner, but again somewhat slower than the maximum midsummer rate. The germ-caps had flattened on the yolk, but there was neither germring nor embrj^onic shield formation yet visible. The Bi and Ci lots had all divided once or twice before cooling down to the 5°C. temperature. Every egg in both vessels was alive and in the 2-, 4-, or 8-cell stage. In the Ci lot almost all were 8 cells. In many the 8 cells were arranged into two groups of four (fig. 9). Lot B2 were, as a rule, in the same condition, all eggs being alive, the great majority in the 8-cell stage, with a few showing the 4-cell stage. The B3 lot, after 24 hours at 9°C., were practically all developing at a very slow rate and had reached about the 64- or 128-cell stage. They seemed normal and in good condition other than for their very slow progress. Forty-six hours after fertilization, the control showed every egg developing, the germ-ring having grown almost completely over the yolk-sphere, the embryonic body was well formed, but the optic outpushing had not yet arisen. STEUCTURE AND DEVELOPMENTAL RATE 149 In lots Bi and Ci the eggs had divided once during the last 24 hours and were now almost all in the 8- and 16-cell stages, while a few were irregular 32-cell stages. Much cellular disorganization had taken place and the cell groups were broken and irregular, often with large unsegmented protoplasmic masses. Lot B2 were in somewhat similar conditions, all showed more or less irregular 8- and 16-cell masses. Many also showed large unsegmented protoplasmic areas with a few cells around the periphery (fig. 10). Lot B3 at 9°C. were all developing somewhat faster than the above, and now presented well-arranged high-segmentation caps. They were normal in appearance up to this tune. When 4 days old, the control showed a perfect condition with not one egg having failed to develop. There- was a vigorous heart beat and a Fig. 9 A cell group 24 hours old, having been in a temperature of 5°C. since three hours after fertilization. The 8-cells are peculiarly arranged into two groups of 4 each, such specimens may give rise to ordinary single individuals. Fig. 10 A large unsegmented protoplasmic mass with blastomeres around the periphery. A frequent specimen in lot B2, experiment 902, when 46 hours old after 43 hours at 7 °C. good circulation fully established. They were thus developing considerably faster at this time of the season than did the control of experiment 901, which was fertilized 10 days earher. These 901 embryos had not developed a heart beat or established a circulation when 4 days old. Lot Bi, after 4 days at 5°C., showed a few regular cleavage caps of about 64 cells. The majority, however, exhibited very irregular cleavage arrangements and some were almost amorphous protoplasmic masses, although all were translucent and alive. The eggs had, therefore, developed at an extremely slow rate, but had not completely stopped. These specimens were now placed at room temperature. In lot Ci the majority had, after 4 days at 5°C., rather regular 64- or 128-cell caps. This lot was from a different group of eggs than the B series, and its control was going in a manner exactly similar 150 CHARLES R. STOCKARD to the B control. These eggs, however, may be individually more resistant. This lot was also now returned to room temperature. Lot B2, after 4 days at 7°C., showed some eggs with regular cleavage caps of 64 cells and more, but the majority showed caps of irregular cell masses. These were now placed at room temperature. Lot Eg, after 4 days at 9°C., had all reached a high segmentation stage comparable to about the condition of the control at 18 or 20 hours old. All of these eggs had a distended l)ubble-like segmentation cavity similar to that described by me ('06) as resulting from treatments with LiCl solutions. Every egg was developing and furnished a particular!}^ fine lot for an experimental test of this sort. These also were now placed at room temperature. At 5 days old, the B and C controls were perfect with all embryos developing well. In lot Bi the great majority had failed to resume development after being 24 hours at room temperature. The segmentation caps were breaking down and becoming disorganized. The few specimens that had resumed development showed the germ-ring formed and about one-half over the yolk-sphere. The lot B2 were in very nearly the same condition as Bi. In B3 the great majority were developing and the germ -rings were here also about half over the yolk-sphere. Lot Ci showed many stopped in development, but here the majority seemed well and showed the germ-ring about one-quarter over the yolk. When 6 days old, the treated groups had been at room temperature for 2 daj's, the Bi lot presented the following condition: Eight embryos had formed, there was one yolk-sac with scattered cells, and 33 eggs had died or failed entirely to resume development. All eggs in this lot had originally begun development and the control from the same group of eggs was perfect, thus the low temperature for 4 days had caused a very high mortality. Only about 22 per cent of the eggs resumed development. In lot B2 49 had formed embryos, 6 of these were short, lacking a complete formation of their caudal ends, the others were well-formed specimens with optic vesicles present. Fifty-eight did not resume development, although all had begun before being placed in the cold, thus there was a mortality' of 54 per cent in this group. In lot B3 practically all formed embryos which now showed optic vesicles and body somites clearly formed. This lot was about as good as the control in respect to the number of eggs developing. Thus a 4 days' sojourn at 9°C., with an extremely reduced developmental rate did not prevent the possibility of again resuming a development of normal rapidity. This extreme slowing at a slightly higher temperature is not nearly so fatal or injurious to later development as the almost complete stop caused by the lower temperatures of 5° and 7°C. Lot Ci similarl}^ treated at 5°C., but consisting of eggs from another parental pair, contained at this time 21 embryos with optic vesicles forming, 7 short embryos with the germ-rings not completely over the STRUCTURE AND DEVELOPMENTAL RATE 151 yolk, and 13 had died or failed to resume development. Therefore, in this lot 66 per cent were able to resume development, which is a somewhat better record than the B series. This difference may easily be due to individual variations between the two lots of eggs from the two different pairs of fish, yet both lots of eggs were unusually fine, as was shown by the perfection of the B control as well as the C. When 10 days old the controls were going perfectly and seemed about at the point of hatching, having grown long with the tails curved around to cover the side of the heads, j^et the yolk-spheres were still rather large. In lot Bi 7 of the 9 living eggs showed embryos almost normal in appearance with good circulations, one was badly deformed and had a pulsating heart, but no circulation, while the one yolk-sac without an embryo had not progressed in development. Lot B2 showed 36 strong embryos with good circulation, though one of these was slow, with eyes abnormally close together. Four specimens Avere badly deformed, one with a circulation of the blood and three without. There were two yolk-sacs with blood and pigment cells present and two others did not develop. Thus 42 eggs were still alive, of which 7, or 16| per cent, were grossly deformed. All eggs in lot B3 seemed normal and well, although far behind the control. In lot Ci 15 specimens seemed normal in structure, though two of these were slower than others in development. Ten specimens, or 40 per cent of the total, were deformed, 8 showed grossly malformed heads and bodies, one embryo being represented by an amorphous mound of tissue on the yolk-sac, and two other specimens had only deformed heads with a fair circulation of the blood. Thus in this lot where the mortality following removal from the cold was low, the percentage of deformed specimens is two and one-half times greater than from the B2 lot that had suffered a high initial mortality. When 16 days old, the majority of both control lots had hatched, though none of the inhibited ones had. When 17 days old, one in lot Bo and 3 in lot B2 had hatched, though none in Bi and Ci. At 18 days old, the controls still had a few unhatched. In lots Bi 6 were hatched and 2 were not; in Bo 21 were hatched and 20 were unhatched; in B3 33 were hatched and 27 were not; in Ci 13 were and 12 were not hatched. When 24 days old, lot Bi contained one badly abnormal specimen still unhatched. In lot B2 17 were still unhatched, 5 of these were grossly deformed. In lot B3 12 were unhatched, though seemingly normal in structure. These were all far behind the control in time and manner of hatching. In lot Ci 8 were deformed and unhatched, and one, slightly abnormal in gross appearance, partially succeeded in freeing itself from the egg membrane. Thus really 9 of these were deformed and unhatched. When 29 days old, one individual in the B control had not hatched though the others had been free swimming for 10 days. This was the 152 CHARLES R. STOCKARD only lack of perfection in this control of more than 50 individuals. In Bi there was one unhatched monster. In Bo 10 were still unhatched, though 6 seemed normal and ready to hatch; therefore, the cold treatment greatl}' reduces the strength and delays the hatching moment of these embryos. In B3 3 were unhatched. In Ci 8 were unhatched, 7 of these were deformed, one being a twin specimen and one almost normal. This series of experiments further shows the possibility of almost stopping, or reducing to an extreme degree, the rate of development during the earliest cleavage stages and again resuming a more or less normal rate on the part of a few individuals. An almost complete stoppage at an early cleavage stage results in a very high mortality ranging from as great as 78 per cent and 54 per cent, down to 34 per cent. However, the reduction in rate brought about by a less severe temperature of 9°C. does not cause so great a mortality and does not prevent the resumption of development of almost normal rapidity. It is clearly show^n, how^ever, that although certain specimens may resume a fairly normal developmental rate after such treatments, the early arrests have had injurious effects upon the quality of the resulting embryos. A considerable percentage of gross abnormalities occurs in all of the groups, and even those embryos which appear on close examination to be normal in structure are extremely slow in hatching and are not in all cases capable of typical swimming reactions and perfect behavior as young fish. A point of particular importance is that in such a series as this w^hich had been arrested during an early cleavage stage, the monsters resulting are not limited to any particular type, but exhibit, in a series of sufficient extent, almost all known types. There may occur double monsters of varying degrees, from separate twins, fused but with complete bodies and tails, to double bodies and single tails, and finally different degrees of doubleheadedness on single bodies. There are specimens exhibiting anophthalmia, monophthalmia, microphthalmia, cyclopia, and all types of malformed eyes. The brains may be slightly asymmetrical, irregular, tubular wdth no primary ventricles, or deformed in various ways. The mouth and branchial region may STRUCTURE AND DEVELOPMENTAL RATE 153 exhibit almost any known defect. The fins may be poorly developed and the bodies ill-shaped and twisted. The tails may be short, bifed, and undeveloped due to a slow or arrested descent of the germ-ring. And finally there may be such minor defects as would escape observation until the hatched embryos were found to be unable to right themselves and swim. These are the defects to be seen on simple external examination, the internal structures are as frequently abnormal. The latter fact is borne out by numerous examinations of these monsters in sections. I have studied a great many of the sectioned specimens during the past number of years. The reason for this great variety of monsters following arrests during cleavage stages is that the development of all organs or parts must subsequently take place and all may thus become arrested and deformed. When eggs are treated at later stages, as at the beginning of gastrulation, no double monsters will occur, their moment has passed, though the various brain, branchial, and other defects mentioned may exist. When treated after the embryonic axis is visible, it is most difficult to get any gross eye defects and so on. Thus it may be said that the earlier the arrest the more numerous will be the type of defects found and the later the arrest the more limited the variety of deformities, since there are fewer organs to be affected during their rapidly proliferating primary stages. The same treatment that causes a gross deformity when applied during an early stage, will during a later embryonic stage often give only a minor effect. The further records of experiments will render these statements more fully certain. Here I wish simply to call attention to the great variety of gross deformities resulting from these early arrests. The contrasts in detail between these and the later treatments will be shown in the following pages. I hasten, however, to caution any experimenter who may in the future find a double monster or cyclopean monster, for example, in a group of eggs arrested or treated during late developmental stages, not to assume that this is due to the late treatment or that it disproves the standpoint stated above. For 154 CHARLES R. STOCKARD such an occurrence is simply accidental and due to the fact that the specimen was already arrested or defective in an early stage as might by chance happen in any normal lot of eggs. It is clearly true, as I shall show beyond, that only very early and carefully regulated treatment can artificially produce twins and double monsters, a phenomenon which must happen about the stage of gastrulation. Therefore, the treatment must be applied much before this time. Cyclopia may be induced by slightly later treatments, but only during a rather limited time, and quite early at that. Other experiments will later be considered in order to illustrate the difference in response on the part of these eggs following treatments similar to those above, but applied as nearly as possible at certain particular developmental periods. d. Differences in effect between greatly reducing the developmental rate and actually stopping temporarily the process In the foregoing review of experiments attention was frequently called to the fact that in certain of the low temperatures employed an almost complete stop in development was actually obtained, while at the somewhat higher degrees the progress of development was reduced to an extremely slow rate, but not actually stopped. A more specific comparison between the effects resulting from actually stopping and greatly slowing the rate of development may now be made. Three groups of Fundulus eggs when in the two-cell stage were placed in temperatures of 5°, 7°, and 9°C., respectively, as reviewed under experiment 901, B series. The first two temperatures wxre sufficiently low to almost completely stop development, so that after twenty-four hours of such exposure the eggs were still in the two- or four-cell stage. The group at 9°C., however, developed very slowly and attained either sixteen- or thirty-two-cell stages within the first twenty-four hours. In other words, at this temperature three or four cell divisions occur per day. When all had remained for three days in these low temperatures, they were removed from the refrigerator and the following results ensued: STRUCTURE AND DEVELOPMENTAL RATE 155 The two groups that had been completely stopped in development suffered very high mortalities. In each a considerable majority of the eggs failed to resume development at room temperature, and during the early days of development very many of the survivors appeared abnormal in structure. These, however, later showed some ability to recover, but finally at an advanced stage about 33 per cent of them were still deformed. In contrast to this, the eggs that had developed slowly at 9°C. suffered only a low mortality on return to ordinary temperature and there was not nearly so high a percentage of abnormalities. At a late stage only 14 per cent were deformed as against over 33 per cent in the two other groups. The slowed group also hatched earlier and with a better record than the two stopped groups. Similar differences in records between such groups of eggs were often even better shown, as is indicated in the results of experiment 902. In this case three lots of eggs in the two- and fourcell stages were placed at 5° and 7°C. for four days, after which interval they had divided four or five times and were all in about the sixty-four-cell stage. They were almost, though not actually stopped, accomplishing only one cleavage per day. On return to room temperature, one of the lots from 5°C. suffered a mortality of 78 per cent, only 22 per cent of these eggs being able to resume development, although every one was developing when first placed in the cold temperature. The lot from 7°C. showed a mortality of 54 per cent. Another group of eggs from the same parents and accompanied by the same control were placed at 9°C. at the same time and for the same interval as the above lots. These eggs developed slowly at 9°C., so that after their four-day sojourn they presented high segmentation caps, similar to the condition of the control after eighteen or twenty hours of development at normal temperature. On return to room temperature, these slowly developing eggs resumed a normal rate and practically all formed embryos. Thus, in respect to the number of embryos that were developed, their record compared favorably with the control and contrasted acutely with the only 22 per cent which resumed development after the 5°C. interruption. The number of de 156 CHARLES R. STOCKARD formed embryos was decidedly less from the 9°C. slow lot than in the groups from 5° to 7°C. which had been almost stopped in their development. The 9°C. group also hatched earlier and somewhat better than the other inhibited lots. It is thus seen that during the early rather critical stages of development an ahiiost complete stop is much more severe in effect than a decided slowing, on both the resumption of development and its later progress. An egg developing very slowly but still continuing the process during the early cleavage stages apparently possesses sufficient powers of adjustment or regulation to take up a much more rapid development either gradually or rather abruptly. When, as a result of low temperature, development actually stops during the cleavage or pregastrular stages on raising the temperature, it is frequently stimulated to start again, but the start is so irregular and so out of normal rhythm that many specimens are unable to continue development. These undergo a cellular disorganization followed by death. A considerable percentage of the specimens that do succeed in reestablishing development, still fail to obtain a proper adjustment and balance of developmental activities among their parts. Thus numerous arrested and defective organs are found. This lack of developmental balance among the various parts and the resulting defects are again not so common with eggs that have maintained a continuous development, although for a time it may have been slowed down to an extreme degree. In nature development rarely or never stops during the active early cleavage stages, though slight temperature changes may frequently cause considerable slowing. The natural interruptions usually occur later, as among the birds, just after gastrulation has been well established. The experiments in previous sections also contain data bearing on the effects of stopping and slowing during these later developmental moments. Experiment 905 shows the record of two series of eggs both stopped and slowed when twenty-three and twenty-seven hours old, respectively. In both cases the germ-rings were about formed and gastrulation was well on its way. The lots Ci, C2, and C3 after being twenty-three hours old, or in gastrula STRUCTURE AND DEVELOPMENTAL RATE 157 tion, were almost completely stopped for two days. Their condition when returned to room temperature was about the same as when placed in the refrigerator. Development was very promptly resumed at room temperature and only a slight mortality resulted from the stopping. Only a few of the embryos showed slight defects, but they were behind the control in time of hatching, on account of the two days' arrest. Lots C4 and C5 at twenty-three hours old were placed at temperatures of 9° and 10°C. in which they continued their development at very slow rates, so that after four days the germ-rings had descended over about one-half of the yolk-sphere. During these four days they had advanced in development to a stage usually attained in about twelve hours or were going approximately at a developmental speed of one-eighth of the control rate. On return to room temperature these lots quickly resumed the normal rate, suffered no mortality on account of the retardation, and developed into normal specimens which hatched somewhat later than the control. These slowed embryos possibly had some real advantage over the completely stopped groups Ci, C2, and C3, though it was only slight if any. The D series stopped and retarded at twenty-seven hours old, when in gastrular stages, gave exactly similar records. There was no noticeable excess mortality and no later injurious effects. It may be generally stated that stopping or slowing the development of Fundulus eggs after the gastrular stage, with the temperatures here employed, have no appreciable effect upon the quality of the young fish up to the time of hatching. The moment after gastrulation is established seems generally to be a particularly passive stage at which neither stopping nor slowing the rate of development is followed by injurious results. The question now arises whether after stopping development during the gastrular stage there is any difference in result if it recommences rapidly or slowly. When twenty-four hours old, eggs were stopped for one day by cooling to 5°C. They were then allowed to resume development very slowly by being brought into a temperature of 9°C. They developed at a very slow rate for three days and were then brought into the room temperature 158 CHARLES R. STOCKARD and resumed a normal rate of development. Such a procedure introduced at this given developmental stage seemed to have no effect other than to throw the lot of. eggs several days behind the control in their degree of development and time of hatching. In order to determine the ability of the embryo to initiate certain functional reactions when developing at an extremely gradual rate, specimens three days old, just prior to the establishment of a heart beat, were placed in the refrigerator at 5° and 8°C. These temperatures do not seem to inhibit changes in the late embryo to the same extent as they do the early cleavage processes. The group in 5°C. still had no heart beat after being chilled for five days, so these specimens may be said to have been almost completely stopped. After five days the lot at 8°C., however, had developed a very slow and feeble heart beat. Thus these had definitely progressed at such a temperature and had established the functional activity of the heart muscle. Both groups on being returned to room temperature recovered completely and hatched in a normal fashion. Therefore, neither stopping for five days nor slowing to an extreme degree the development of these three-day-old embryos produces noticeable effects on their subsequent development and hatching ability. If abnormal development is simply the result of developmental arrests, why should not eggs which have been decidedly slowed in their developmental rates by lowering their temperature give rise to monsters as frequently as do those eggs which have been actually stopped in development at critical stages? When eggs are treated with alcohol, other anaesthetics, or a great variety of chemical substances, their development is not necessarily entirely stopped in order to induce monstrous results. These specimens, however, act during later development in a manner much more comparable to that shown by eggs actually stopped by refrigeration than like specimens in which the developmental rate was simply greatly reduced. The explanation of this fact is probably as follows: Specimens which are caused to proceed at a greatly reduced though continuous rate of development by simply lowering their temperature apparently adjust the developmental progress of STRUCTURE AND DEVELOPMENTAL RATE 159 their several parts to the slow rate in such a manner as to maintain the normal differences in rate of activity among the several parts. The developmental rhythm of the parts is retained and the proper system of balance is unchanged. On resumption of the normal rate the parts all respond in their usual accord. After a complete interruption in development at a critical stage, on resuming the process those parts or organs that were formerly developing at a rate in excess of the parts in general are unable to start up again with their original excess or advantage and other parts have an opportunity to compete equally with them and may thus cause their reduced or arrested expression. That organ developing at the most rapid rate or having the highest degree of metabolism or oxidation at the time of the stop is less able to initiate its original rate when the moment of resuming development occurs than are those parts that were developing more slowly. The production of abnormal development and malformation of organs by treating eggs with strange chemical materials is brought about in a similar manner to the abnormalities following stopping. The part or organ developing at the most rapid rate is inhibited more decidedly by the treatment than are less rapidly developing parts and is, therefore, most affected or modified in its development. For example, at certain stages, the formation of the optic outpushings from the neural tube is the most energetic process taking place in the embryo. Any injury to the egg at this time works to the particular disadvantage of this process and results in underdeveloped or deformed eyes. If the injurious element is then removed, all other parts may continue their development normally, since they were not sufficiently active at the time of injury to be affected in particular. In other words, all of the other parts were affected similarly and no one was any more inhibited than another. The results of slowing and stopping development may be stated very concisely as follows: On slowing development, all parts and organs lower their rates in a somewhat relative fashion, the faster-going parts, even though more decidedly slowed, are still progressing at a faster rate than the slow-going parts. On 160 , CHARLES R. STOCKARD Tesuming a normal rate, the more rapidly developing parts still maintain their necessaiy supremacy. On completely stopping development at a critical stage, that is, when certain parts are progressing at excessive rates, as compared with the rate in general, the rate of all parts is reduced to zero or equality. On resuming development from such a condition, the differential rates are not again established with sufficient promptness and certain parts or organs are suppressed, poorly expressed, or deformed in structure. On stopping devel' opment at an indifferent stage, that is, when important inequalities in developmental rate of the different parts are not occurring, it matters not if the entire rate be reduced to zero. On resuming development the parts all begin at about equal rates without the necessity of a prompt establishment of differences and no particular arrests or suppressions occur. e. The types of arrests or deformities following a stop or slowing in the rate of development Only a general statement of results from a few experiments have been given in the previous pages without going into particulars regarding the variety of deformities occurring. At this juncture I should like to enumerate in a very brief way the kinds of abnormalities which have occurred in all of the experiments where development has been stopped or slowed by a reduction in temperature. In the first place, there were produced a nmnber of doubleheaded, double-bodied, and twin individuals which will be fully considered in the following section. Along with these were single individuals with all varieties of eye defects, anophthalmia, microphthalmia, monophthahnia, cyclopia, etc. These defects were present in heads with either structurally normal or variously malformed brains. The mouth and branchial arrangements were frequently deformed. The otic vesicles were occasionally suppressed to various degrees or developed abnormally during the later stages. A number of specimens were short bodied, some with bifed caudal ends. The general body form and the shape STRUCTURE AND DEVELOPMENTAL RATE 161 of fins showed frequent peculiarities. Extreme cases arose in which amorphous masses of embiyonic tissue were present on the yolk, but no definite embryo was formed. There were simple yolk-sacs with blood-cells and chromatophores scattered irregularly through them. Along with these variously defective indi• viduals were almost invariably certain specimens which in gross structural appearance were normal and succeeded in hatching and swimming freely about. Others were almost normal with a pulsating heart, but without a circulation of the blood. Further more detailed conditions need not be mentioned. This list of defects is sufficient to show that the types and actual individual conditions resulting from a simple interruption of development by reducing the temperature are all identical in character with those induced by treating Fundulus eggs with various chemical solutions (Stockard, '07, '09, '10, '15, etc.) during their early developmental stages or actually with the results of certain mechanical operations upon these (Lewis, '09) and other eggs (Stockard, '13). Furthermore, deformed hybrids resulting from crosses between distantly related species also present exactly the same structural peculiarities (Newman, '15). And finally the progeny derived from male guinea-pigs that have been chemically treated for long periods of time occasionally exhibit exactly similar deformities of their eyes and other parts (Stockard, '13; Stockard and Papanicolaou, '15 and '17). It seems difficult to imagine that the deformities occurring among the eggs that have been merely interrupted by being placed in a refrigerator temperature could be interpreted as other than simple arrests in development resulting from the slow progress which had taken place at certain critical times. It seems equally as certain that the comparable conditions following the other experimental procedures have resulted from a similar cause, simply a lowering of the developmental rates of certain parts at critical moments in their origin or developmental history. In the several sections to follow I shall give much crucial evidence bearing on such an interpretation. 162 CHARLES R. STOCKARD 5. EXPERIMENTAL PRODUCTION OF TWINS AND 'DOUBLE MONSTERS' BY AN EARLY ARREST OF THE DEVELOP.MENTAL RATE One of the earliest accomplishments in experimental embiyology was the production of two embryos, or twins, from a single egg (Driesch, '92; Wilson, '93; Morgan, '93; Zoja, '95; Loeb, '95; Schultze, '95, and others). This phenomenon was first produced by separating the two primary blastomeres so that they were no longer in their usual intimate relation, each then developed independently and produced a complete individual. In the light of this striking experiment, the occurrence of twins and double monsters under natural conditions was readily explained as being the result of an undue separation of the two blastomeres during the first cleavage. Such a separation might have been caused in a mechanical way, the two cells being pressed or squeezed apart, or something unusual in the chemical nature of the environment may have reduced the normal degree of cohesion between the first two blastomeres, allowing them to fall abnormally far apart and finally to become entirely separated from one another. This clean-cut experimental production of twins and its ready application and acceptance as an explanation of the modus operandi for a well-known natural phenomenon, has undoubtedly held back our real understanding of the phenomenon and strikingly illustrates the dangers of directly interpreting occurrences in nature on the basis of results from experiments. Almost at once evidence began to accumulate which questioned the general application of the separate blastomere explanation of twin formation. Such evidence was not always appreciated in this connection, but from our present point of knowledge its bearing is more readily seen. The discovery was very soon made (Wilson, '04; Conklin, '05, and others) that on separating the primary blastomeres in certain species of eggs complete twin embryos do not result. Yet there is no reason to believe that in nature twins and double monsters do not at times arise from the eggs of such species. Twin formations are certainly not due to the separation of the first two blastomeres in these particular species, since each of these blastomeres developing independently STRUCTURE AND DEVELOPMENTAL RATE 163 gives rise to a partial and not an entire embryo. Such eggs have an early differentiation and localization of 'organ-forming stuffs' and these stuffs are unequally distributed to the blastomeres even at the first cleavage. The individual blastomeres are, therefore, not totipotent, but only capable in their later development of giving rise to certain parts of the embryo and not the whole. The eggs of a number of worms and molluscs present this very early localization of differential stuffs, yet in some of these various types of double individuals are not uncommon. These double individuals I believe, in the light of evidence contained in the literature along with that presented here, are the results of a simple process of budding. Again it was shown by Enders and later by Spemann ('03) that double specimens not only resulted from the separation of blastomeres, but the late blastular and gastrular stages could mechanically be caused to develop into double instead of single individuals. The degree of duplicity depended somewhat upon the extent to which the eggs were constricted in a given plane. This was evidently a case of dividing or separating into two parts the growing region of a single individual and thereby establishing two new growing points instead of the original one. The division of a single growing bud into two may be illustrated on plant buds, embryonic animal limb buds, etc. The interpretation of the two separated regions as being the exact derivatives of the two original blastomeres, as Wilder has suggested, is in many cases entirely implausible. Doubleness in nature is probably due to a modification of a budding process, and double monsters and actually identical twins, like all other abnormalities, may result from an arrest or inhibition in development. To state that twins and double individuals are induced by a developmental arrest seems at first thought almost absurd; for how could an arrest serve to give a formation structurally exceeding the normal in extent? One might accept developmental arrests as explanations for many deficiencies in structural expression, but such an explanation of excessive conditions or double-headed and twin individuals. would scarcely be suggested. In the present consideration, however, it 164 CHARLES R. STOCKARD will be very conclusively shown that double conditions and twinning in nature are the result of an unusual budding process produced by an early interruption of developmental rate, and are not connected with a separation of the primary blastomeres except under experimental procedure. Before entering into the particular points of the present experiments, it may be well to explain in some detail the writer's conception of embryo formation and the general process of budding in plants and animals. It has long been known that the notches around the border of certain plant leaves, such as Bryophyllum, have the power under certain conditions to bud and give rise to an entire new plant. It is observed, however, that the new shoots, as a rule, arise from only one or two notches instead of from many. Loeb ('16) has performed most elucidating experiments on the budding phenomena in these leaves. In the first place, although' in nature only a few notches on any one leaf send out shoots at any one time, yet Loeb has shown that there is a potential ability present in every notch to form a shoot. This fact is demonstrated by cutting the leaf into parts in such a way as to isolate each notch. Following such an operation a tiny shoot grows from every one of the isolated notches. It becomes evident, therefore, that not only does each notch possess the potential ability to form a shoot, but under ordinaiy circumstances this shoot-forming ability is suppressed in most of the notches by ths growth of shoots from only one or a few notches. It was further found that almost any notch on the leaf could be selected and forced to bud at the expense of the other notches by simply suspending the leaf so that the selected notch dipped into water. This suggests, of course, that ordinarily the conditions for bud formation are not equally favorable in all notches and, therefore, only a few shoots arise from a leaf instead of one in every notch. These few then tend to suppress the origin of buds from other notches. Does any such set of comparable conditions exist in a developing egg or blastoderm before the initial line or axis of the embryo arises and begins development to form a cornplete animal? STRUCTURE AND DEVELOPMENTAL RATE 165 The periphery of the blastoderm in the eggs of the bird and mammal or the germ-ring in a teleost's eggs is probably in some sense comparable to the notched order of the budding leaf. At a certain place along the germ-ring in the fish's egg a peculiarly rapid cell multiplication begins and the embryonic shield with the axis of the embryo buds away from this place. There is already evidence for believing that more than the one place may be capable of embryonic axis formation, and much is added to such evidence by the experiments now to be presented. There are many potential points around the germ-ring at which an embryonic axis might arise. Here again, as in the plant, when one bud or embryonic axis has arisen, it tends to suppress the potential ability of other points to form an axis, and normally only one individual is developed from the egg. We are entirely unable to state the reasons why a certain point along the germ-ring should form the bud and not another. One can only imagine that this point has some peculiar advantage of position which gives to it a higher power of oxidation and a temporarily more rapid rate of cell proliferation than is possessed by other points, just as the notch which is dipped below the water surface possesses a budding advantage over the other notches around the leaf. Can the advantage of position possessed by a particular point on the germ-ring be reduced so as to equalize the budding tendency of several points and thus allow them all to express their ability to form embryonic axes? Could such a condition be brought about double embryos, twins, triplets, etc., would be produced. The use of the word bud or budding in connection with double embryo formations as employed by Patterson ('14) has been criticised by Assheton, who suggests fission as the better word for the process. Such a discussion seems devoid of value and I employ the word bud to mean what is indicated above. 166 CHARLES R. STOCKARD a. Arresting development by low temperature and the production of double embryos and twins in Fundulus A number of years ago I occasionally found a double embryo or a twin condition in Fundulus eggs that were arrested in their development by being kept in solutions of jMgCl2 (Stockard, '09, figs. 22, 56) and 57) . Such specimens, however, were so extremely rare that their occurrence was never associated with the experimental procedure. Chidester ('14) also found a twin among Fundulus eggs arrested in ether solutions, and reported one other in an egg which had developed in a crowded condition. The eggs of Fundulus heteroclitus are extremely hardy and twins or double monsters are practically never found among these eggs developing under ordinaiy conditions. During fourteen spawning seasons many hundred control embryos have been examined and I have not found among them a twin or double specimen. While on the contrary trout eggs are known to be rather sensitive, and must be developed under very carefully regulated conditions. In the trout hatcheries double embryos and twins are very often found and have at times been collected and studied in large numbers (Windle, '95; Gemmill, '00, and others) . Recently I have found strong evidence of a causal relation between slowing development and the formation of twins in trout, this will be discussed beyond. The evidence led me to experiment with Fundulus eggs in order to determine whether here also there was a direct connection between arresting development or slowing its rate and the origin of double individuals and twins. During the past three spawning seasons, a number of experiments have been performed and the general results of these may be reviewed. Two methods of slowing the rate of development have been employed; lowering the temperature and reducing the oxygen supply. The latter method will be considered along with the occurrence of duplicities in trout eggs. It was soon learned that double embryos and twins could be induced, but only by treating the eggs during a limited develop STRUCTURE AND DEVELOPMENTAL RATE 167 mental period. Either stopping development or greatly reducing its rate during cleavage stages and before the germ-ring has formed, that is, at periods preceding gastrulation, frequently serves to cause doubleness in the subsequent embryo formation. Specimens subjected to any degree or kind of treatment after the gastrular period never produced double or twin embryos. Subjecting Fundulus eggs to low temperatures during early cleavages, the four-, eight-, or sixteen-cell stages, not only arrests the cleavage process, but on later resuming development many eggs fail to establish a normal rate and balance for some time and the early processes of gastrulation would seem to be disturbed. The majority of eggs after a stoppage of cleavage are completely unable to resume development and may live for a few days in an almost stationary condition and then die. Other arrested cleavage caps undergo a breaking-down or falling apart of the individual cells before the death of the eggs. A small minority of these hardy eggs after an arrest during cleavage stages succeed in finally readjusting their development to a sufficient extent to give rise to apparently normal free-swimming young fish. The individual variations in resistance and developmental ability shown among Fundulus eggs are remarkable in all experiments performed on them. Our present consideration is to be centered on that group which is sufficiently viable to continue development, but not so resistant as to be able to completely readjust its developmental processes following the early interruption. Not only does the entire experimental lot become divided into the three above crude classes, but the members of our selected group which is not completely capable of normal readjustment by no means all develop in a similarly defective fashion. These discrepancies again are due to individual variations in the manner of resuming development. Certain specimens after removal from low temperatures resume their cleavages with a fairly normal rhythm and form a typical embryonic shield, but later the larger diverticula from the interior parts of the central nervous system fail to arise in a usual manner, or other processes requiring a high degree of developmental energy are not sufficiently expressed and various de 168 CHARLES R. STOCKARD fects become evident. Other individuals resume their cleavage processes, form a typical blastoderm and begin the formation of a germ-ring, which indicates the commencement of gastrulation, but just here the degree of energy necessary for normal developmental processes is insufficient and a single embryonic bud is not formed with that normal rate of growth which suppresses the appearance of other embryonic buds. Therefore, instead of the one point proliferating at a disproportionate rate to form the embryonic shield, two such points are established with more or less equal rates of proliferation, both of which may be somewhat less active than the single one should be. The formation of two embryonic shields or the initiation of two points of rapid gastrulation away from which will grow the axes of the embryos is in fact the initial or primary step in double formations. The phenomenon is exactly the same as when two buds arise from two notches on the leaf border instead of one bud growing from a single notch. Every notch is a potential bud-forming point, and in the same way many potential invagination points exist on the blastoderm, and when more than one such place begins to grow we have double formations. In this sense it may be appreciated that the intrinsic conditions which give rise to double monsters or twins exist in all eggs and are not produced by the experiment. The experimental modifications of the external conditions simply serve to allow more than the one growing point to express itself. The actual results of several rather typical experiments may be given as better illustrating the occurrence of the double individuals. Experiment 903. A particularly fine lot of eggs was obtained from a large female and fertilized by a single male on July 5, 1919, during the height of the spawning season. Three groups of these eggs were selected, one serving as a normal control and the two others, A: and A2, at three and one-half hours after fertilization when dividing into the 8-cell stage, were placed in temperatures of 6° and 8°C., respectively. The outside temperature was unusually warm and the control eggs developed at a vigorous rate. When 22 hours old the germ-rings were from one-third to one-half over the yolk-spheres in all the specimens the embryonic shields were well formed with the embryonic axes already indicated in the midline. Ever}^ egg in the control lot was developing. STRUCTURE AND DEVELOPMENTAL RATE 169 The two lots in the refrigerator at 22 hours old had as a rule undergone only one cleavage further than when placed in the cold. All were in a rather typical 16-cell stage. The low temperatures had not quite completely stopped development. At 48 hours old, the control embryos were in a very advanced condition. They were large in size with fully formed optic cups and lenses, about 10 to 12 pairs of somites, the pericardium distended and the heart formed, although not yet pulsating. Chromatophores were present and though small had already differentiated into the red and black types. Five hours later the hearts were pulsating, but the bloodvessels were not fully connected and there was no circulation. One familiar with these embryos will realize that such a condition of development is rarely attained in less than 70 hours, thus this control group was developing with unusual rapidity. The eggs composing the Ai lot when 48 hours old at 6°C. were in about 32- or 64-cell stages. Many of the blastoderms were discs of irregular cell arrangement and some presented large uncleaved protoplasmic portions. The A2 lot were in a closely similar condition. The control specimens when 72 hours old had a vigorous blood circulation, with the vessels already mapped out by the migrating chromatophores. During the cooler, earlier part of the season a similar condition was not reached in less than four days of development. The eggs in lot Ai, after being 69 hours at a temperature of 6l^°C., all showed irregular segmentation caps, the cells of which seemed to be in a large vesicle or bubble-hke formation. The caps appeared to contain approximately 64 to 128 cells loosely arranged and in every case located within the bubble-like area, which seemed to prevent the normal flattening down of the cap upon the yolk-sphere. There seems to be a clearly marked surface film between the yolk and the region containing the cleavage mass. It is as if the cleavage mass existed in a drop of more transparent highly refractive fluid. The drop is not in a segmentation cavity, but probably consists of accumulated fluid such as normally exists in the cavity, but here located between the cell mass and the yolk, possibly on account of some peculiar osmotic effect. The specimens of group A2 kept at 8°C. were at 72 hours old in a closely similar condition to those of Ai. Both groups were removed from the refrigerator and placed at room temperature after this 69-hour exposure to the low temperatures. After being out of the refrigerator for two days, many eggs in the Ai and Ao lots had failed to resume development and had died. When 8 days old, or 5 days after removal from the low temperature, many more, 41 of the remaining 99 eggs in lot Ai, were dead and many of those living were grossly deformed. In lot A2 a few more were dead, many were not developing, a number were grossly deformed, yet some were apparently normal. When 10 days old, the eggs were all very carefully examined to determine as nearly as possible the exact nature of the abnormalities 170 CHARLES R. STOCKARD which had occurred. The control consisted of 114 eggs, each of which contained a normal well-formed fish. In the Ai lot 4 more had died, and thus the total mortality in this group after removal from the cold was very high, a little over 70 per cent. In all 54 individuals had survived to develop embryos, and of these 16, or 30 per cent, showed gross abnormalities. Five of the 16 abnormal ones showed double conditions. One was a complete twin, two were double-headed and two had double anterior halves with single tails, Y embryos. Thus 9.3 per cent of all surviving embryos were specimens exhibiting some degree of doubleness, and 33 per cent of the deformities which occurred were duplicities. When we consider the very delicate degree of arrest and the particular developmental moment that must be affected on the basis of our explanation of double monsters, the above result is a remarkably significant one and is as good as any I have obtained by this method during the past three seasons. In the A2 group at 10 days old 2 others had died and 88 were now alive. Among the 88 survivors eleven individuals, or 12.5 per cent of all, were grossly deformed and many others were pale in color and far behind the average in their degree of development. Two of the 11 grossly deformed specimens were double, one showed a slight degree of anterior duplicity and the other was a twin with the two embryos 180° apart on the yolk. One of the twin components was large, well developed and normal in structure, the other was a short embryo with almost no body but with a well-formed head containing eyes and a pulsating heart and good blood circulation. In this group only 2.3 per cent of the surviving embryos were double specimens, but almost 20 per cent of those actually deformed were of this type. When 25 days old, man}^ of the normal specimens in both the Ai and A2 groups had hatched, although all of these were far behind the control, which had begun hatching when 12 days old. The actual percentage of double individuals induced by this experiment is not really large, yet it is comparatively very significant. From a long experience with these eggs I would venture to believe that under normal developmental conditions there is only a small chance for finding one double specimen among a thousand. During the past three spawning seasons a great number, certainly many thousand, of Fundulus eggs have been arrested in their development by being placed in low temperatures after the germ-ring had begun to form. These specimens were all examined with such care in connection with the various problems being studied that no double specimen could have escaped record. Yet among all these late arrests not one double individual existed. STRUCTURE AND DEVELOPMENTAL RATE 171 In comparison with such facts, the occurrence of 9.3 per cent in lot A] and even the 2.3 per cent of doubleness in group Ao would scarcely warrant any other interpretation than that such conditions had in some way been induced by the experimental treatments. There can be little doubt that the embryonic axis is initially expressed during a very critical and comparatively brief developmental moment. When the axis is once expressed, common observation teaches us that in some way it prevents the occurrence of other axes or other embryos on the same blastoderm. Doubleness very probably, as will be more fully discussed below, results from the almost simultaneous occurrence of two embryonic shields instead of one, and this is further due I believe to the probability that neither of the axes possesses the advantages which normally suppresses the expression of other potential budding points. To further illustrate the occurrence of doubleness in Fundulus following treatment with low temperature, we may briefly summarize one other experiment. Experiment 890. These eggs were developed during the early cool part of the season and the control itself progressed rather slowly. The lot Bi was placed in a temperature of 5°C. 3 hours after fertilization when in an early 2-cell stage. Twenty-four hours later the control had developed high segmentation discs which had not yet flattened to cap down upon the yolk-sphere. The night had been unusually cool and these eggs were thus considerably retarded in their development. This amount of retardation is not, however, particularly injurious, as is shown by the later development of the eggs. It would seem that Fundulus eggs were sufficiently resistant not to be noticeably deformed by the retardations in development induced by the degrees of low temperature which might occur during their spawning season in this climate. Nevertheless, embryos developed during the early cool part of the season are not so large in size or vigorous in behavior at the time of hatching as are those being developed during the warmer days to follow. The eggs of lot Bi after 20 hours at 5°C. are in 2- and 4-cell stages, they are, therefore, almost completely stopped, having divided only once during this time. • When 2 days old, the control had the germ-ring only about onefourth over the yolk sphere, with the embryonic shield beginning to form, a stage not more than one-half as advanced as is usual for this age. Group Bi contained eggs in the first, second, and third cleavage stages with many very irregular arrangements of the cells. These 172 CHARLES R. STOCKARD eggs were now returned to room temperature, and many of them very soon began again to develop. The control at 3 days old showed the embryos well formed, although the germ-ring was not entirely over the yolk-sphere. The Bi lot, after being out of the refrigerator for 24 hours, had high segmentation discs which had not begun to flatten down upon the yolksphere. There was no indication of the germ-ring or embryonic-shield formation. After 24 hours more the germ-caps had flattened and grown about one-half over the yolk-sphere, the embryonic shield was well formed in most of the specimens and the line of the embryo was visible in the shield. Thus within the first 48 hours after removal from the low temperatures many of these eggs have attained about the same condition as was shown by the present control specimens when 50 hours old. Many of the eggs after refrigeration failed to recover, and died during the first two days at room temperature. When 6 days old, the control embryos were twitching and moving their bodies and were in all respects normal in condition. The Bi group contained small embryos without a blood circulation, many of them were abnormal at the head end, and many were short. Thus after developing for 4 days at room temperature they are far behind a usual four-day embryo. The Bi group were carefully surveyed for deformities when 9 days old. Four eggs had yolk-sacs containing blood-cells and chromatophores, but without formed embryos. Six eggs still had an early cell mass at the upper pole which had not developed, although even at 9 days it was translucent and alive. There were 10 deformed embryos without a circulation, and 4 deformed but with a circulation. The majority, 45, of all living specimens seemed normal, with vigorous circulations. Thus more than 34 per cent of the specimens which survived the low temperature were grossly abnormal. Three of the 10 eggs which contained abnormal specimens with circulating blood showed double embryos. One was two-headed, and two were double throughout their anterior halves, each having two heads and two bodies with a single caudal half. The control embryos were with two exceptions all fine normal specimens. Two of the 86 individuals were small and considerably behind the others in their stage of development, although their structures were normal and they later succeeded in hatching several days after their fellows. This experiment again shows a pronounced difference between the modes of development in the normal control lot of eggs and in a similar lot which had been inhibited by lowering their temperature before the time of gastrulation. More than 4 per cent of the eggs which survived the inhibition contained double embryos, and one-eighth of all the gross abnormalities was of this STRUCTURE AND DEVELOPMENTAL RATE 173 nature. Here again it would seem to be strongly indicated that a connection of primary importance existed between the retardation of development and the origin of the double specimens. A number of similar experiments with low temperature arrests could be reviewed, but they would differ little in their general results from those above. We may, therefore, pass to an analysis of another type of experiment before undertaking a general consideration of the significance of the results. b. Arresting development by reducing the oxygen supply and the occurrence of double individuals and twins in the trout and Fundulus Cellular proliferation which is so important an element in development is a great energy-consuming process. No doubt the interruptions in cell proliferation which were described in the preceding section as due to low temperatures are actually caused by a lower rate of oxidation which takes place at such temperatures. In nature development is not only interrupted at times by indirectly lowering the rate of oxidation through temperature changes, but also by directly reducing the oxidation rate through a lack of free oxygen. In the present section we may review some of the consequences of lowering developmental rate by directly reducing the available oxygen supply. The methods employed have been extremely crude, just such methods as nature might frequently use. With such methods the results are, of course, more variable than might be obtained from highly refined manipulations, yet the variations themselves are quite instructive. Experiments with Fundulus eggs may first be considered. 1 . Results with Fundidus. The eggs of Fundulus are demersal and are supplied with long thread-like processes which normally serve to entangle them on the blades of sea-grass or other objects among which they are deposited by the female. This arrangement serves to keep the eggs near the surface, and to insure contact with a better oxygen supply than might be obtained should they lie in the sand or silt of the bottom. When these eggs are developed in the laboratory they are kept in small glass 174 CHARLES R. STOCKARD dishes, ordinary 'finger-bowls/ containing about 60 cc. of water. The thread-like processes from the egg membranes become entangled and cause the eggs to cluster together in bunches of from a few to even as many as one hundred or more. It is a well recognized fact that in such clusters the conditions for development of the individual eggs are not equal, and the egg group fails to present a uniform mode of development. The common practice is to separate the eggs in a dish so that they lie apart and are not clustered together. The permanent separation of the eggs requires care and attention, since they may again become bunched by the agitation of the water. When they are properly kept apart the entire lot in a dish will develop with remarkable uniformity. No control group of Fundulus eggs should serve as a standard for development unless the individual eggs are kept completely free from contact with one another. In my experience, under such conditions only the most insignificant percentage of developmental abnormalities ever occur. I am convinced that the high percentage of abnormalities recorded by certain experimenters among their control sets are due to a failure to properly separate the eggs. The clustered condition also vitiates the results obtained from experimental groups of eggs. Advantage was taken of this tendency to become entangled into clusters in order to study the developmental reactions of eggs with more or less access to a free oxygen supply. The eggs about the outside of such a cluster are in contact with fresh surrounding water and a sufficient amount of oxygen for normally rapid development. Those specimens lying deeper and deeper in the cluster are more and more removed from a freely changing water supply, and, therefore, experience various degrees of a stagnating environment. Such eggs not only lack a constant oxygen supply, but no doubt exist in an environment containing an excess of waste products, such as the CO2 given off by their neighbors. The developmental perfection attained varies directly with the distance from the center of the egg cluster, the further removed from the center the more perfect the development. STRUCTURE AND DEVELOPMENTAL RATE 175 In many of the experiments the available oxygen supply was further reduced by first boiling and driving the air out of the sea-water into which the eggs were to be placed. The central eggs of large clusters in this boiled water frequently had their development stopped in various stages, while other specimens progressed at an extremely slow rate. One group of such experiments will be briefly reviewed as illustrating the general results from all. Experiment 915. A large number of eggs, from three females, was fertilized by a single male. After 3 hours they were almost all developing and presented the typical 4 -cell stage. About 75 of these eggs were placed in ordinary sea-water and separated apart on the bottom of a dish to be developed as a control., The other eggs were divided into three lots. Two lots were placed in dishes containing sea-water that had been boiled, and the third lot was put in ordinary sea-water. The eggs in the three dishes were then moved gently around until they became clustered into large groups of about 100 or more. After 2 days of development, the control contained well-formed embryos with the optic vesicles prominently shown and with 8 to 10 pairs of somites present. Many eggs on the outer parts of the clusters in the unboiled sea-water wxre equally as far along, while others near the center of the clusters were still in segmentation stages, and still others were in various degrees of arrested development. The two lots in l)oiled sea-water were in closely similar conditions. When 8 days old, the entire experiment was carefully examined and the following conditions found. The control eggs all contained normal embryos except for the fact that 3 specimens were smaller than the others and somewhat delayed in development. These, however, later succeeded in hatching. The clusters in unboiled sea-water contained many dead eggs. The more superficial eggs of the cluster contained in general normal embryos, though some were behind the control in their degree of development. Almost all of the more centrally placed eggs of the group were several days slower than the control in their developmental stages. These embryos were small and pale with poorly expanded chromatophores, and 15 of them, or 13 per cent of the small embryos, showed gross abnormalities. They possessed narrow undeveloped heads, defective eyes, deformed hearts with no circulation, and other common defects, while 2 of the larger better developed specimens were double-headed embryos. This dish contained a few more than 200 eggs, thus only about 1 per cent developed double conditions. The first group that had been clustered in the boiled sea-water showed a somewhat better record than the preceding. Here also many of the eggs had died. There were again a number of normal embryos in the superficial regions of the cluster. The more centrally 176 CHARLES R. STOCKARD located specimens were small and far behind the control in their rate of development, but here only about 10 per cent of them were actually grossly deformed, and there were no double conditions at all. The second group in boiled sea-water was more decidedly affected than any. Man}^ of the eggs died. Many superficial ones were almost up to the control in their state of development. But the great majority of specimens were small, pale, and poorly developed, being several days behind the control. Almost 16 per cent of these small specimens were considered to show gross defects. Twelve specimens had no circulation of the blood; 10 had decidedly defective eyes, minute in size and poorly developed or deeply buried in the head, and two were cyclopean. Four specimens that were near the surface of the clusters and very well developed presented double conditions. One egg contained separate twins, both embryos being fully developed. The 3 other eggs showed different degrees of anterior duplicity. Therefore, more than 2 per cent of the entire number of specimens developing in the dish were double; this is much the highest record that was obtained among ten similar experiments. The other experiments with low oxygen supply gave closely comparable results to the three above, and need not be reviewed in detail. Only a very small number of double specimens occurred in any of them. In all cases, the double individuals were among those of fairly normal development and were not extremely small and highly defective specimens. This, in my opinion, is a fact of considerable importance, and is to be explained somewhat as follo\vs. The origin of two embryonic axes or growing points on the germ-ring of the fish probably results from a rather mild or slight reduction in the normal developmental rate at the time of gastrulation or embryonic-shield formation. It is probably more important to obtain the reduction in rate at an exact and very limited moment than to have a definite degree of reduction. That is, the reduction in rate may be little or much, but it must occur during a very limited time and not continue for long after the doubling has once been accomplished. Should the arrest continue, it is possible that one of the buds, even though it had begun to develop, might be suppressed, and the more vigorous or more favorably placed one might later continue as an apparently single individual. STRUCTURE AND DEVELOPMENTAL RATE 177 Certain delicate or sensitive eggs will probably respond more readily and give double conditions more frequently than hardier eggs. The eggs of Fundulus are very hardy, and it may be that a treatment when acting in a delicate manner affects favorably for our purpose only the more sensitive eggs, while the large majority are too resistant to respond. Should the conditions be more severe they would act too harshly to obtain a double response from any. These speculations will appear to have a stronger foundation after we have reviewed the very remarkable tendencies on the part of the delicate eggs of the trout to give double and twin embryos. 2. Double embryos in trout eggs. The eggs of the trout unquestionably possess a stronger innate tendency to form double and twin individuals than do those of Fundulus. Twinning and double formations, like all other unusual developmental phenomena, are not simply and entirely due to the action of an ususual environment, but also depend upon the internal structure of the given egg and its peculiar manner of development. An environmental stimulus which would frequently induce double formations in one type or species of eggs might be completely ineffective in its action on the eggs of another species. The burden of evidence for the cause of twin formation as well as the means of artificially inducing it indicate an accessory budding or double blastopore formation as the primary step, and it is obvious that the early morphology of certain eggs more readily lends itself to the establishment of accessory blastopore formations than does that of others. Not only is this morphological difference to be expected, but from what we know of the physiology of budding, it is also logically probable that in certain eggs the bud for the embryonic axis will arise with higher powers for dominating the entire budding region than in others. Different degrees of dominance of the apical bud in different plants is a well-recognized fact. In some plants the terminal bud grows to form a long slender stalk, without producing axillary or lateral shoots, while the terminal shoot of other plants grows to a limited extent only before axillary buds and branches make their appearance. The THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 178 CHARLES R. STOCKARD embryonic axis in the vertebrate egg may be compared with the terminal shoot of a plant. A second embryonic axis formation is roughly comparable to the occurrence of an ordinary lateral or axillary plant bud. A better comparison, however, is made between the animal egg and the budding leaf, such as Bryophyllum. In both of these, the egg and leaf, we may recognize an area of potential budding capacity, and we know that not only one, but several equal buds may arise simultaneously from either of these two stocks. There are doubtless certain kinds of budding leaves which are more prone to form multiple buds than others. From such leaves instead of one shoot arising in a certain notch, several notches are equally capable of budding and several shoots are formed. It would seem that certain animal eggs also normally possess a disposition to produce several buds. Such eggs develop more than one embryonic axis and give rise to several individuals instead of the usual single embryo from a single egg. This is probably the case in the Texas armadillo. The eggs of Fundulus and those of the trout, very probably illustrate two different degrees of capacity to form several instead of one embryonic bud. This much of the twinning process is truly inherited, and variations in the tendency may occur not only among the eggs of different species, but probably also exist among the individual eggs of the same species. For example, certain mothers may produce eggs highly inclined to give rise to two embryonic buds or twins, and such an inclination may be transmitted or inherited by her daughters or even through her sons. Davenport ('20) has recently found in a study of human twins that the males of a family carry or transmit the twinning tendency in equally as evident a manner as do the females. Double and twin individuals are also of much higher frequency in certain human families than in the community as a whole. All this indicates that the eggs of certain individuals are more inclined to form twins than are those from others. In the human cases the present consideration refers, of course, only to so-called identical and not fraternal twins. The latter, truly speaking, are not actually twins. STRUCTURE AND DEVELOPMENTAL RATE 179 There can be little doubt from the experiments recorded above and the results to be given below that the environmental conditions or external factors are of greater importance than the internal tendencies in twin formation. It is evident that eggs, although capable of producing more than one embryo, rarely ever do. The number of twin formations in a given lot of eggs may experimentally be increased so greatly in excess of the natural occurrence of such individuals, that we are forced to believe even in the cases before mentioned of the armadillo and the excessive occurrence of twins in certain families; as cited by Davenport, that the en\'ironment may in these instances also be the actually direct cause. A peculiar uterine reaction may be inherited in the armadillo and in certain human families which prevents a ready or rapid placentation and thus primarily brings about an initial slowing of development. There is much evidence of a slow placental formation and a peculiar uterine condition in the armadillo which will be considered more fully beyond. This brief estimate of the internal and external developmental factors concerned in twinning has been given just here in order that the reader may appreciate more fully the very different reactions shown by Fundulus and the trout. He may form for himself some idea as to whether this is due to a difference in morphological pattern of the germ-rings or potential budding regions in the two species or to differences in the physiological reactions to the environment or finally to a combination of both. Double and twin trout are classical objects, they often occur in the hatcheries in various parts of the world and have been frequently figured and described since the early studies of Lereboullet by Rauber, de Quatrefages, Klaussner, Gemmill, and others. All of the double individuals and twins recorded have been surprisingly well developed and normally formed. From figures and descriptions, it would seem as though the trout egg possessed a rather normal tendency to form double embryos, and the causes necessary to give expression to this tendency were so slight as not to be further injurious to the development of the individual 180 CHAELES R. STOCKARD embiyos. In other words, some very small and simple chemical or physical irregularity in the developmental environment is sufficient to cause two embiyos to grow from the germ-ring, but is not so injurious as to induce a deformed or abnormal development in the young fish. When either component of these double specimens is deformed, the cause of such deformities may be more reasonably attributed to conditions other than the surrounding environment (see beyond). Several years ago I obtained a large number of young trout, many of which were twins and others presented different degrees of doubleness. Since then I have visited several trout hatcheries and have found in all that double specimens very frequently occur. The practical fish culturists in two of these hatcheries thought that such abnormal double specimens were caused by early development under too crowded conditions, or in sluggish water where the eggs did not obtain sufficient aeration. Such views are very probably correct, since all of my experimental studies with fish eggs has indicated that some retardation in rate or interruption of development was the simple cause of unusual structural responses in the embryo. Only recently, however, could a satisfactory explanation of double conditions be worked out on this basis, and the trout specimens gave the key to the situation. The foregoing experiments with Fundulus were then conducted to further substantiate the conclusions. The artificial production of double trout embryos is no doubt rather difficult to bring about, since evidently only a slight slowing of the rate of cell proliferation at a particular moment is favorable. Plates 1 and 2 illustrate a series of double trout which are selected from the large number of such specimens that have been obtained. The series shows the various degrees of double formation, beginning with a partially double-headed condition, and passing through the double anterior regions on single bodies, to double bodies with single tails, and on to the condition of complete doubleness but with the two components jointed more or less intimately together. The final specimen shows two com STRUCTURE AND DEVELOPMENTAL RATE 181 pletely developed twins attached to the common yolk-sac. It will be noted at once that each of the final twin individuals is equally as large and perfect in form as is the single specimen at the beginning of the series. This fact is of importance in showing that up to this stage of development and growth there is no question of available food, since the amount to be had in each egg is here demonstrated to be sufficient to form two full-size perfect young trout instead of the usual one. In studying the graded series of duplicities illustrated by plates 1 and 2, the question immediately presents itself as to why the two components in the several specimens show the different degrees of separation? What conditions or arrangements determined that the specimens in the upper part of the series should be double headed, while those at the end of the series are completely double bodied? Gemmill ('12) in his monograph on the teratology of fishes, considered these propositions and gave an explanation for the varying degrees of doubleness which I believe my studies completely confirm. On the other hand, Gemmill failed to give any explanation of the initial or actual cause of doubleness. In accordance with the view that has often been suggested, the germ-ring was recognized by Gemmill "as a stock, able to give rise vegetatively, so to speak, to more than one embryo." The embryonic axis or body begins to form in the embryonic shield which arises from certain places along the germ-ring. When two shields arise, the degree of duplicity of the resulting double fish "varies directly with the original distance between the two centers of embryo-formation." When the centers of embryo formation are close together, only 5° to 10° apart on the germ-ring, the embryonic axes very soon become united so that a double-headed specimen with a single body finally develops. It may be stated generally that when the original buds are less than 90° apart the specimens formed will exhibit various degrees of double anterior halves on single posterior parts. When the distance between the initial buds is greater than 90° and on up to 180°, the resulting specimens will show the double condition 182 CHARLES R. STOCKARD not only involving the anterior half, but extending into the posterior part of the body. Finally, at 180° apart, the two embryonic shields give rise to two completely separate twin individuals. The accompanying diagram may serve to illustrate the manner in which such processes operate. In figure 11 the diagram on the left shows two early embryonic shields arising about 20° apart. When the germ-ring has descended further over the yolk-sphere, the dotted line indicates 'how the two embryonic 11 Fig. 11 A series of diagrams illustrating the manner in which the degree of duplicity in embryos is determined by the original distance apart of the two embryonic shields on the single germ-ring. The solid lines indicate the eai'ly germ-rings with the two embryonic shields, and the broken lines show the resulting body outlines of the former embryos. The figure on the left has the embryonic shields less than 90° apart on the germ-ring and the dotted outline of the resulting embryo indicates it to be a double-headed specimen. In the central figure the embryonic shields are a little more than 90° apart, and the resulting duplicity extends throughout the upper half of the body. In the figure on the right the embryonic shields are 180° apart, or opposite one another, and two complete twin individuals result, as the dotted lines indicate. axes become united or common in the body region, and such a condition would finally give rise to a single-bodied fish with two heads. The middle diagram in figure 11 illustrates similar steps in the history of a 'Y monster,' or individual with two heads and bodies and a single tail. The right diagram of figure 11 shows two embryonic shields arising 180° apart, or opposite one another on the yolk-sphere, each of these has an entire half of the germring to develop from, and complete twins are produced. STRUCTURE AND DEVELOPMENTAL RATE 183 In studying the early embryos of Fundulus these several steps have actually been observed. An observation of further importance in this connection has also been made, but unfortunately at present on very few specimens. In attempting to discover the earliest stages of doubleness from great numbers of eggs, I have selected all specimens seeming in any way to possess two early embryonic shields. On two occasions a fair number of such specimens were apparently found, one lot of seven such eggs and another of five. These seemingly double-embiyo formations were isolated and observed during later stages, with the result that from among the seven specimens only two double-headed individuals arose, while the remaining five formed typically single embryos. The second group of five seemingly early double shields gave rise to five perfectly single specimens. There would appear to be only one interpretation for such a phenomenon: two initial buds may sometimes appear, but later one is completely suppressed by the other, or the two possibly fuse completely and only one normally single individual is developed. Therefore, it would seem that initial multiple buds are much more common than the resulting double specimens indicate, and that many secondary buds are suppressed or lost during early development. A comparison of the two components in older double monsters which is undertaken in a further section of this paper makes still more probable such deductions. I wish to present these observations on the early double specimens with the chances of error fully in mind. In the first place, I succeeded in isolating a very few such probable earl}^ specimens, twelve from the many hundred eggs examined, and from these twelve only two actually showed double conditions during their later stages of development. The early embryonic shields were irregular and not strongly expressed. On the other hand, it seems to me significant that from the twelve specimens which were isolated two of them definitely developed double embryos, while it is recalled that among the great numbers of Fundulus eggs experimented on extremely few double specimens actually occurred. However, when one selects early specimens thinking them to be of a definite type and they later develop into indi 184 CHARLES R. STOCKARD viduals of another type, his confidence is considerably shaken in the vaUdity of the selection. I have had similar experiences in attempting to isolate from large numbers of eggs those showing the earliest indication of the cyclopean defects. There are no doubt processes of regulation which may tend to correct and obliterate an early unusual arrangement, yet in spite of the recognized probabilities for mistake, I nevertheless feel that the foregoing indication of suppression of early buds has some real value, since two actually double specimens were certainly selected, as later development showed. Kaestner ('98-'07) has figured very early double primitive streaks in the chick and Assheton ('08) double embronic shields in the sheep. Kopsch ('95) has described in Lacerta agilis, the European lizard, one blastoderm with two blastopores, and thus showed that a double gastrulation had taken place. From this observation he agreed with O. Hertwig that all twin formation as well as all anterior duplication arose from a double gastrulainfolding or proliferation. This position leaves, as Kaestner ('99) has stated, the question of doubleness or twins merely moved back to an earlier stage before the origin of the two blastopores, it remained to be answered why the double infolding takes place, and w^hy it is so rare? In the present study it is felt that both questions are answered. A developmental arrest does away with the normal advantage of the usual growing point and permits a double gastrulation; the condition is rare for the same reason that the apical or dominant bud rarely fails to grow. Returning to the consideration of the actual case in the trout, we may judge indirectly by the degree of separation of the two components in the several individuals as to the probable distance apart of the original embiyonic shields or embryonic axes on the germ-rings. Gemmill ('01), has found a rather high proportion of complete twins among something more than seventy double trout specimens that he examined, while Windel ('95), had found only nine complete twins among 117 eggs containing double trout, or a proportion of one to thirteen. Among my double trout specimens there is one case of complete twins for every eight. From these observations it may be concluded that STRUCTUEE AND DEVELOPMENTAL RATE 185 when two embryonic shields arise from the germ-ring they occupy positions about 180° apart, or are opposite one another on the yolk-sphere, in nearly 10 per cent of the cases. A further question bearing on the relative position of the embryonic shields on the germ-ring suggests itself. If the germring is actually a potentially budding stock, why does not triplet and quadruplet formations appear almost as frequently as the double or twin condition? This question can at least be answered with a probable explanation, if not with a completely satisfactory one. In the first place, the extreme tendency of the eggs to form only a single rather than a double individual is important in this connection. There is certainly only a slight chance to form double specimens. The single bud is almost always capable of suppressing further expressions in the blastoderm. When this capacity is in any way lowered and a second bud arises, the stock is then still further dominated or preempted by the presence of the two, and the chance for still a third embryo formation is decidedly reduced. Yet among a hundred multiple cases one triplet may be found. Gemmill found one case of three embryos in a trout egg as against over seventy doubles. This specimen had one almost perfect embryo and the other two were very abnormal and poorly developed. Among something less than 150 double fish embryos seen during the past few years I have observed only one triple specimen. This arose from a Fundulus egg that had been inhibited during early development by a weak solution of alcohol. One embryo, almost normal, was on the same blastoderm with a double-headed specimen. I have never observed, nor found record of a fish's egg containing more than three embryos. The conclusions seems warranted that one point of gastrulation, or embryo formation, has an extremely high tendency to prevent or suppress the existence of any other such point of excessive proliferation.' When a second point is capable of expression the two almost without fail completely dominate the growth capacity of the entire germinal region and triplets are the rarest exception. 186 CHARLES R. STOCKARD The relative conditions of the individual components in the double trout are of considerable importance and will be discussed in connection with their several particular bearings in the pages beyond. c. An explanation of the frequent occurrence of twins and double chick embryos It is extremely rare among birds for a double-headed or otherwise double individual to hatch from the egg ; a few such irregular cases have been recorded. I have never, however, found record of complete twins hatching from the hen's egg. On the other hand, when the earlier stages of chick development are studied in the laboratory, one rarely fails, even in a limited experience, to meet with double and twin embryos. The prevalence of these early specimens has long furnished material for studies on twinning in the chick. Among many such investigations are those of Gerlach ('82), Burckhardt, Dareste, Klaussner, Erich Hoffman, Mitrophanow, five somewhat more recent studies by Kaestner ('98, '99, '01, '02, and '07), and most recently the description of several double chick embryos by Tannreuther ('19). The almost abundant occurrence of double specimens among the limited numbers of eggs developed in the laboratoiy and the well-known high mortality among incubating eggs of the poultry farm, makes it highly probable that double and deformed embryos are not uncommon under natural conditions, but that they usually die during the early days of development. From a survey of the literature on double monsters in the various vertebrate classes, it would be impossible to form anything like a correct estimate of the comparative frequency of such individuals in these several groups. It would be simply speculation to claim that doubleness was more frequent among the embiyos of birds than among those of mammals. Yet the double condition in birds is just here of particular interest as probably being due to a somewhat definite and uniform cause arising out of their peculiar mode of development. The double bird embryos are very probably the result of a rather easily followed natural experiment. STRUCTURE AND DEVELOPMENTAL RATE 187 It is a well-known fact, as mentioned in the early pages of this discussion, that the eggs of birds normally have a discontinuous mode of development. Fertilization takes place in the upper part of the ovdduct and the egg begins its development in the high temperature of the maternal body and continues to develop as it travels down the uterine tube and becomes surrounded by its several accessory coats. Finally, at the time of laying, the blastoderm has passed the gastrula stage. The fall in temperature experienced on leaving the body of the mother causes development to stop in this early postgastrula condition, and the egg remains quiescent until the temperature is again raised to about that of the bird's body. From the evidence given in preceding sections regarding the developmental time of inducing double embryo formations, it is seen that the bird's egg at laying has just passed the critical moment for causing double invagination or double blastopore formation. Since these double invaginations may be brought about by either interrupting development or slowing its rate before gastrulation, it would seem that the bird's egg had been piloted beyond this danger period within the body of the mother. How, then, is the frequency of double and twin chicks in the bird's egg to be accounted for? In studies on the early stages of development in the bird's egg, it has been found by Patterson ('09) and others that the process of gastrulation takes place very close to the actual moments of laying. The time relationships between the moments of laying and finished gastrulation are, however, in general slightly variable, and the eggs of certain females, as I learn in conversation with Professor Patterson, differ decidedly from others in their tendency to be deposited at an unusually early stage. There would thus seem to be a strong probability that all eggs of the bird have not reached or passed the gastrulation process before the time of laying. This is a most important probability, and is believed to be true by some of those who have studied these early stages very extensively. On the basis of my own experimental results, this probable variation in the moment of laying is entirely sufficient to account 188 CHAHLES E. STOCKARD for the double individuals and twins among the chick embryos. It also accounts most satisfactorily for the apparent frequency of such occurrences. The interruption of development following a fall in temperature at laying and before gastrulation has begun prevents the single gastrulation process from beginning at a rate sufficient to dominate the growth conditions of the entire blastoderm as it normally does. A second gastrular infolding or blastopore formation is established and thus two embryo formations are begun. The usual interruption in the development of the bird has, with slight variations, been introduced at a most fortunately passive stage, just following gastrulation. This is a moment at which developing fish eggs may be stopped with impunity for considerable lengths of time and injurious results rarely ever follow. It is a moment following which no important embryonic structure need arise for a considerable length of time. After gastrulation only the linear growth to establish the embryonic axis immediately occurs. None of the highly energetic folding processes resulting from a localized excessive or unequally rapid proliferation take place until after a considerable interval of slow growth has passed. This interval of slow cellular proliferation following gastrulation is the fortunate occurrence that has preserved the birds as a class among present-day vertebrates. Had birds been so constructed that the egg was laid and allowed to discontinue its development before gastrulation had taken place it is conceivable that this condition could have eliminated them from the animal kingdom. There would have followed such a high proportion of deformed and defective specimens from eggs interrupted before gastrulation, that the individuals of a class having its eggs stopped at this time would very soon become so generally deformed as to be unable to maintain their existence. The important matter of a few hours' difference in egg-laying time lies between the successful class of birds and a hopelessly unfit monstrous condition. Obviously, the evolution of the developmental environment has been of equally as great importance in the survival of a species, as has been its constant structural fitness. Nature's experi STRUCTURE AND DEVELOPMENTAL RATE 189 merit of temporarily lowering the surrounding temperature and stopping the developmental progress of the bird's egg has not proved fatal simply on account of the fortunate fact that the development is usually stopped during a very passive stage. The slight individual variations in egg-laying time which cause certain eggs to be interrupted before gastrulation very probably furnish the material for the many descriptive studies of double avian embryos. On the other hand, it is a most significant fact to note that in spite of the many experimental studies on developing hen's eggs by Dareste, Fere, the writer, and others no double monster or twin conditions have been produced. ' This absence of double productions would naturally be expected, since the eggs were experimentally treated only after having been laid. They had thus passed gastrulation or the time after which double conditions cannot be induced. Gerlach ('82) long ago thought that he had probably induced experimentally double anterior ends in chick embryos. His results were most uncertain, and have been interpreted as accidental by subsequent writers. He made injections over the biasderm so as to get fusions with the overlying shell. With such experiments he obtained double indications at the forward end of the embryos in two cases out of sixty eggs. Gerlach realized that conclusions could not be drawn from these meager results, but believed that if this method were perfected, it would yield more convincing results. Such experimental efforts to produce doubled conditions in hen's eggs are very probably futile, since the evidence at hand would indicate that there is only the rarest chance of the experimenter's striking an egg in the proper developmental condition to make possible the production of twin or double individuals. Should such specimens be obtained among the eggs employed in an experiment, there would always be the possibility that the natural interruption in development occurring in an egg laid at an unusually early stage was the cause of the doubleness, and not actually the experimental procedure. 190 CHAELES R. STOCKARD d. An explanation of polyemhryony in the armadillo On examining the uterus in two pregnant specimens of a South American armadillo, von Jhering in 1885 discovered that each contained eight fetuses enclosed within a single chorion. He correctly concluded that all of the fetuses in each mother had been derived from a single egg by some process of division into separate embryonic rudiments. After this valuable discovery and interpretation, the study of the armadillo's development lapsed and nothing of importance was added for almost twenty-five years. Two series of investigations were then begun simultaneously, one on the South American species by Fernandez ('09) and the other on the Texas armadillo by Newman and Patterson ('09). The growth and expansion of these twin studies has brought our understanding of the phenomenon of polyembryonj^ in the armadillo to a considerable state of maturity. These authors readily agreed that in most species of armadillo the individual members of a litter, usually four in the Texas species and eight in the common South American form, are all derived from a single egg. It required considerable effort, however, to obtain the material that would furnish the morphological stages of the proccess by which this polyembryonic development was accomplished. We are finally indebted to Patterson ('13) for the very thorough and satisfactory manner in which he has collected and studied the early embryonic conditions; and particularly for having shown the first stages of the budding process through which the single blastocyst gives rise to four distinct embryonic areas, each exhibiting a typical primitive streak region. In connection with the idea constantly advanced in the present study that twins and double vertebrate embryos arise from accessory growths or invagination points around the blastoderm, it now becomes important to ascertain exactly what degree of development has been attained by the armadillo blastocyst at the time the budding process begins. And since, according to our interpretation, these buds should arise at the time of gastrulation or blastopore formation, it becomes necessary to consider very briefly the germ-layers and gastrulation in mammals. The STRTJCTURE AND DEVELOPMENTAL RATE 191 decidedly precocious and highly modified method of forming the primary germ-layers in the mammalian blastocyst is not strictly comparable to gastrulation or the method of germ-layer formation found among the other vertebrates. On the other hand, the embryonic line or primitive streak of the mammalian egg is exactly comparable to the blastopore and embryonic process formation in the simpler forms. The blastocyst of the armadillo has already, by a process of cell migration and delamination, separated off the primary entoderm from the ectoderm and further modified these layers before the budding which forms the embryonic primordia has begun. But it is in the primordia that the invagination of the entoderm forms the secondary entoderm of the gut and the embryonic mesoderm arises from a typical primitive-streak region much as in lower vertebrates. The precocious cell migration and splitting into layers in the mammal's egg is associated with the early implantation of the embryo upon the uterine wall of the mother,, and the later primitive-streak formation may be interpreted as. related to the actual gastrulation or blastopore formation away from which the line of the embryo always develops. Whether the validity of the above briefly outlined interpretation of the germ-layer formation is admitted or not, we have in the armadillo a process of budding taking place from the blastoderm and associated with accessoiy or extra blastopore formation in much the same way as are the accessory embryos along the germ-ring in the egg of the bony fish. These buds also accord with Kopsch's ('95), description of a double gastrular condition with two blastopores in a blastoderm of Lacerta agilis, from which he concluded that twin formation as well as anterior duplication arises from a double gastrula — Einstiilpungen. And, further, Assheton has described a similar condition in a blastodermic vesicle of the sheep. He, however, imagined the condition to have been due to a sphtting during the morula stage. The double primitive streaks in the hen's egg and other forms all lend themselves to strengthen the interpretation that double embryo formation first asserts itself by a double gastrulation or blastopore formation, which is initially a process of double 192 CHARLES R. STOCKARD instead of single bud formation. Patterson's description of the origin of the quadruplet buds in the Texas armadillo furnishes the most striking case in the study of these conditions. And we may conclude that the budding or accessory embryo formation in the egg of the armadillo is exactly the same developmental process as that which gives rise to twins and double individuals in other vertebrate eggs. However, the very important question yet remains to be answered: Why does this accessory bud formation occur so constantly in the Texas armadillo in contrast to the single embryo formation of mammalian eggs in general? Patterson ('13) failed entirely to answer this question, but he supplied some veiy significant data which Newman ('17) has appreciated as being intimately connected with the occurrence of polyembryony. In connection with the collection of material Patterson ('13) discovered a 'period of quiescence' of the embryonic blastocyst. Regarding this he states: The fact was first made apparent in 1911, when, after I had started collecting two weeks earlier than in the preceding year, I failed to obtain the cleavage stages, although judging from the condition of development in the vesicles collected in previous years, one would naturally expect to find these early stages during the period of my first collection in 1911." The following year be began collecting still two weeks earlier and again had a similar experience. "Practically all of these vesicles lie free within the uterine cavity, either in the horizontal groove or in the region of the attachment zone (placental area)." "It is evident from these data that the embryonic vesicle remains for some time lying free "within the uterine cavity. Just how long this period lasts, I am unable to state; for practically every old female taken at the earliest date (October 15th) at which I have collected, possesses a free blastocyst. How long such blastocysts have been in the uterine cavity it is, of course impossible to determine; but I should judge not very long, because two vesicles taken from the fallopian tubes show a development almost as far advanced as that of some vesicles taken from the proximal parts of the horizontal grooves. Taking all the facts into consideration, I estimate the 'period of quiescence' to last STRUCTURE AND DEVELOPMENTAL RATE 193 about three weeks; that is, from about the middle of October to the third or fourth of November. ... Of the thirty-four free blastocysts obtained in 1911 and 1912, twenty-eight of them were secured within this period." In a study of sections no mitotic divisions were found to occur in the blastocysts during the 'quiescent period.' The only point of interest cited by Patterson in connection with this peculiar phenomenon of interruption in development, was the fact that in no other mammal except the deer, had such a condition been found. Bischoff ('54) had long ago reported a 'period of quiescence' lasting for some weeks during a so-called morula stage of the deer embryo. Newman ('17) has recognized the importance of Patterson's discovery of the 'period of quiescence' during the early development of the armadillo, and states in a discussion of twin formation that this 'period of quiescence' probably "holds the clue to the physiological explanation of polyembryony." In this position Newman is, in my opinion, largely right, but this is as far as the data led him, and he finally remarks: "The problem is to locate the factors responsible for the slowing down of the developmental rhythm. Whatever these factors may be, and we have no definite knowledge of them, the result of retardation is polyembryony." Newman thus fails to appreciate the second point in Patterson's discovery, and that is, that the blastocysts always lie free in the uterus during the 'period of quiescence.' This fact enables us to go one step further since the lack of attachment and, therefore, lack of oxygen supply are very probably "the factors responsible for the slowing down of the developmental rhythm." The armadillo egg, like that of most mammals, undergoes its early development in the Fallopian tube and is, therefore, capable of reaching the blastocyst stage on its initial oxygen supply. After this time however, it must become attached to the uterine wall for a further source of oxygen. For some reason, in the armadillo the reaction between the blastocyst and the uterine wall is postponed and the blastocyst is incapable of further developmental progress until this reaction is established and the necessary supply 194 CHARLES R. STOCKARD of oxygen becomes available. In exactly the same way the development of the blastoderm in the fish's egg is experimentally retarded or stopped by reducing the available oxygen and is again made to resume its development by supplying oxygen. In the case of the fish egg, the supply of ordinary nutriment is not involved, and reactions similar to those of the armadillo egg are only obtained as responses to changes in temperature or rate of oxidation. I do not believe the retardation in the armadillo egg is of the nature of a starvation phenomenon, since we see nothing of the kind in other forms. Temperature changes are ruled out, since the temperature of the uterus is more or less constant. The absence of oxygen necessary for the energetic process of cell division is, therefore, in all probability the arresting cause, and the retardation results in polyembryony. Thus Patterson has found the developmental interruption to exist, and he has also shown the blastocyst to be disconnected from the uterine wall and its necessary oxygen supply during this time. However, he has furnished no data bearing on the reason for the delay in uterine reaction and the consequent failure of immediate implantation of the blastocyst such as normally occurs in other mammals. The consideration of the armadillo egg up to this point has taken account only of the external factors influencing its mode of development. It must now be remembered as a fact of serious importance that the production of quadruplets from the single egg of the Texas armadillo is an almost constant occurrence, while the experimental attempts to produce twins and double individuals in fish eggs and other forms have given at best only small percentages of such individuals among the large groups of eggs treated. It is also recalled that all eggs do not furnish equally favorable material for artificial twin production. The eggs of the trout seem unquestionably more disposed to give rise to twin formations than do the eggs of Fundulus. Thus some eggs would seem to have an hereditary or truly innate predisposition toward polyembryonic formations. There is much reason to believe that, aside from the external factors discussed, the STEUCTURE AND DEVELOPMENTAL RATE 195 armadillo egg itself is highly disposed toward the formation of accessory embryonic buds. There is the possibility, of course, that this natural experiment with the armadillo egg has become so exactly regulated as to influence the developmental processes precisely the same way each time, yet this is highly improbable for several reasons. The armadillo egg is not a case of simple twin growths from the blastoderm, but, as Patterson finds, there are primarily two buds, and then very promptly two secondary ones arise making the four, and after this the budding process ceases. In the South American species, however, it would appear as though a tertiary budding occurred giving the usual eight embryos; and in rare cases still another budding occurs from a few of the existing buds, giving a total of as many as twelve. It would certainly seem as though the blastoderm in these species passes through a stage of agametic reproduction or budding of a nature unknown among other higher vertebrates. But the possibihty for such expression might only exist on account of the delay in implantation of the blastocyst and consequent shortage of the oxygen supply necessary for the rapid formation and growth of the single embryo. It is important to keep in mind that there are species of the armadillo which produce only a single offspring from one egg. It is not known whether their embryos have a 'period of quiescence,' but if they have the period either occurs at a different developmental stage or the egg does not possess the inherent budding tendency of the other species. It remains now to account for the fact that although the egg of the deer has a 'period of quiescence' during its development it does not give rise with any degree of frequency to twin individuals. In the first place, it is entirely uncertain from the scanty accounts as to what time in development the quiescent period occurs. Assuming that such a period does exist, it might occur at some indifferent stage when no peculiar result would be expected, for example, after gastrulation as it does in the bird with no subsequent effect. In the light of the experimental production of double individuals, it is readily understood that even though the egg of the deer is interrupted in its development at an early 196 CHARLES R. STOCKARD stage, it might still be capable, on resuming development, of giving a normal single embryo. The egg of the deer may possess only a very slight tendency toward accessory embryo formations. A study of the experimental production of twin and double individuals among fish leads one to be surprised at the case of the armadillo and to expect the reaction found in the deer. The constant interruption occurring in the development of the birds and other animals at indifferent developmental moments with no subsequent ill effects renders commonplace the fact that the deer successfully withstands an interruption during its development without noticeable modifications in structural response. In conclusion we may summarize the cases as follows. The development of the armadillo is interrupted on account of a failure to become promptly implanted on the uterus and a consequent exhaustion of available oxygen supply. The interruption occurs at a critical period just preceding the primitivestreak and embryonic-line formation. The internal qualities of this egg gives to it a decided tendency under conditions of arrest to form accessory embryonic buds. As a result of the interaction of these external and internal forces polyembryony is prpduced. In the case of the deer only one probable fact is known, and that is that a 'period of quiescence' occurs. It is uncertain at what stage the arrest takes place, but it is probably due, as in the armadillo, to a delayed implantation of the blastocyst. Either on account of the stage of arrest or a lack of tendency to form accessory embryo buds, a typically single individual arises from this egg. The external factors may be the same as in the case of the armadillo, but they interact with different internal factors or different developmental moments to give a very different result. STRUCTURE AND DEVELOPMENTAL RATE 197 e. ^Alternation of generations' and twins in vertebrates Among plants and lower animals, particularly the coelenterates, there commonly exists a so-called alternation of generations. A given species at one time reproduces sexually by the union of gametes, egg and sperm cells, and the individuals derived from such gametes then give rise to a number of other individuals by a growth and fission or a budding process. Finally, sexually mature individuals again occur to reproduce another generation from germ-cells. In general this phenomenon is thought to be limited to these lower forms. The suggestion has frequently been made but without sufficient emphasis that the blastoderm may be looked upon as a stock able to give rise asexually to more than one embryo. Since the natural process of budding to form four or more embryos in the armadillo is recognized, and accessory individuals may be produced experimentally from other vertebrate eggs, it becomes evident that even man and the highest animals may actually at times exhibit an alternation of the sexual and asexual processes of reproduction. In a subsequent section of this paper the origin of various organs of the individual's body will be considered as arising initially through a budding process exactly comparable to the initial embryonic axis bud on the blastoderm. These buds may also be suppressed or inhibited in their expression in much the same way and by similar experimental methods as was described above in the case of the embryonic axis or initial embryo bud. From a general biological standpoint the adult body of higher animals may be very correctly considered to be derived from a sexually produced embryonic axis the stock which gives rise by an asexual method of budding to the various special organs. The vertebrate body is thus composed of a group of different zooids, the organs. There are seeing, hearing, excretory zooids, and so on, comparable to the zooids of a siphonophore colony. Alternation of generations is here considered a phenomenon, not limited as is generally taught to lower forms, but occurring throughout the animal kingdom. 198 CHARLES R. STOCKARD 6. STRUCTURAL DIFFERENCES BETWEEN THE TWO COMPONENTS IN CONNECTED TWINS AND DOUBLE INDIVIDUALS As illustrated in plates 1 and 2, the components in connected twins and double individuals exhibit various degrees of separateness from partial double-headedness to completely double individuals. It has also been brought out in the previous section that the degree of doubleness shown by any such specimen depends upon the original distance apart of the two embryonic shields along the germ-ring of the fish's egg, as illustrated in the diagrams of figure 11. As Morrill ('19) has pointed out, the different extents of doubleness are in no way connected with different times of origin of the condition as was suggested by Newman ('17, p. 17-18), since every extent of doubleness is shown in this fish series and the time of origin from the developmental standpoint is the same in each case. Irrespective of the degree of doubleness or the distance apart of the two components, there is a most significant competition, so to speak, between the components themselves, just as exists among several buds growing from a common stock. It is the results of this interaction or competition between the two components which we wish to consider in the present section, and their bearings, of very general importance, will be analyzed in the sections following. Of. Double individuals with identical or equal-size components The two components in each of the specimens photographed in plates 1 and 2 are practically of equal size. The first plate illustrates the young trout from a dorsal view and the second plate shows the same individuals arranged in the same order from the ventral aspect. On comparing the two views of every specimen, it will be found that all heads are perfectly normal in appearance, each having two fully developed eyes, a perfectly formed mouth and branchial structures and a perfectly developed bilateral brain with its general contour clearly visible below the skin. On further comparing the two views in a given specimen, the body regions of the components are also found to be about equally STRUCTURE AND DEVELOPMENTAL RATE 199 developed, except that in one or two of the cases one component is more decidedly twisted than the other. This twisted condition in some cases causes one component to appear considerably larger than the other. This, however, is only an appearance, -and examination of the actual specimen shows the components to be very closely equal in size. Correctly speaking, none of these components are structurally deformed. The application of the term 'double monster' to such individuals as these is actually a misnomer, since there is nothing whatever deformed or monstrous about their structures. The condition of being double is a perfectly normal result of the growth of two buds from a single stock. However, these individuals have arisen from unusual conditions acting on the developing egg during a particular interval and exhibit, therefore, unusual and modified developmental results. Similar conditions affecting other developmental periods are responsible for the production of all types of structural deformities and so-called monsters. The double series is, therefore, similar in so far as its causal origin goes to the ordinary monstrous forms, yet one could scarcely term two perfectly developed identical twins such as those shown by the last specimen of the series as monsters. A study of the series here illustrated in addition to a large number of similar double specimens not only of fish, but of other animals as well as man, leads to the general conclusion that, When the two components of a double individual are equal in size they are both normal in structure. This means simply that such components are as strongly inclined to be normal as is a single individual and not that they are never deformed. All figures of double specimens in the literature furtheii illustrate this point. One may deduce from these facts that if there was a competition of any kind between two such components, the advantages of each in the struggle have been equal. When the advantages are unequal, it will be found that a very different state of affairs results. 200 CHAELES R. STOCKARD STRUCTURE AND DEVELOPMENTAL RATE 201 b. Double individuals with unequal components In every extensive collection of double specimens we not only have those with components of similar size, but also a number of double individuals presenting two components of different size. The discrepancies in size between the two components may be arranged in a graded series beginning with only a slight size difference and finally ending with a very small mass attached to the larger component. Figures 12 to 17 illustrate such a series in cases of anterior duplicities, and figures 20 to 27 show various size differences between the components in completely double specimens. Associated in all cases with these size differences are strikingly noticeable and important structural differences between the components. Figs. 12 to 17 A series of double-headed trout specimens some time after hatching, and ilhistrating the fact that when the two components of a double individual are unequal in size the larger component is normal in structure and the smaller component is invariably 4efective. Fig. 12 The two heads in this individual are equal in size and both are structurally normal. Fig. 13 The left head is slightly smaller than the right, and the right eye of the smaller head is defective with a wide coloboma. The right head is entirely normal. Fig. 14 The difference in size between the two heads is more marked than in figure 13 and the smaller head is also more decidedly deformed. Its right eye is entirely absent and the left eye is extremely defective, being only a small choroid body with a protruding crystalline lens. The mouth and gills are unopened with considerable structural distortion. The larger left head is in all respects perfectly normal. Fig. 15 The left head is normal in size and perfect in structure, while the smaller right head is completely deformed with a twisted irregular shape and no definite outer indications of mouth and gills. The right eye is absent and the left eye is defective. A somewhat different view of the smaller head is shown immediately below the entire figure. Fig. 16 A double specimen with the left head still smaller in size and more completely deformed. It has a cyclopean eye, and a narrow tubular brain, and the branchial parts are entirely distorted. Fig. 17 Completes the series with a perfectly formed larger component, while the smaller left head is represented by an amorphous mass as seen from surface view. Should this specimen have attained adult size, it would probably have been a normal trout with a small nodule representing the lesser component projecting from its body wall. 202 CHARLES R. STOCKARD 1. Condition of the larger component. Whenever the components of a double individual are unequal in size, the larger component, with one exception in more than seventy such specimens that I have studied, is invariably normal in structure. A careful examination of a large number of illustrations of such specimens through the literature, without exception confirms the above fact. It would seem to be a rule, that the larger component of a double individual is no more likely to be defective inform or structure than is a single individual of the same species developing under a similar environment. 2. Condition of the smaller component. Whenever the components of a double individual are unequal in size the smaller component, in all cases examined, is always abnormal inform and structure. A survey of the figures in the literature also shows this to be constantly the case. A study of the types of deformities and defects exhibited by these smaller components is most instructive, and is further extremely suggestive in an analysis of the causes underlying all abnormal development. Examining first the cases of anterior duplicities, figure 12 shows two heads of equal size, both structurally normal. In figure 13 the left head is only slightly smaller than the right. The right head is normal, but the right eye in the left head is small and defective in form, with a ventral coloboma and a protruding crystalline lens. The size difference between the two heads is slight and the abnormalities shown by the smaller are not of an extreme type. In figure 14 one head is decidedly larger than the other, the larger head, as usual, is normal, the smaller is very abnormal. There is only one minute deeply buried eye, E, and the structures of the mouth and branchial arches are peculiarly distorted. Figure 15 shows a still more marked size difference between the two components, and the smaller one here is decidedly twisted, with two poorly developed eyes almost in apposition on the ventral surface of the head. Mouth and gill formations are superficially suppressed, but there are certain contorted structures representing these parts. The brain lacks its usual bilaterality and has a STRUCTURE AND DEVELOPMENTAL RATE 203 twisted tubular shape. The small figure immediately below the defective head represents the opposite view of this head. In figure 16 the smaller component presents a typical Cyclopean eye beneath the anterior tip of a narrow, almost solid brain. Here again the mouth and gill structures are grossly deformed. Finally, figure 17 shows only an amorphous mass representing the smaller head on a perfectly normal larger component. This head mass contained no ophthalmic structures at all, the brain was entirely distorted and the mouth* was completely absent, with the gill structures greatly deformed. Behind this head mass the pectoral fins were fairly developed and a short anterior body portion representing the rest of the component is shown in the figure. Figures 18 and 19 give two views of the only case observed among these individuals in which the larger component was also deformed. In figure 18 the larger left head is seen, in dorsal view, to have a left eye, but no right. The ventral view shown in figure 19 illustrates the normal left eye and also shows the normally well-formed mouth and gill arrangements in the superior component. The right head, or smaller component, is shown from both views to be much more decidedly deformed than the left. It is completely anophthalmic and the brain, mouth, and gill structures are clearly abnormal. Since in all the other specimens the larger component is normal, we may claim with justification that the larger component in this specimen simply happens to be deformed as any single individual might chance to be. But the smaller component is more decidedly deformed than the larger, and the deformity in this instance no doubt results from the same reasons which have brought about similar deformities in all other smaller components of the entire group of double specimens studied. It is only to be expected that the larger component developing under somewhat modified conditions, such as those necessary to induce the initial doubleness, will occasionally be further affected and present some structural deficiency. Such abnormalities are not uncommon among those members of the experimental group which are not induced to double formations, but continue to develop as single 204 CHARLES R. STOCKARD individuals. In other words, monophthalmia, cyclopia, anophthalmia, deformed brains, and branchial structures occur among single specimens developing along with the double ones. We may now consider the condition of the smaller component in double specimens in which the components are two complete individuals, or conjoined twins. . Figs. 18 and 19 Two views of a rare double specimen in which the components differ slightly in size yet both components are deformed. The left larger head has only one eye, the left; it is otherwise perfect, as the figures show. The right smaller head is completely eyeless and grossly deformed in the anterior portion. In this case the larger left head is by chance defective just as any single individual might be. Figure 20 illustrates normal equal-sized identical twins attached to a common yolk-sac. The development of a teleost embryo on a large yolk-sphere and the structure of its yolk-sac prohibits a free separation of identical twins and they always remain joined as shown in this figure. STRUCTURE AND DEVELOPMENTAL RATE 205 In figure 21 the lower component is somewhat smaller than the upper and there is a complete absence of one eye. A drawing of the opposite side of this head shown below figure 21 represents the other eye with an extreme coloboma, its entire ventral part being deficient. This component seems otherwise normal. Two views of another double individual are illustrated by figures 22 and 23. The larger component is perfectly normal except for the fact that its tail is somewhat unusually bent. The smaller component is completely anophthalmic and its brain presents a very abnormal contour. In figure 24 the smaller component is still more reduced in size as compared with the larger normal member. Here also the extent of deformity is still more marked than in the two foregoing specimens. There is one small deeply buried eye in a more or less shapeless head. The mouth and gills are distorted and poorly developed and the brain is deformed. The body is small and abnormally developed. The specimen shown in figure 25 carries the condition a step further. A normal well-formed trout has attached to its ventral surface a greatly coiled and twisted twin. This small component shows a minute almost buried eye, E, and the head is in many ways, grossly deformed. But for the extreme coiling, the body would present almost as good an appearance as that of the smaller component in figure 24. In figure 26 the small twin has a still more malformed head with no eye, but a more or less anteriorly protruding crystalline lens just beneath the skin. The body here is shorter than in the figure above and has only a single twist. Finally, in figure 27, the last of the series, the large component is a splendidly developed young fish with little more than a nodular twin attached to the ventral portion of its yolk-sac. The little component has one small eye deficient ventrally, no external mouth or branchial formations, the brain is tubular and the entire head knob-like in shape. The middle body portions are suppressed and only a conical stump-like tail end is shown. The entire growth of the lesser component has been but a small fraction of that attained by the larger member. One might 206 CHARLES R. STOCKARD STRUCTURE AND DEVELOPMENTAL RATE 207 readily imagine that if this specimen had grown from its present length of 3 cm. up to a size of 30 cm., the small component would have been so outgrown by the larger as to appear a tiny almost unnoticeable nodule on the ventral surface of the large fish. The little component might possibly have become entirely included within the ventral body wall of the larger one. A twin inclusion would thus be formed. 3. The small component and the frequency of double or twin individuals. The frequency of double and twin individuals is probably much greater than realized. No doubt such specimens as the last one considered in the foregoing section might often attain the adult state without being suspected of their twin nature. It is also likely, in view of the fact that a graded series of reductions in the size of the smaller components in double specimens can be arranged down to the conditions here illustrated, that still more decided reductions exist. There probably are specimens with merely a trace of the smaller component, or it is possible that the small component might entirely disappear. Thus an individual appearing as a typically single specimen might in truth partake of the qualities and nature of the major component of a double individual. In connection with such probabilities the condition of situs inversus viscerum is of interest. Morrill ('19) has found in an examination of certain of these double fish that a reverse arrangement of the viscera occurs in one of the components with a far greater frequency than has ever been known to occur among any group of single vertebrate individuals. The reverse arrange Figs. 20 to 23 A series of united twin trout, some time after hatching, further illustrating the principle that in double individuals with components of different size the larger one is normal structurally and the smaller is deformed. Fig. 20 Twin trout, both of equal size and normal structure. Each twin is fully as large as a single specimen of the same age. Fig. 21 The upper individual is the larger and is structurally normal, the lower specimen is slightly smaller with no eye on the right side and the left eye, shown in the small accompanying figure, is deformed with a decided coloboma. Figs. 22 and 23 Two views of the same united pair. The upper larger individual is structurally normal, and the lower smaller twin is eyeless and somewhat further deformed, with a twisted caudal region which also causes a twist in the tail of the larger specimen. 208 CHAKLES R. STOCKARD ^'^^.^^ STRUCTURE AND DEVELOPMENTAL RATE 209 ment of the viscera in one component, though it by no means always occurs, would seem in some manner to be associated with the double condition. This reversed visceral arrangement also occurs very rarely among man and other mammals in single individuals. Its remarkable frequence among these double specimens would lead one to suspect very strongly that when a reversal of the visceral arrangement occurs, the apparently single individual is in reality a twin. All such specimens should be carefully examined for twin or embryonic inclusions as positive evidence of their double nature. Failure to find such inclusions would not, however, disprove the above suspicion, since the inclusions might be represented by structures so minute as to be readily overlooked. 4. The small component and certain theories of teratoma. Another much-debated problem may be somewhat illuminated by this study of double specimens. I refer to the various ideas of the possible origin of so-called teratomata or embryonal tumors. Such formations occur with greatest frequency in the lower abdominal or pelvic region. Certain pathologists have thought them to arise from a development of misplaced or arrested blastomeres, others have thought it possible that they might arise through some form of parthenogenetic development, and still others have looked upon them as a type of twin inclusion. The Figs. 24 to 27 A continuation of the twin trout series shown in figures 20 to 23. In this group the smaller member is still more inhibited in size and more completely defective in structure. The larger component is perfect in all. Fig. 24 The smaller twin is little more than half the size of the larger with an amorphous head containing one defective eye and the body is twisted. Fig. 25 The smaller twin is here greatly twisted or coiled, its head is deformed, possessing a large defective left eye, and the right eye consists of a tiny choroid vesicle indicated by the dark spot, E. Fig. 26 The lesser component is still smaller in size, short and twisted with a considerably suppressed eyeless head. Fig. 27 The larger twin is a beautifully normal specimen, while attached to the opposite surface of the yolk-sac is a small individual represented by a badly deformed head with no mouth or gills and one defective eye. Almost the entire body of this component is absent and the tail is represented by a conical mass with no caudal fin. Should such a specimen attain adult size the smaller individual would be attached to the ventral abdominal wall of the larger as a nodular twin. THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 210 CHARLES R. STOCKARD frequent occurrence of teratoma in the pelvic regions was in line with any of these explanations. Misplaced blastomeres might readily be in this portion of the body and twin inclusions or partially deformed twin bodies are frequently connected with the pelvic region. The parthenogenetic theory which has received considerable support would necessitate the occurrence of all such formations in close proximity to the gonads and therefore would practically limit their occurrence to the pelvic region. It so happens, however, that teratomata occur with considerable frequency in the head and neck regions. This is most difficult to explain on the basis of a parthenogenetic origin. It might be possible, though not so probable (for the blastomere theory), that misplaced blastomeres arrested in the early egg might develop in such cephalic positions. On the other hand, if teratoma arises from early twin inclusions, one would, on the basis of our series and a general survey of recorded double individuals, readily recognize that the head and neck regions should be places of frequent occurrence for these structures. The double specimens arrange themselves roughly into two groups, the anterior duplicities or double-headed lot, and the completely double specimens. Should the smaller components of the first group become greatly inhibited and included within the larger components, we should have the inclusions in the head region. Should these then give rise to teratomata, such growths would not be expected to contain tissues found in the caudal regions of the body, such, for example, as nephric, gonadal, lower intestinal structures, etc. The rather frequent occurrence of teratoma in the head and neck would be what one would expect. The inclusion of the smaller component of the completely double specmien would in most cases occur in the lower abdominal region. This would account for the great frequency of pelvic teratomata. Such teratomata in contrast to those of the neck region, may be found to contain tissues characteristic of any portion of the body, since these are inclusions of a possibly complete twin and are not limited to structures of either the STRUCTURE AND DEVELOPMENTAL RATE 211 anterior or posterior regions. Nevertheless, from what we have seen of the tendency on the part of the smaller component in the completely double individuals to possess poorly developed head ends, it would not be surprising to find that ophthalmic tissue and other cephalic structures were frequently absent from pelvic teratomata, though such structures might in certain cases be particularly evident. 5. Types of defects exhibited by the smaller component. We may now return to a brief consideration of the types of deformities shown by the smaller components in the unequal pairs and decide whether these defects are similar in kind to those which occur in nature, as well as those experimentally induced, among single individuals. In the first place, it is noted at once that ophthalmic deformities are particularly frequent. The illustrations show complete anophthalmia, monophthalmia, and typical cyclopean conditions as well as various degrees of imperfection in the individual eye, such as coloboma and reduction in size of the retinal region. Duplicities produced by any method such as the mechanical constriction employed by Spemann, as well as those occurring in nature, show in the smaller component the same ophthalmic defects as are found among these double specimens induced by development in low temperatures or with insufficient oxygen supply. The brain in the smaller "components shows various abnormal contours or may be simply tubular in shape without a normal expression of bilateral diverticula or hemispheres. The mouth is often deformed and frequently absent and the operculum and branchial arches are distorted in shape. The fins are often small and underdeveloped. The general body shape may be variously modified, the caudal end being short and stumpy or absent. The heart may be poorly developed and pulsating feebly so that the blood fails to circulate and becomes massed in various regions of the body. It is unnecessary to do more than enumerate these defects and examine the illustrations to convince anyone familiar with the commonest developmental anomalies that the structural 212 CHARLES R. STOCKARD modifications and defects of the lesser components are in every sense identically the same as the defects which have been recorded and illustrated as occurring in single individuals. The types of defects most commonly found, such as those of the eyes, are also the most frequently observed anomalies in single individuals. Therefore, not only the kinds of defects, but the frequencies of their occurrence are the same among these lesser components as among single deformed specimens. The fact that these malformed components are developing in intimate union with larger normally formed components makes it evident that the causal factors for the malformations are to be sought in some difference that exists between the developmental processes of the two. And, further, since the malformations of the one component are identical with those in single specimens, the difference in conditions found between the larger and smaller component may also furnish the clue to causes of malformed structures in general. We shall attempt beyond, in section 7, to give a logical explanation of abnormal structure from this standpoint. c. The components in double human specimens In order to demonstrate that the conditions above described as existing in double specimens of fish are in no way limited to this class of vertebrates, I wish briefly to consider several very interesting degrees of double development in human specimens that have recently come into my laboratory. All of these specimens have been examined by my colleague Doctor Morrill for conditions of situs inversus viscerum, and the left component in one case of anterior duplicity, as reported by him ('19), shows a reversal in the position of the viscera just as was found in certain of the components among the double fish. The three specunens seen in plates 3 and 4 to form again a graded series. The series begins with a double individual presenting two heads and anterior portions on a single pelvis, seen in the upper photograph, plate 3, and passes on to a completely double specimen with the components strongly united through STRUCTURE AND DEVELOPMENTAL RATE 213 their ventral surfaces, the lower figure, and finally ends with the entirely separate unquestionably identical twins illustrated in plate 4. The case of separate twins differs in many ways from the other two and will be considered alone. The first two specimens are in general alike. In each the two components are practically of equal size, and all of the four components present entirely normal structures. These specimens follow exactly the rule stated in reference to the double trout; that is, when the two components of a double individual are equal in size they are both normal in structure with almost the same frequency as a single individual would be. In the first specimen, of plate 3, each head shows a perfectly formed face with all the sense organs fully developed. A dissection of this body shows the vertebral columns to be separate down to the sacrum. The pelvic skeleton is single with the normal single pair of lower extremities arising from it. The median arms of the two components have their soft parts fused or pecuharly arranged, a synbrachium, the details of which are being studied by Mr. H. B. Sutton. Each arm possesses a complete skeleton. Further details of the visceral structures are of no importance in the present connection. This specimen is in general comparable to the fourth case of anterior duplicity shown in the equal component series of trout (plates 1 and 2) . The second human specimen, in plate 3, is a case of full-term united negro twins. There are two completely formed components with a very wide ventral union into which the two livers and other viscera are drawn. The babies are females and each would weigh more than 5 pounds. A careful examination shows all organs and parts to be perfectly formed and of normally large size. This specunen is comparable to the last ones shown in the equal component series of trout (plates 1 and 2) . Here again we are warranted in attributing the different degrees of doubleness to the different distances apart of the two original embryonic lines or axis as they appeared on the blastoderms. In the first case the primitive streaks were not far apart and in the second case they were in positions almost 180° 214 CHARLES R. STOCKARD apart on the blasto-disc. Such an interpretation would certainly seem proper in the case of the fish and the bird. The third case, that of the separate twins, plate 4, differs from the others in that both babies are deformed. Yet the deformities and other peculiarities of this case make it unique in value. It has been seriously questioned, on the basis of psychological and other studies (Thorndyke '05), whether actual cases of human A 28 B Fig. 28 Drawings of the ventral surfaces of the hands and feet of the identical human twins shown in plate 4. A, from one individual, and, B, from the other. The four hands are all polydactylous, having an accessory finger on the ulnar side, and the four feet are similarly polj'dactylous, all having an accessory toe on the fibular side. The polydactylism is practically identical in the two individuals. identical twins do exist. The structural conditions of the two male twins in this case renders it practically certain that they arose from a single fertilized egg. There are six fingers on each of the four hands, as shown in plate 4, and more distinctly in figure 28 A and B; there are also six toes on each of the four feet, as the illustrations show. Such a polydactylous condition is known to be derived from a peculiar germinal complex and is not produced by the developmental environment. The chance is one against thousands that two fertilized eggs carrying exactly STRUCTURE AND DEVELOPMENTAL RATE 215 the same capacities for polydactylous development should occur at one time in this mother. The expression of Polydactyly in a family in which it is hereditary, is most variable. Neither the father nor the mother of these twins were said to have polydactylous hands or feet, and it was claimed that they had another child with an ordinary number of fingers and toes. This, however, was a case of a 'seven-month' stillbirth in a New York hospital, and it proved impossible to obtain a family history of positive value. These twins show further deformities that are almost identical in the two. All of the four kidneys are cystic and the left kidney in each individual shows the cystic condition in a more exaggerated form than does the right. The two heads have posteriorly protruding meningoceles, one being slightly larger than the other. The meningeal hernias are probably due to the action of some environmental influence that produced closely similar responses in these two individuals of identical germinal composition. In this case both members of the pair are malformed in addition to their unusual characters of genetic origin. In every way these twins are structurally about the same with the exception that the one on the left is smaller than the one on the right to just the extent carefully shown in the two figures. This is a most positive case of identical human twins and would certainly seem to leave no reason for question as to the occurrence of such individuals. From these examples it is probable that the two equal components in double human individuals are in about the same relationship to one another as are the equal components of other double vertebrate specimens. The double human specimens seen in many musemns as well as those illustrated in the literature in which the two components differ considerably in size also follow the rule found for similar fish specimens. In double human specimens the larger component is usually normal in structure and the smaller component is always deformed. Extreme cases of this type are exhibited among the freaks in 'side shows.' In these living specimens the larger component has a well-formed human body with the 216 CHARLES R. STOCKARD smaller component represented by a malformed partial body, attached to or protruding from it. The cause of doubleness and twinning in these human specimens is in all probability the same as in the other cases discussed. The rate of development of the egg was probably arrested during an early stage, and perhaps on account of some interference or delay in implantation on the uterus. Very recently I have obtained a specimen through the kindness of Dr. Frank Erdwurm, of New York, which is of great value in an understanding of twin development in man. The specimen secured by Doctor Erdwurm is shown by photograph in plate 5. A living female baby weighing 6| pounds was enclosed within the upper membranes seen in the photograph. The cord from this baby is connected to the upper placenta near its lower border. After delivering the child, a second chorionic sac ruptured and discharged its fluid to the surprise of the observer. Later the two dead fetuses seen in the picture were delivered along with the placental mass. The fetuses proved to be identical twin girls enclosed within a common chorion and attached by their cords to a common placenta. The photograph clearly shows the single membranous sac in which they were enclosed and the positions of attachment of their cords to the placenta. The size and other conditions of the fetuses indicate a stage of about six months' development. They had evidently been dead for a long time, probably about three months. The heads and bodies were somewhat macerated and shriveled and the blood-vessels had broken down in their placenta so that this no longer had any circulatory communication with the uterus. The two umbilical cords had become so wound around each other and knotted, as to completely cut off the connection of the fetal bodies with the placental circulation. The two fetuses were no doubt asphyxiated after six months of development. This structural evidence is substantiated by the behavior of the mother. She had passed through the first six months of pregnancy in a normal fashion and then became greatly disturbed, so that it was feared that her pregnancy might be inter STRUCTURE AND DEVELOPMENTAL RATE 217 rupted entirely. The severe condition gradually subsided and she was able to carry the living child to full term. The reaction of the mother was doubtless due to the death of the two fetuses and the cut-off in the placental circulation. The uterus was able to adjust itself to the condition and the fetuses remained aseptically enclosed within their membranes. This was the mother's second pregnancy; exactly twelve months before, lacking one day, she had given birth to a single normal child. My interpretation of this triplet condition is as follows: The mother liberated from the ovary two eggs, both of which became fertilized and began development. One became implanted slightly before the other and developed into the single living girl. The second egg was not so favorably implanted as the first; this is indicated in the specimen by the lower placenta riding up over the larger one. The delay in implantation, due to the presence of the first egg, caused a slow rate of development at an early stage in the second and two embryonic buds arose instead of one, just as was described on the germ-ring of the fish. In this human specimen there is fortunately present the physical cause that might have produced the delay. The woman gave birth to triplets, two of which were female identical twins derived from one egg and the other was a single sister individual derived from another egg. Doctor Erdwurm furnishes a further very important record. This woman's mother had eleven pregnancies, nine of which resulted in living single births and two in abortions during the first half of pregnancy. One abortion, the tenth pregnancy, consisted of twins, and the eleventh pregnancy resulted in the abortion of triplets. The nature of these twins and triplets, unfortunately, is not known. Evidently there is here a family tendency to ovulate more than one egg at a time. This may be due to simultaneous ovulations from both ovaries, from two follicles of one ovary, or from the rupture of a single follicle containing two or more ova. 218 CHARLES R. STOCKARD 7. THE DOUBLE INDIVIDUAL WITH UNEQUAL COMPONENTS AND AN UNDERSTANDING OF THE CAUSE OF ALL MONSTROUS DEVELOPMENT The most valuable material that falls into the possession of the investigator attempting to analyze the causes of abnormal development is furnished by the united twins and anterior duplicities where one component is fully developed and perfectly normal in structure, while the other component presents in a series of such forms various degrees or combinations of malformation and deformities. There are practically numberless attempts, from the time of Aiistotle until now, at theoretical explanations for the cause of monstrous development, but none of these, as far as I know, have recognized as crucial the condition presented by the combination of a larger normal twin developing in actual union with variously deformed lesser individuals. Certainly, all theories that conflict with this condition of fact may be discarded as being inadequate in general. And, as mentioned before, the explanation of abnormal development probably lies in the differences between the factors operating on the development of the two connected individuals. In the first place, one could scarcely state in the presence of these specimens that the abnormalities of the lesser component are of germinal origin. Yet similar deformities in single individuals have been interpreted in Wilder's ('09) theory of 'Cosmobia' as always being of such origin. The larger and smaller members of the double complex have both arisen from a single fertilized egg. There is no trace of either direct or collateral evidence to indicate that the hereditary factors are not equally distributed in the cells of both components. The germinal origin of one component could in no way be different from that of the other, since the entire specimen was a single individual up to about the stage of gastrulation. Further, when the two components are of equal size their identical genetic composition and character is evident. Obviously, then, defects similar to those enumerated as occurring in the smaller component are not in general of germinal origin. STRUCTURE AND DEVELOPMENTAL RATE 219 It is further evident that 'identical twins' and the components in double individuals need not necessarily exactly resemble each other as is commonly thought. The two members of the pair may be structurally very different ; in extreme cases one may be normal and the other actually deformed. What influences could act on the smaller component that do not also act on the larger? Evidently there can be nothing in the external environment that would not come in equal contact with both components, since they are intimately united and enclosed within the same egg membrane. There can be no case of injurious substances inducing a 'blastolysis' in the one component and not acting on the other, or causing an early 'cellular disorganization' (Kellicott, '17), which would not affect both components. There is also no question of an insufficient supply of nutriment of the ordinary type, since it is shown by many specimens that two normal embryos equally as large as the usual single one may develop on the yolk-sac of the fish's egg, compare the first and last specimens shown by photograph in plates 1 and 2. There must, however, be some sort of competition between the two components other than a competition for the appropriation of ordinary yolk material. Much evidence suggests that an interaction exists between the components similar to, if not identical with, the interaction between two plant buds growing from a common stock. When the growing tip is cut from the shoots of certain plants, e.g., the ordinary privet, Lagustrum, as a rule the axillary buds of the two leaves immediately below the cut give rise to growing shoots. In many cases two shoots grow at equal rates and are about equal in size, in other cases one of the shoots evidently possesses some advantage and grows much faster and becomes larger than its companion. Finally, in a few cases a single vigorous shoot arises from one of the resting buds and the opposite bud is entirely unable to grow. There is here involved a factor in addition to available food material, just as Loeb has found in the Bryophyllum leaf, and whatever this factor may be, through it the growing parts exert an inhibiting influence on one another. In the first case cited for the plant, the growing impulse was balanced between the two upper axillary 220 CHARLES R. STOCKARD buds and they grew equally, in the second and third cases one bud occupied a position of advantage over the other; this advantage may have been due to a slightly more favorable exposure to sunlight, heat, or moisture, or to a better flow of sap material on its side of the stem. Its more rapid rate of growth in some way imposes an inhibiting influence on the expression of the other bud, causing it to be smaller, sometimes ill-formed or suppressed entirely. If the larger bud be pinched away in any of the cases, the smaller immediately improves its condition" and grows large, provided it has not been held back too long (figure 29 A, B and C). The advantages of certain positions on a stem over others is strikingly shown by privet branches growing in dense shade. These branches are slender shoots with long intervals between the pairs of leaves until finally they reach the sun. After a certain length of the stem has grown into the sunlight, the axillary buds of a particular pair of opposite leaves grow into shoots. Later, when still further in the sun, the two axillary buds immediately below those that grow first, now grow to form the second pair of shoots. Still later the axillary buds from the leaf pair immediately above the first shoots send out the third pair. I have observed this exact order of growth in nine cases of shaded stems. The first shoots to appear have an advantage of position over the second and third on account of a proper exposure to sunlight and at the same time occupying a certain distance away from the growing tip. The second buds then come into sufficient sunlight and grow out despite the inhibiting influence of the first pair, and finally the third pair of buds now grow on account of having become more mature and further removed from the growing tip. The two embryos developing from a single blastoderm compete in a comparable way, and the results of the competition are also similar to the case of the plant buds. In the present state of our knowledge, it is impossible to say what the primary cause is that gives one of the growing parts an advantage over the other. We may merely express this in a non-committal way as an 'advantage of position.' It would in no sense relieve our ignorance of the situation to state the likely probability that one of the growing points has a higher rate of metabolism or a more rapid oxidation STRUCTURE AND DEVELOPMENTAL RATE 221 than the other. No doubt this is a fact, and should it be demonstrated, we still have the question: why is the rate of metabolism or oxidation higher? Why does this difference in rate of oxidation exist in some instances and not in others? What is there in these apparently similar points around the germ-ring that brings Fig. 29 Outlines of branches from the common privet in which the terminal portions had been cut away, as indicated at the upper ends of the stems. In the first and usual case, A, following removal of the tip each of the upper axillary buds have given rise to equal-size shoots. In B the shoot from the right axillary bud is large and strong, while the shoot from the left bud is slow-growing and small. In C the right shoot is normally expressed, but the left upper bud has failed to grow entirely, yet if the right shoot were pinched away the left bud then readily grows out. In A the advantage of position in the two upper axillary buds was equal, but in B the right bud had an advantage in growth position over the left bud, and in C this difference in growth advantage was still greater. In both of the latter cases the growing shoot exerted an inhibiting influence over the opposite bud. 222 CHAKLES K. STOCKARD STRUCTURE AND DEVELOPMENTAL RATE 223 about this higher affinity for oxygen at one point than at another? Certainly, we do not at present know! This unknown factor acting between the growing buds or embryos when out of equihbrium, inhibits to an unusual degree the rate of oxidation and through this probably the rate of cellmultiplication, and certainly the rate of development in one of the components. All the defects observed in the inferior component are simply due to a slowing of its developmental rate or are strictly what I have always termed developmental arrests. This problematical factor, then, simply tends to lower the rate of development in the one component and thereby does w^hat the experimenter is able to do in various ways with any developing single individual. I ('06, '07, '09, '10 a b, '13, etc.) have experimentally produced in single embryos all of the deformities seen in the smaller components by arresting the developmental rate of eggs with a large number of different chemical and physical treatments. Newman ('17) has observed exactly the same types of monsters among slowly developing hybrids. As Newman very correctly points out, one obtains similar monsters by any method, either treating the eggs with injurious chemicals, strange physical conditions, or by heterogenic hybridization. Each of these methods simply lowers the rate of metabolism and the rate of development. Newman, in agreement with my position, recognizes all of the monsters as being primarily due to a lowering of developmental rate and, therefore, generally speaking, they are actually developmental arrests. From an extensive study of monstrous individuals, Dareste ('91) long-ago believed that all developmental abnormalities were in general arrests, yet he lacked proof for such a position. The present study, however, enables me to state the case in far bolder and more definite terms than it has been possible to do before. In the first place, every type of developmental monster Fig. 30 The terminal portion of a long privet stem which had grown upward in a shaded position. On reaching direct sunlight the axillary buds three leaves from the bottom of the figure grew into lateral shoots. Very soon after this the axillary buds of the pair of leaves immediately below the first shoots give rise to the second pair of lateral shoots, and finally the buds of the leaf pair immediately above the first shoots grow into a third pair of lateral branches. In nine cases this budding sequence was invariably followed. 224 CHAELES R. STOCKARD known in the literature may be produced by one and the same experimental treatment. For example, simply by lowering the surrounding temperature or by treating with a weak ether solution all monsters may be produced. Secondly, the same structural abnormality may be induced in the embryos of various species by a great number of different experimental treatments. Thirdly, in all cases the initial effect of the experimental treatment is a lowering of the developmental rate, and the resulting deformity is always secondarily due to this slow rate of development. Fourthly, the type of monster or deforrnity is determined by the developmental period during which the slowing in rate is experienced. An early slowing will induce the growth of accessory embiyonic axes or duplicities, while a similar reduction in rate at later periods may produce anophthalmia or cyclopia, simple tubular brains, malformed otic structures, deformities of the mouth or branchial arrangement, etc., depending upon the time at which the rate of development was slow. Slowing by a number of different ways if done at the same developmental time will give closely similar defects. We may finally state a common law of both normal and abnormal development as follows : Structural quality may be affected by many things, but always depends directly upon the rate of development of the part or of the individual. I have many reasons for believing that this law equally applies during postnatal growth and change in higher animals, as well as during their prenatal development. In a study of a number of different embryos it will be observed that a particular structural modification is more common in one species of egg than in another. With trout eggs for example, duplicities more readily occur than in the egg of Fundulus. While, on the other hand, cyclopia and certain eye defects are more common among deformed specimens of Fundulus than among those of the trout. It would seem as though particular moments and localities were more susceptible to modifications in one egg as a result of slow development than in another. Further, certain eggs are in general much more sensitive than others and, as is well known, more frequently deformed. Their developmental rate is less strongly regulated than is the case in the more resist STRUCTURE AND DEVELOPMENTAL RATE 225 ant types. These facts are due to the different hereditary backgrounds on which the modifying conditions act. Finally, if one admits the above generalizations to be true, studies and descriptions of individual monsters and deformities lose much of their interest and so-called value. It is evident from the present standpoint that a single deformed specimen, whether human or lower vertebrate, must be considered as resulting from an arrest or slowing of its developmental rate during a particular period. Observing the nature of the deformity or the parts involved enables one to estimate the developmental period during which the arrest was effective. Such individual monsters in no way supply evidence to determine what the initiating cause of the deformities may have been, since we know that the same type of deformity may be experimentally caused by many different treatments. We can estimate simply that the exciting cause acted to lower the rate of development during a definite interval. The great number of descriptions in the literature of isolated monsters have added very little to our understanding of the causes of abnormal development. The writer believes after a prolonged study of this subject that the only benefit to be derived from examinations of such isolated specimens is possibly to obtain aid in studying the normal sequence of development. This of course is valuable and only to this extent are such descriptions worthy of record. Detailed descriptions of monsters occupy the same level of scientific value as do records of ordinary structural anomalies observed in the dissecting room. Having stated the above conclusions derived from an extensive study of abnormalities in single individuals as well as the double specimens under present considerations, it is not deemed necessary to enter into a general discussion of the various views regarding abnormal development with which morphological literature abounds. Many of these positions have been discussed in my previous papers. I shall here only attempt to consider briefly the last contribution by Mall ('17) who devoted so much study and masterly consideration to this subject. The investigations by Mall are far the most valuable that have been made on human material. 226 CHARLES R. STOCKARD In his last study on the frequency of locaUzed anomalies in human embryos and stillborn infants, the data from a thousand specimens are recorded. Mall's method of arranging this material may be considered in brief in order to attempt fitting it into our above conclusions. The material was primarily divided into normal and pathological specimens. Some of the 'normal' may possess localized anomalies, such as cyclopia, etc., and a study of the pathological group Mall believes justifies this inclusion of localized anomalies among the normal embryos. The pathological group was then subdivided into seven classes of specimens: first, those consisting of only degenerate choi ionic villi ; second, of only the chorion with the extra embryonic coelom ; third, of the chorion and amnion; fourth, of the embryonic membranes containing a nodular embryo; fifth, of cylindrical embryos; sixth, stunted embryos, and, seventh, dried and deformed or soft and macerated specimens. The series thus begins with the most degenerate conditions and passes on to those specimens which maintained their integrity fairly well, though evidently malformed and dead for some time before being aborted. Unquestionably, all members of such a series have suffered developmental arrests of the severest types. In some cases the arrest has come at an early stage and been followed by a disorganization or cytolysis and subsequent absorption of the embryonic material. In guinea-pigs one frequently finds similar stages of embryonic degeneration in utero, and here also the placenta and membranes are the last parts to disappear. In almost all of these cases portions of the pregnancy must have remained in the uterus for some time after the embiyo died before being discharged. If these pathological specimens are primarily due to developmental arrests, what, if any, evidence is there that conditions may have existed which could probably have induced such arrests? Or, is there evidence that human embryos are affected very readily by strange conditions? Very valuable data bearing on both of these questions are supplied. In the one thousand specimens considered, about 33 per cent of the ova and embryos from the uterine lot w^ere pathological, while as many as 66 per STRUCTURE AND DEVELOPMENTAL RATE 227 cent of the ectopic specimens were of this nature. The double frequency of pathological specimens in ectopic pregnancies shows at once the influence of unfavorable environment. These facts are of primary importance and Mall discusses them in a most instructive way. He states, to account for human monsters : It would have been quite simple to conclude that the poisons produced by an inflamed uterus should be viewed as the sole cause, but when it is recalled that pathological ova occur far more commonly in tubal than in uterine pregnancy, such a theory becomes untenable." It is then stated further: "For this reason (meaning the records from ectopics) I have sought the primary factor in a condition buried in the non-committal term, 'faulty implantation.' " The faulty implantation acted to injure the development, in Mall's opinion, on account of supplying insufficient nutriment. I should be inclined to accept the faulty implantation as the primary factor, but the injm-ious effects of such an arrangement are due to an insufficient oxygen rather than food supply. This difference in interpretation is only of academic value. Malnutrition effects developing individuals in a general way causing a condition of undersize, while insufficient oxygen decidedly slows the rate or may completely interrupt development and thereby induces various structural deformities. Mall in this paper is inclined to drop the cruder term 'nutrition' and admits that, "Probably it would be more nearly correct to state that change in environment has affected the metabolism of the egg." This would be entirely in accord with the interpretation e! arrest as being due to lowered oxygen supply. Again, Mall reaches significant conclusions when considered in connection with the foregoing general principles of abnormal development. For, on page 72, he states: "Accordingly, when an embryo through changed environment is profoundly affected, the development of one part of the body may be arrested, while the remaining portion may continue to grow and develop in an irregular manner. In very young embryos, tissues or even entire organs may become disintegrated, as can easily be recognized by the cytolysis and histolysis present, and the resultant disorganized tissue cannot continue to produce the normal form of an 228 CHARLES R. STOCKARD embryo. If this process (evidently meaning disintegration) is sharply localized, for instance, in a portion of the spinal cord or in the brain, spina bifida or anencephaly results. To produce a striking result, as in cyclopia, a small portion of the brain must be affected at the critical time." The one position with which we are entirely unable to agree is that the arrested development must so constantly be "associated with the destruction of tissue." This tissue destruction is not at all essential to the production of such defects as spina bifida or anencephaly. It may be demonstrated in many experimental cases that the tissues fail entirely to arise or differentiate without there being any indication whatever of a previous destruction. As stated in the beginning. Mall included localized anomalies among his normal specimens, yet such anomahes occurred about twice as frequently among the pathological individuals as among the normal. This is closely in accord with what has been found for the abnormal fish embryos. After this review of monstrous development in general, and an analysis of its causes from the conditions found in the smaller components of double individuals, we may consider in the following section the interaction, if such can be observed, among the early organs in the single individual. It may be possible that these organs are related to one another in their development in somewhat the same manner as are the components of double specimens. A further test, therefore, of the correctness of my interpretations for abnormal development may be had in an analysis of the relationships among the developing organs in the single individual. 8. THE DOUBLE INDIVIDUAL WITH UNEQUAL COMPONENTS AND AN ANALYSIS OF THE DEVELOPMENT OF ORGANS IN THE SINGLE INDIVIDUAL By way of introduction, we may further consider certain conditions in developing plants on account of their apparent simplicity and also their very striking suggestiveness in connection with an analysis of the origin and growth of organs in the vertebrate embryo. The imaginary elements involved in comparisons of STRUCTURE AND DEVELOPMENTAL RATE 229 plant conditions with animal development I very fully recognize. There is, however, evidence of certain actual similarities which, along with deductions from my experiments on embryos, may serve to elucidate the problem of organ formation to a considerable extent. Particularly suggestive is an examination of — a. The growth influence of the apical or primary hud over the secondary and potential buds in plants It is commonly observed that when a number of beans or other seeds are planted in a row under similar* conditions of soil and moisture, the initial bud from each seed sprouts upward and grows to a definite extent and then temporarily stops. On examining the row of young shoots, each with two horizontally spread terminal leaves, it is generally found that all are very nearly of the same height. Should a certain part of the row occur in a more favorable environment than another the sprouts in this part may grow higher than in the others, or should certain seeds have been defective or their environment in the row unfavorable, the sprouts in such cases are lower and smaller than the average. These low small plants seem as a general rule unable to overcome their inferior condition during later growth and either die or form very poor specimens. The small sprouts would appear to have suffered an arrest during their early development in consequence of which they generally fail to be normally large fruiting plants. The original shoot is entirely formed from the food contained within the cotyledons and the water of absorption. After attaining a definite length, it stops or slows its progress until the roots become sufficiently established to obtain further food and moisture from the soil. On becoming properly rooted the apical bud then grows upward from a point between the two original leaves and from this the development of the plant proceeds. We thus have an interruption, after the formation of the original sprout, similar to that found in the development of many vertebrates and from a somewhat similar cause. Here the plant could not continue to grow until certain substances were supplied by 230 CHARLES R. STOCKARD the roots, through the assimilation of which, cell multiplication was made possible. In the birds and in the experiments with fish eggs, the initial development is interrupted by a sudden lowering of temperature and through this the chemical processes necessary for cell multiplication are slowed or stopped and development ceases. Although the stuff is available, the conditions prevent its use. The case of the mammal is more closely analagous to that of the plant. Here the fertilized ovum within the Fallopian tube begins to develop and continues until it exhausts its initial supply of oxygen, though there may possibly be here also an exhaustion of nutriment as in the plan. Following this, the development of the embryo is either stopped for a considerable time, as in the extreme cases of the deer and armadillo, or it is temporarily interrupted or slowed until the membranes have become established or embedded in the uterus of the mother and a further source of oxygen and nutriment is thus acquired. The placentation of the mammalian ovum and the rooting of the plant in the earth as a mother, are comparable processes. Any lack of perfection in the process is either fatal or lowers the supply of necessary stuffs and thus causes an abnormally slow rate of development and growth with a resultant imperfection in structural formation. After the original linear sprout of the plant has rooted, and a certain extent of linear growth has taken place from the apical bud, growth in length gradually slows as if the apical bud had passed bej^ond the point at which it could dominate the growth activities throughout the length of the plant. When this time is reached, the axillary buds at the base of the leaves are able to express their growth capacities and the plant develops its lateral branches. Though all the branches of a plant have a more or less similar function, yet each may be looked upon as an organ, and their origin and subsequent competitive growths are in many respects similar to the origin and growth of organs in the vertebrate embryo. Such a statement of the situation in plant development is rendered further justifiable by a very common experiment. If, instead of allowing the apical bud to gradually exhaust its suprem STRUCTURE AND DEVELOPMENTAL RATE 231 acy by continuous growth, it be injured or pinched away at an early stage, the lateral buds very quickly grow out, showing their liberation from some controlling influence possessed by the apical bud. In other words, each growing hud {also true of the embryonic organs) exerts a depressing influence on the growth of all other buds in the individual plant. As a shoot gradually ceases to grow its depressing influence also gradually ceases. b. The initial linear growths, subsequent lateral buds, and the interactions among the organs of the vertebrate embryo When the first trace of the embryonic body begins to express itself in the blastodermic matrix it appears as a linear growth, the head process extending forward from the blastopore or primitive streak. This very soon becomes surrounded by, or associated with the linear outline of the arising neural folds, the beginning central nervous system. The neural folds indicating the early nervous system are originally of more or less straight outline and their first growth is largely a growth in length. When in a given species the neural groove has attained a certain length, it then begins a series of lateral outgrowths, or branches. The first and largest of these are the two optic outpushings and after them follow in a general way, a series of bilateral outgrowths designated the three primary brain ventricles. The initial linear origin and growth of the nervous system is very probably due to an equal rate of cellular proliferation along the entire extent, with perhaps a somewhat more rapid rate at the tip. The lateral outgrowths arise on account of an excessively high rate of proliferation occurring in a given region during a certain time. For some unknown reason the rate of metabolism, or actually the rate of oxidation becomes disproportionately high in a particular group of cells, and these begin to multiply rapidly as compared with the multiplication rate of neighboring regions, and thus a folding or outgrowth occurs to produce, for example, the optic vesicles. Since other portions of the brain seem nof to be proliferating so rapidly at the same moment, it may be that 232 CHARLES K. STOCKARD the growing optic vesicles exert a depressing influence over the growth of other parts. There is indirect experimental evidence for such a statement. The initial moment of high cell multiplication for a particular organ outgrowth is a most critical instant in the development of this organ. Thus, if the general developmental rate of an embryo be reduced by exposure to low temperature or cutting off the oxygen supply at the time when the rapid proliferation of the optic anlage should occur, the disproportionate growth of this region is prevented, and the result of such an experiment will be either the complete suppression of eye development, anophthalmia, Cyclopean eyes, monophthalamia, or some other degree of defective eyes. This result ensues in spite of the fact that after the critical moment for eye origin has passed the embryos may have been again developing at the usual rate in a normal environment. The eye has only one favorable period for its origin, its moment of supremacy so to speak, and when it is unable to express itself at this time, the opportunity is largely, if not entirely, lost. This is probably due to other organ anlagen having arrived at their controlling moments, the optic inhibition being no longer sufficient or capable of suppressing them, but they, on the contrary, now suppress the optic bud. The arrest in development necessary for suppression of the optic vesicle must be induced in the early embryo, before the embryonic shield stage in the teleost, or before the optic anlage is at all visible in the neural plate. This I ('09, '13) have shown by a number of different experiments, and now also find to be true in case of treatments with low temperature and scant oxygen supply. I ('09) have reported a number of experimental cases of fish embryos in which the eye was absent or was cyclopean, while the general brain structures were as usual bilateral and normal. Such specimens are viable and swim actively about. It is evident in these cases that the arrest was limited in its effect to the optic outgrowths and was no longer effective when the primary brain ventricles were forming. STRUCTURE AND DEVELOPMENTAL RATE 233 Specimens have again been recorded from my experiments, and these also may be induced in a great number of ways, showing either two poorly formed eyes, cyclopia, or anophthalmia, accompanied by a narrow tubular brain. Here the arrest or slowing in developmental rate has affected the optic outgrowths only slightly in some cases, in other cases severely, but in all cases it has persisted or continued to act for a longer period and has thereby also suppressed the outgrowths which normally form the series of primary bilateral brain ventricles, hence the final narrow tubular brain. Depending, then, upon the rate of development at a given moment, we may obtain : first, as is normally the case, optic vesicles on a brain with three bilateral primary ventricles; second, no optic vesicles, yet a brain with the bilateral primary ventricles ; in the third case, we may or may not obtain optic vesicles on a brain with no growth of the bilateral ventricles — a simple tubular brain (fig. 31). We may describe the development of the central nervous system in the vertebrate embryo very simply and schematically as follows. At first a more or less straight linear growth takes place until a given length for the given species is attained, then the linear growth possibly becomes slower in rate and lateral branches or outgrowths begin to appear, first the optic vesicles and then the first, second, and third primary brain ventricles. A competitive element is involved in the origin and growth of the lateral outpushings so that should one of these fail to express itself during the usual time for such expression, it is later unable to grow out normally or may not grow out at all (fig. 31). We know from experimental demonstration (Lewis, '04; Spermann, the writer, Leplat, '19, and others) that the optic vesicles are derived from a definitely located group of cells in the neural plate of the embryo. When they do not arise from this group of cells no other cells are capable of forming optic vesicles and they do not appear at all. In addition to our knowledge of this definitely located optic anlage in the embiyonic brain, I have now to contribute a fact of equal importance in the development of the eye which may be stated thus. When the optic vesicle does not grow out from the 234 CHARLES R. STOCKARD brain at a definite developmental moment, it is subsequently unable to grow out and develop normally or it may be unable ta grow out at all. I have definitely inhibited development during this period in a large number of experiments and have either suppressed or modified the development of the eye. It may be concluded that, such an organ as the eye is not only derived from a 31 Fig. 31 A series of diagrams indicating modifications in the lateral outgrowths or budding processes of the anterior region of the central nervous system. A, outlines the normal case with the optic-outgrowths shown above and followed by the first, second, and third primary ventricular outgrowths of the brain. B, shows the outline of a brain in which the optic outpushings were suppressed, but the three brain ventricles succeeded in their lateral expansion. C, indicates the opposite case in which the optic outpushings were expressed, but the three brain ventricles were suppressed. This is a narrow tubular brain with eyes developed from it. D, outlines the condition of complete suppression of all lateral outgrowths, there being neither eyes nor bulging brain ventricles. A simple tubular brain. definitely located primordia, but must also be derived during a limited moment of development. This time-limited opportunity for origin is probably due to a growth competition between organs. The eye, not attaining a maximum growth rate at its proper moment, may permit an excessive growth to commence in a neighboring part and such a growth may then further prevent the initial growth of the eye. STRUCTURE AND DEVELOPMENTAL RATE 235 There is also a possible chemical interpretation of the limited moment. The great activity and high oxidation rate of a given group of cells might result from the formation of certain specific compounds of a highly labile nature within these cells. Should the available oxygen be insufficient or the temperature be too low at the moment of origin of such molecules, they would be unable to produce the usual cellular activity, and on account of their labile nature would soon become changed. The opportunity for unusual growth activity of the specific kind on the part of the given cellular group would be lost. No doubt some such peculiar chemical process must be taking place during the different stages of cellular growth and differentiation in a complex vertebrate embryo. When such labile compounds do break down we may also imagine that a more generalized chemical condition of the cell is produced. And such cells may subsequently take part in the formation of the more general tissues and may not necessarily be lost on account of not having succeeded in giving rise to the specific tissue intended. Certainly, one does not find necrotic and disintegrating cells in all brains of anophthalmic embryos. Ralph Lillie ('17) has described structures simulating organic growths arising from electrolytic local action in metals. He also shows the formation of filaments from one metal to be inhibited by contact with another metal. The inhibitory influence of zinc upon the formation of ferricyanide filaments from iron may be shown as follows: a straight piece of thin bright iron wire some centimeters long, one end of which is w^ound with a small strip of zinc, is placed in a 2 per cent Kr-FeCye solution in dilute egg-white. Filaments put forth rapidly from the zinc, especially near the iron, but the iron itself remains perfectly bright and bare, and may show no development of filaments for hours. If then the wire be cut in two by scissors, the part remaining in connection with the zinc remains unchanged, while the isolated part quickly develops the characteristic blue-green filamentous growth of ferrous ferricyanide. Evidently this growth had previously been repressed by the influence of the zinc . . . ." Or when the zinc becomes completely covered by a growth of zinc ferricyanide the growth of ferrous ferricyanide will begin. 236 CHARLES R. STOCKARD Such reactions resemble in general the inhibiting effects of one growing bud or organ over the growth of other buds in the plant or organs in the embrj'o. The consideration up to this point has been limited to the developing nervous system and its organs. Does a similar relation of linear and lateral growths and evidence of a similar competition among organ buds exist in other systems of the embryo? And, further, is there any evidence of a wider competition between the different systems of the embryo? The development of the foregut from which is derived a large portion of the alimentary tract in the vertebrate embryo is closely similar in many ways to that outlined above for the nervous system. The initial anteriorly directed conical evagination of the entodemi first undergoes a linear development or growth, simply becoming longer. When a certain length has been attained by this early tubular foregut, here again lateral outgrowths begin to appear, and a series of them is formed in order from the anterior end backward in much the same way as the early neural tube gives off the optic vesicles followed by the three primary brain ventricles. The first and largest of the early foregut outgrowths is the pair of mandibular pouches, in association with which the mandibular arches arise to form the lower jaw. This pair of outgrowths is soon followed by the hyoid pair and this by the series of branchial pouches associated in later development with the several gill arches. An outline scheme of these growths is simply represented by the three accompanying diagrams in figure 32. The further development of the alimentaiy canal also shows in a very definite way a continuation of this process of lateral outgrowths or buds to give rise to other organs. The lungs in higher animals bud away from the floor of the entodermal canal immediately behind the branchial pouches. And again in the branchial region the thyroid and other glands arise by a definite budding process from the epithelial wall. The development of the stomach itself is due to an excessive proliferation or diffuse budding in a limited region, giving finally the local sacculation in the otherwise narrow tube. Fol STRUCTURE AND DEVELOPMENTAL RATE 237 lowing closely behind the stomach, the canal buds off its most striking secondary growth. This begins as an evagination following rapid cell multiplication, the excessive growth becomes too great to be longer retained by the wall and the liver pushes out, always maintaining the original connection through the bile-duct, its old stalk. This large liver bud generally contains some cells Fig. 32 A series of outlines indicating the primary linear, or cephalad, growth of the foregut, and the subsequent lateral branches or outgrowths from it. A, outlines the simple forward growths of entoderm to form the foregut. B, lateral outgrowths have begun from the forward end to form the mandibular pouch. C, a series of lateral branches, following the mandibular, now grow out to form the hyoid and branchial pouches. >l> not exactly of its own kind, and these later begin to increase and again bud away from the wall of the bile-duct as the ventral pancreas. Other cells of a similar kind are left in the wall of the tube, and these now grow^ out as the dorsal pancreas. This is the behavior of the pancreas in higher forms, while in lower animals it may arise from more than two separate buds or may fail entirely to grow away from the tube, and remain as scattered masses of 238 CHARLES R. STOCKARD cells in the gut wall of this locality. The pancreas in different vertebrate groups illustrates the phylogenetic steps in the development of a budding outgrowth from the wall of a linear canal. The entire alimentary tract in the lowest vertebrates was no doubt originally a simple tube and the lateral outgrowths or buds are the highly specialized organs that have become so excessively developed as to necessitate their separation from the tube. Thus in phylogeny as well as ontogeny of the vertebrate gut it would look as though the primary growth was linear and its complexity has been added by lateral buds and offshoots. The above being the general state of affairs in the development of the foregut, we come now to the point of experimental importance in the dynamics of these organ-forming processes. And that is, that each of the organs derived from the entodermic wall is in its critical or sensitive stage at the moment of its outgrowth, or at the time of the excessive cell proUferation in its region of the wall. Should any condition be introduced which would lower the general developmental rate, that organ will be most affected which happens at the time of the arrest to be in or nearest its critical moment. Thus an arrest during very early development will inhibit the growth of the mandibular pouch and through its malformation distort the formation of the mandibular arch, causing deformed and strangely developed mouths (see figures of the deformed fish). Since the hyoid and other branchial pouches arise so nearly at the same time as the mandibular pouch, they, with later accompanying structures, are likewise almost invariably deformed along with deformities of the mandibular structures. Such deformities as these may, however, exist in individuals with perfectly normal stomachs, livers, etc. In these cases, a normal or fair rate of development had again been established before the critical moment in the origin of the latter organs had arrived. It would thus seem possible that an experimenter might inhibit at will the rate of development during particular intervals and thereby succeed in suppressing and deforming the mouth and branchial structures and leave the more caudally situated organs uninjured. Or, reverse the experiment and obtain normal mouth and gill structures in an embryo with suppressed and underde STRUCTURE AND DEVELOPMENTAL RATE 239 veloped liver and pancreas. I have repeatedly succeeded in performing the first experiment with Fundulus embryos by early arrests. The second experiment is more difficult and is not yet completely perfected, though among a large number of cases certain specimens arise in the experiment with underdeveloped livers, but normal mouth and branchial regions. The experiments with the alimentary organs are more difficult than those on the eyes and brain, since the former are more difficult to observe and are not all so decidedly expressed in the young embryo. The study of the liver and pancreas must also be largely limited to examination of microscopic sections, while the mouth and branchial arrangements and the eyes and brain are very readily examined in total specimens after some experience. The experiments on the nervous and alimentary systems as they now stand make very probable the correctness of the following proposition. The organs arise as a series of buds which bear a relationship to one another very similar to that existing among the buds of a growing plant. A given bud is dominant or has an 'advantage of position' for a limited time during which its rate of oxidation and cell proliferation is higher than that of other potential buds in the system. It grows at this moment and continues to dominate the situation until it has exhausted the advantage, when its proliferation rate decreases and another region attains the advantage and begins to bud to form another organ. If the entire embryo be depressed or has its developmental rate reduced at a moment when a certain bud is proliferating at its height, this bud is more decidedly reduced in its rate than any other portion of the embryo. On resuming a more rapid rate the other slow-going parts are able again to attain their ordinary rates, but the bud in question is unable to regain its extraordinarilj'high rate and therefore loses its exceptional advantage. This bud may be subsequently unable to express itself, since other parts now arrive at the stage of advantage. The problem is then to locate the given critical moments for the several developing organs. By depressing development during a period which would cover a definite moment one might be able to suppress any given organ at will. With sufficiently re 240 CHARLES R. STOCK ARD fined technique we could get not only embryos otherwise normal, but without ej^es, without normal brain hemispheres, without normal mouth and branchial structures, and without ears, as can now be done, but also simply without a liver, without a pancreas, etc. In many of the arrested embryos it would seem highly probable that the total number of red blood-cells in the yolk-sac and body was greatly reduced. The red cells may be considered as a diffuse organ, and this organ seems at times reduced in size as a result of arrest. It cannot be positively stated that the entire embryo in such cases is not proportionately reduced. Thus the probability of having specifically arrested the early blood formation is still questionable. The development of one other organ, however, may be frequently interfered with by arresting the rate of development of Fundulus embryos for a time immediately following the early embryonic-shield formation. Specimens so arrested by low temperatures, treatment with alcohol solutions (Stockard, '10), or by reduced oxygen supply, often show various abnormal conditions in the development of the otic vesicle. The vesicle on one side may be absent and the other normal or poorly formed. Or the semicircular canals may not arise and only the ampullae or small cysts may represent the entire ear. In such cases, as I pointed out and illustrated in 1910, the cartilaginous capsule representing the hard parts of the inner ear forms immediately around and exactly fits the defective membranous arrangement. The cartilage development would seem to be regulated by the membranous portion of the ear. The details of these experiments may be more fully presented when a larger number of these critical moments in organ origin are more exactlj^ located by a further refinement of the experimental method. It is most difficult to apply a treatment that is not permanently injurious in such a way as to have the rate of development very low at a given brief interval of time and again restored to the normal rate shortly following. The crudeness of the experiment necessitates bringing on the arrest some time before the particu STRUCTURE AISD DEVELOPMENTAL RATE 241 lar moment desired and it later continues until further critical stages are interfered with. In this way, as a rule, we obtain specimens with several regions or organs deformed and rarely secure a specimen simply defective in respect to the state of a single organ or part. In plants the conditions are far more simple, and it is possible to suppress or bring out a given bud at will. In spite of the fact that we may not understand exactly how it is accomplished, it is definitely the growth of one bud in a plant that prevents the growth of another particular one. Similarly in the embryo probably the growth of a given organ holds back the initial growth of another organ until the first organ has exhausted its power of suppression. The two components of a double individual interact on one another in a way which would strongly support the foregoing interpretations. When the components arise in positions of equal advantage on the germ ring their interaction is balanced and both develop normally and are equal in size. When one component possesses an advantage over the other, its growth tends constantly to suppress the growth and development of its fellow, and the inferior component is, therefore, deformed and arrested in its development. When a growing shoot of a plant, such as the common privet, has finally exhausted itself, the terminal bud goes into a dormant or resting stage and stands only a little above the axillary buds of the two uppermost leaves. After a certain interval of rest the sh6ot may again begin to grow, and then one of several possibilities may occur. In the first place, the terminal bud generally possesses an advantage or occupies a more advantageous growth position. It again shoots up continuing the line of the original shoot. Its advantage is so complete that the uppermost axillary buds are unable to express their growth potential and remain dormant (fig. 1, plate 5). In the second place, the terminal bud may again shoot up, but its growth is not so pronounced and it fails to completely suppress the two uppermost axillary buds. One of these being in some way more favorably located than the other, also begins to grow a shoot in a direction at an angle to THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 ' 242 CHARLES R. STOCKARD that of the shoot from the terminal bud. The other top axillary bud, however, was not so fortunate as its fellow and was not capable of overcoming the inhibiting influence of the terminal bud and so remained dormant, as is shown in figure 2, plate 5. Finall3\ the third possibility very rarely occurs and all three of the uppermost buds are able to grow. In this case both of the axillary buds had a potential or tendency of growth sufficiently strong to successfully compete with the inhibiting influence of the terminal bud (fig. 3, plate 5). We may imagine that the growth of the terminal bud in the second and third cases was not normally vigorous. For some reason the advantages of the two or three potential buds were equalized and we find twin and triplet shoots growing out. Similarly, we may imagine the potential buds around the germring of the fish's egg to vary in their degrees of supremacy, ordinarily only one grows and suppresses the growth tendency of all other potential points. But if the developmental rate be slow, the one bud fails to suppress all other points on the blastoderm, and twin or triplet buds may become capable of expressing themselves. In conclusion, these experiments and observations make it seem highly probable that influences similar to those acting between a growing plant bud and its resting buds, or between the stronger component in a double vertebrate embryo and the smaller component, are also acting between a rapidly growing organ bud and other potential organ buds in the embryonic individual. Such a conception has the decided advantage of being of practical scientific value. Since on this basis the experimenter has a logical working scheme for a study of the abnormal and through this, the normal development of a given organ. Such a method in an analysis of the development of the eye, for example, has been most valuable. Summarizing the present status, I have succeeded in locating more or less definitely the critical moment of origin in the following individual developmental processes : 1. The growth of the primary embryonic axis : If this be slowed by arresting early cleavage stages or pregastrulation stages, the STRUCTURE AND DEVELOPMENTAL RATE 243 usual single axis does not arise with sufficient influence to suppress the origin of other axes, and twins or double individuals result. Twins and double monsters are, therefore, types of developmental arrests. 2. The suppression of the eyes or modification of their structure : When an arrest is induced later than the above, but before the origin of the embryonic shield in the Fundulus embryo, all known modifications in the structure of the eyes may result in otherwise normal individuals or in further deformed specimens. 3. Suppression of the primary brain ventricles inducing subsequently deformed or tubular brains: Arrests induced before and just about the time of first appearance of the embryonic shield result in malformation of the embryonic brain ventricles. These do not express their usual bilateral outgrowths and are frequently of a simple tubular outline. The periods of arrest necessary to induce the eye and the brain modifications are so close together or so nearly the same, that one generally finds combinations and mixtures of the defects among the same experimental group of embryos. Arrests at the earlier moment give a majority of eye conditions, many without brain involvements, while arrests at the slightly later stage give a majority of brain modifications, a few with fairly well-formed eyes. The individual variations in developmental moments among the embryos of a group also tend to contaminate the results and give mixtures of the two classes of deformities. 4. Modified or contorted mouth and branchial systems: Arrests during the embryonic shield stage, and earlier, frequently cause deformities of the mouth and branchial regions of the Fundulus embryo. In a few cases these deformities have existed in individuals otherwise normal. They, therefore, must possess a critical moment occurring at a time more or less distinct from that of the other organs. The close association of mouth abnormalities with those of the eyes is probably not due to identical critical moments of origin in the two cases, but more likely to the fact that when a slow rate of development exists during the eye moment it is rarely completely overcome and the normal rate reestablished before the critical moment for the bilateral outgrowths of the mandibular pouch is reached. 244 CHARLES R. STOCKARD There is also probably some overlapping between the critical moments of origin for the ectodermic organs, for example, those of the central nervous system and the entodermic organs of the alimentary canal. There is, further, the possibility that the interaction or inhibiting influence existing among the buds of one system may not extend to the organ buds of a different system. I believe, however, that this is not the case, and it is more probable that the growth of each organ affects to a degree the growth of all other parts in the entire embryo. The degrees of effect exerted by one growing organ over the others may differ, for example, the growth of the optic vesicles very probably affects the primary brain ventricle growth more strongly than it does the growth of the branchial organs, etc. 5. Modifications in size and structural outline of the inner ear: Arrests during a closely similar stage to that in case 4 sometimes show their effectiveness on a different bud. In such cases the ear as well as the mouth and gills becomes affected. The otic vesicle may be completely suppressed or develop into a simple tiny cyst with no outgrowth for the semicircular canals. 6. Faulty development of the liver and pancreas : Later arrests, after the embryonic line is distinctly seen, in the Fundulus embryo may cause an abnormally small outgrowth representing the liver. Such conditions make it appear as though the primary liver bud had been inhibited in its outgrowth from the intestinal wall or the later rate of multiplication of liver cells had been reduced. The pancreas evidently arises and has its critical moment at a somewhat later instant than does the liver. Yet the two moments are so close together that it would require a very deHcate difference in time of arrest to affect the one and not the other. The findings in these six attacks on the problem make evident this very important fact: That each organ arises at a definite moment during embryonic development and not during widely different moments just as truly as that an individual organ arises from a very definite embryonic area or anlagen and from no other. The organ defects found in nature further confirm the results of experiments on the Fundulus embryos. It is well known from STRUCTURE AND DEVELOPMENTAL RATE 245 the studies by Mall ('17) and many others that localized anomalies are quite frequent in both normal and pathological human specimens. The localized anomaly may involve only the eye, only the bilaterality of the brain, only the ear, only the mouth structures, only the kidneys (I have dissected two fetuses at term neither of which possessed any evidence of a kidney, but one of which was otherwise structurally normal), only the genitalia, etc. It is evident that such anomalies could not occur unless there was a certain moment of specific and peculiar susceptibility on the part of each organ during which any unfavorable condition would act on it in a selective way. Of course, the specific action of certain substances on certain embryonic buds would give a possible explanation, but there is so much strong positive evidence in the present study as well as from other sources against this once attractive possibility that it can well be discarded. A strong point of evidence is the fact that a typical defect of one organ can be induced by a great number of treatments, and different defects of many different organs can all be induced by one and the same treatment. In every case the result depends upon the developmental time during which the treatment acts and not upon chemical or physical properties of the substance used. As repeatedly shown, the primary effect in all instances is simply a slowing in the rate of development. c. Developmental rate and postnatal changes The relations between the rate of growth and particular developmental moments found in the embryo probably continue to be of importance during postnatal development as well. The numerous studies on inanition in rats and other mammals bear on the same general problem as that considered in the present report. The one important effect that inanition might have on the subsequent history of a developing individual would be the malformation and arrest in development of certain tissues or parts. After birth a number of important organs and tissues still have a considerable degree of growth and differentiation to accomplish, and very probably the same rules apply to this activ 246 CHARLES R. STOCKARD ity during the postnatal period as are found to apply in the embryo. The important question at once arises as to whether there are periods during which starvation might produce no subsequent ill effects, alternating with more or less critical periods during which a similar treatment might be followed by considerably more serious results. For example, if a given treatment be administered to one group of animals from the second to the fifth week after birth, and to a similar group from the fourth to the seventh week, the results might be the same or they might be very different. The results would depend very largely upon whether significant tissue changes susceptible to variations in developmental rates had occurred during the time intervening between the two experunents. The interaction or competition among the growing and developing organs found in the embryo certainly continues during postnatal development. The suppression of development in certain organs and tissues by the activity of another organ is splendidly illustrated by the glands of internal secretion. The further development of certain secondary sexual characters, such as hair and plumage, after subdued activity of the gonads is a case in point. The difference in importance between two developmental moments in the postnatal individual is, however, of far less significance than in the early embryo, just as arrests during early cleavage stages are of more far-reaching consequence than similar arrests after gastrulation has occurred . Differences in developmental rate during postnatal periods incline to affect the finer features or the type of the individual rather than cause actual malformation or pathological deficiencies in the tissues. Such effects are readily observed in many of the arrested, status, and infantile human types. A fuller conception of the significance of developmental rate and rhythm in the determination of human types and appearances will be given in a separate communication. STRUCTURE AND DEVELOPMENTAL RATE 247 9. CONTINUITY OF THE SERIES FROM MONSTRA IN DEFECTU THROUGH THE SINGLE NORMAL INDIVIDUALS TO MONSTRA IN EXCESSU AND FINALLY IDENTICAL TWINS It has long been recognized that certain types of monsters exhibit their characteristic defect to varying degrees. The Cyclopean series, for example, may present individuals not only with a single median eye, but with a bilaterally wide eye, hour-glass eyes, and finally closely approximated separate eyes. The series of diplopagi likewise exhibit all degrees of doubleness, as illustrated in plates 1 to 4. In studying monsters belonging to these groups. Wilder ('08) went a step further and called attention to the fact that the socalled series of defective monsters passed by degrees up to the normal individual and continued from there through the excessive series on to identical twins. He was impressed by the 'orderly development' of the members in such a series and termed these individuals 'Cosmobia.' The treatment of the series as variations about the normal as a standard was a most important advance in an analysis of their structural conditions. Wilder further emphasized the important fact that monstra in defectu and monstra in excessu are both due to the same kind of cause and should be considered together in any general treatment of the subject, especially concerning cause. However, after enunciating this clear arrangement of the problem. Wilder was entirely misled in his interpretation of the cause of these individual anomaUes. The fact of their 'orderly' and symmetrical structure, and the further evident fact that normally formed identical twins represent the termination of the diplopage series, led him to consider all such forms as due to a definite germinal variation. It seemed to him more probable that orderly deviations from the normal would arise in the germ-plasm than that they should occur as a result of some modification during individual development. The burden of evidence, however, is unfortunately against such a proposition, and weighs decidedly more at the present moment than when Wilder published his account. 248 CHAJILES R. STOCKARD From what we know of germinal variations and mutations, they do not necessarily give rise to individuals that gradually grade away from the usual type. There may be wide structural breaks between the parent stock and the mutant. On the other hand, we now know that unusual environmental conditions tend to modify the normal course of structural development to varying degrees and give rise to the exact series of defects on which Wilder's conceptions were based. The present contribution clearly demonstrates the underlying factors and the very probable cause of this orderly series of beings deviating from the normal individual as monstra in defectu and monstra in excessu. The idea is entirely correct that double monsters and twins are due to the same cause as cyclopia. And both may be experimentally produced by an identical physical change in the environment, lowering the temperature. Both conditions also result from a slowing of developmental rate, but one differs from the other because of the difference in the developmental periods during which the slowing in rate was effective. 10. THE NECESSITY OF A CONTROLLED OR REGULATED ENVIRONMENT IN WHICH TO DEVELOP HIGHLY COMPLEX INDIVIDUALS From the foregoing considerations it has become evident that normal development of the vertebrate embryo depends acutely upon the stability of certain factors in the environment. Changes in the conditions of moisture, temperature, or oxygen supply are the most frequent causes of embryonic death as well as monstrous development. Any degree of actual dryness is fatal to the vertebrate embryo, and sudden lowering of the surrounding temperature and reductions of the oxygen supply interrupt development with the significant consequences discussed above. A normal amount of ordinary food materials is not, however, so acutely necessary for perfect structural expression. The rate of development under malnutrition is slow, but the depression does not come on suddenly nor is it often sufficiently complete to cause serious structural anomalies. Vertebrate animals are faced with the problem of the necessity of a regulated environment in which to develop their eggs into STRUCTURE AND DEVELOPMENTAL RATE 249 the free living individual. The lower vertebrates are almost entirely aquatic and their eggs undergo only a short embryonic development before reaching the swimming larval stage. The birds and mammals, however, at the moment of birth or hatching have, as a rule, attained a complexity of structure greater than that of the adult stage in fishes and lower forms. The period of their prenatal development is extremely long, offering far greater opportunity in time for changes in the environment and, therefore, necessitating some means of control on the part of the parent generation. The marine and fresh-water fishes live in a more or less homogeneous medium which rarely undergoes sudden or marked changes during the spawning seasons. Their eggs are deposited in the water in instinctively chosen places during definite times when the conditions of oxygen and temperature are generally favorable for the given species. This developmental environment may in unusual cases fail in one or all respects. The water may become so stagnant as not to supply oxygen, or it may suddenly become either too hot or too cold for the welfare of the developing eggs, or in a dry season it may become evaporated or carried off, allowing the eggs to dry. The instinct of the fish helps to guard against such accidents, and the eggs are deposited at a season when the temperature changes are least likely to be harmful, and localities are chosen where the water is properly supplied with oxygen and is sufficient in amount to escape rapid drying. The higher land-living vertebrates have no such surroundings in which to develop their eggs. In becoming terrestrial, these animals must have evolved not only appendages for locomotion on land, but also some means of controlling or providing an environment in which their long embryonic developm'^.nt could take place. The eggs of reptiles and birds, as is well known, are provided with comparatively enormous amounts of food-yolk surrounded by layers of other food and enclosed in protective membranes and shell. These arrangements not only supply food, but insure a moist environment essential to all development and permit free 250 CHARLES R. STOCKARD access of oxygen from the surrounding air. The one element essential for development of these eggs, not yet provided, is a constant high temperature. The reptiles are largely confined to warm regions and deposit their eggs during the hottest periods of the year in sand or other heated places, and in this way the proper temperature is usually provided. The birds, however, with the extremely high temperature of their own bodies, supply in a more definite way the proper amount of heat for the incubation of their embryos. Lack of moisture and oxygen very rarely causes the death or abnormal development of the eggs of reptiles and birds. But failure to maintain a uniform temperature and unfavorable degrees of heat and cold are the chief causes for embryonic mortality and deformity in these animals. The mammals have advanced a step further in perfecting a controlled developmental environment. The internal development of the embryo not only insures a properly moist condition, but the high temperature of the maternal body is sufficiently uniform never to cause interruption of the normal progress of development. The supply of oxygen is derived from the blood of the mother through the placental circulation, and this is the one element in the mammalian developmental environment which most frequently becomes deranged. Faulty placentation cuts down the supply of oxygen to the mammalian embryo and lowers its rate of development, producing as a result prenatal death and all varieties of malformation. Yet we may well believe that the long and highly complex development of the mammalian embryo could not take place unless it was protected by a fairly well regulated environment. Abnormal development in the embryos of birds may very rarely result in nature from poor ventilation on account of a coated egg shell, but more frequently it results from failure to maintain a uniform temperature. While in mammals the temperature changes are eliminated by the internal mode of development, the one great danger to normal development still not completely controlled is the chance of a low oxygen supply brought about by a delayed or poor implantation of the placenta. The great majority of monsters in mammals are very probably due to an insufficient oxygen supply during development, and this results as a rule from faulty placentation. STRUCTURE AND DEVELOPMENTAL RATE 251 The ready manner in which the structures of the developing individual are modified by changes in temperature and oxygen supply makes it evident that the existence of the species often depends upon some means of regulating the developmental environment. We may readily believe that species have been lost during evolution not only on account of failure of their adult structures to fit them for existence, but equally often as a result of failure to obtain an environment in which their embryonic development was possible. No developmental environment in nature is constantly perfect, and this fact is the underlying cause of the frequently occurring malformations and monstrous productions. 11. GROWTH COMPETITION BETWEEN THE TWO COMPONENTS IN DOUBLE INDIVIDUALS AND THE TIME OF OCCURRENCE OF TERATOMA IN MAN It has been clearly seen that in cases where one component of a double individual is larger because of a more favorable location^ the smaller has been inhibited in its growth and development by the presence of the larger. In plants this inhibiting influence is readily demonstrated, since on pinching away a growing shoot the suppressed buds immediately spring into growth. There is much evidence to indicate that a similar interaction exists between two developing organs in a single individual. The alternating moments of rapid growth among the several organs of the embryo is a case in point. With the preceding discussions of these propositions in view, if it be now admitted that teratoma in man often originates as a twin inclusion, we may expect an antagonistic growth reaction to exist between the teratoma and the host. In other words, while the host individual is rapidly growing, the teratoma will be suppressed and when the rate of growth of the host individual becomes slow, the teratoma will tend to grow more rapidly. If such an opinion be correct, there should be a marked correlation between the postnatal growth curve and the time of enlargement or recognition of teratomata. When the individual is growing very rapidly during the first year and a half of infancy, few tera 252 CHARLES E. STOCKARD tomal enlai'gements would be expected; following this period there is a decided fall in growth rate and the teratomata of early childhood may occur. The alternating periods of fast and slow growth should then "continue to correspond with periods of few and many recorded teratomata. Dr. H. E. Himwich has undertaken a careful survey of the teratomata as recorded in the literature in order to ascertain whether any apparent relationship does exist between the time of occurrence of a teratoma and the periods of fast and slow growth rate in man. The results of his investigation are soon to be published. 12. CANCEROUS GROWTHS AND THE GENERAL CESSATION OF ALL NORMAL GROWTH IN THE OLD INDIVIDUAL In an interpretation of the cause of cancer the fact that the condition is so much more frequent in the adult and old individual than in the young is to be recognized as of deep significance. The fact that there is an interaction and especially a growthinhibiting effect exerted among proliferating tissues in the individual is a second point of great importance. In the young rapidly growing and developing person almost all organs and tissues are increasing in amount through multiplication of their cellular constituents. The liver, for example, grows in actual mass until it reaches the adult size. This size, although decidedly variable in a group of individuals, has rather definite limits. The normal human liver is never indefinite or unlimited in its growth. Almost all other organs are similarly of limited size. Thus growth in general tends to cease as the body approaches its adult proportions. Finally, in the old individual, the only remaining cell proliferation becomes almost entirely confined to the germinative layer of the skin, the lining epithelium of the alimentary tract, the testes in the male, and the production of red blood-corpuscles. Even these proliferation processes become feeble with increase in age and new cells are not abundantly supplied. This is the normal course of events. The size and proportion of parts are largely determined by heredity, but may be seriously interfered with by irregularities in the environment. STRUCTURE AND DEVELOPMENTAL RATE 253 A slowing of the developmental rate at particular times may largely suppress the growth of certain organs, rendering them abnormally small in size and insufficient in their function. The normal proportion of things becomes distorted. Again it may rarely happen that one organ takes on an excessive growth and attains a size entirely out of normal proportion. There is thus a frequent lack of proper balance and adjustment among the several organs of the developing body. The properly regulated balance among the organs is to a great extent due to the inhibiting and controlling effects of one growing region or part over other parts. This is readily demonstrated by the modifications which result in size and proportion of certain parts of the body following the experimental removal of other parts. All parts may be thought of as having more or less to do with the ultimate growth results of the whole. On becoming adult, a state of apparent balance is maintained. Growth is considerably reduced and largely confined to the repair of natural loss and the maintenance of this state of adult balance. Under such conditions there still remains considerable regenerative powers following injuries of various kinds. Yet these regenerative processes are not so perfectly accomplished or so well controlled in the adult animal body as they were in the larval or immature condition. This fact may in some way be associated with the absence in the adult of general growth and the wellexpressed regulatory processes which are necessary in the developing individual. The regenerative growth following injuries to the adult animal may become morbid in degree and without regulation, thus giving rise to malignant conditions. Such a growth might rarely occur in the immature body, but in this case one would expect to find the growth proportions among the tissues in general to be abnormal and distorted. Thus, juvenile cancer conditions are rare and are probably associated with other deformities. Cancer in the adult would be expected to occur more frequently in certain families, since the growth balance and proportions are hereditary characters, and on the state of these, the cancerous growth largely depends. Families or persons derived from 254 CHARLES R. STOCKARD similar cytological complexes show more nearly similar growth and tissue reactions than do random groups of individuals derived from non-related parentage. In the old individual with but little normal growth still in existence, there can be, on the basis of my interpretation, but slight inhibition to any regenerative process that might be set up. Such animals naturally on account of their old condition usually regenerate very slowly, but following continued trauma, active regenerative growths are frequently begun, and not being under the inhibiting control of any other active growth processes, this regeneration attains an excessive, distorted, and malignant condition. All very old animals no doubt experience a considerable amount of trauma, and if they lived long enough almost all of them might possess some cancerous growths. The truth of this statement is well illustrated by comparing the frequency of reported cancer in rats and mice with similar growths in guinea-pigs, all constantly used laboratory animals. Rats are very old after three years of life, and actually at two years old may properly be compared, according to Donaldson ('15), with a man at sixty. Mice attain old age even earlier, and at two years are very old. This being the case, it frequently happens that the rats and mice used in laboratories have actually become old individuals, having been kept by the breeders and the laboratory for as long as two years. Cancerous growths are common in these animals. The guinea-pig under favorable conditions does not become old until it has lived for about five years, and we have frequently kept these animals for more than seven years; at this age, however, they are extremely old. Thus, as a rule, the guinea-pigs used in laboratories are really young individuals, generally less than three or four years old. Consequently, cancerous growths are said to be uncommon among these animals. However, among the old individuals in our stock a considerable percentage of cancerous ones have occurred. So it might be inferred that if as great a number of really old guinea-pigs were observed as of old rats and mice, cancer might be found to be almost as common among guinea-pigs as among rats and mice. And finally it may STRUCTURE AND DEVELOPMENTAL RATE 255 be supposed that every mammal would develop some form of cancerous growth should it chance to live until extreme old age. The increased length of life in man may be associated with the increased frequency of cancer. 13. GENERAL SUMMARY In considering the results of the present study it is necessary to recognize the fact that a given animal species passes through its embryonic stages at a specific rate of development, probably dependent upon the rate of oxidation in the protoplasm of the species. This developmental rate varies within certain normal limits; should variations in rate extend beyond these limits, the developmental result frequently becomes modified and distorted. The rate of development is not uniform throughout the entire process, but periods of rapid progress alternate with moments of slow rate or almost quiescence. In spite of these changes in rate, development in most forms does not actually stop after it has once started, but progresses in a continuous manner until the fully formed animal is produced. There are certain animals in which the continuous mode of development has become modified. In these forms development begins and attains a definite stage and then stops completely, to remain at a standstill for days or even weeks, until a change in the environment again permits the resumption of the developmental processes and the completion of the fully formed animal. Such a discontinuous mode of development is universal among the birds and is known to occur in several mammals. With these points in mind, the results of the present study may be summarized as follows: 1 . The continuous mode of development may be experimentally changed into the discontinuous by two very simple methods, temporarily lowering the surrounding temperature and thereby reducing the rate of oxidation and by directly cutting off the supply of oxygen. The effects on subsequent development of interruptions caused by these methods depends upon the stage during which the interruption is introduced. There are stages of apparent indifference to a stop in development. Shortly after gastrulation is completed, 256 CHARLES R. STOCKARD the development of the fish's egg may be stopped for a considerable length of time with impunity, no ill-effects resulting. This is the developmental moment at which the bird's egg is normally stopped on account of the fall in temperature experienced after passing out of the mother's body. There are other stages during which a temporary interruption of the developmental processes will be followed by most disastrous effects. These critical stages are usually moments during which marked inequalities in rate of cellular proliferation are taking place in different portions of the blastoderm or embryo. The period preceding the process of gastrulation is just such a critical moment. 2. There are considerable differences in effect between greatly reducing the rate of development and actually stopping the process temporarily. The development of certain eggs may be slowed down to one-tenth or one-twentieth of the usual rate and be maintained in such a slow condition for days without the majority of specimens losing their power of regaining the normal rate and giving rise to structurally perfect individuals. If at similar stages the development of the same eggs be completely stopped instead of slowed down, they are in' many cases unable later to resume the process and die, in other cases they may resume development in a most abnormal fashion, or finally a few may be capable of resuming the apparently normal process. This difference in results between a severe reduction in developmental rate and an actual temporary stop is to be explained as follows: Slowing does not completely eliminate the normal inequalities in rate of developmental change existing among the several parts. Those parts that were in states of rapid development are depressed in the same proportion as other parts that were developing more slowly and inequalities in rate still exist in the slow-going embryo. When such specimens are allowed to resume a faster development the several portions of the embryo are able again to maintain normal differences in developmental rate and a proper balance is assured. A complete stop in development reduces the rate of all parts to zero and eliminates normal inequalities. On resuming develop STRUCTUEE AND DEVELOPMENTAL RATE 257 ment from such a state, parts that should progress at a disproportionately fast rate are unable to attain such supremacy and all portions of the embryo start at about the same rate. The usual developmental balance and inequalities in rate among the parts are lost and thus the typical form of the individual which actually depends upon these inequalities in rate of growth becomes modified. 3. The types of deformities following a stop in development as well as those occasionally resulting from a slowing of the rate are similar to the defects produced by all experimental methods. Practically any deformity recorded in the literature other than those resulting from germinal variations or mutations may be induced by lowering the temperature and thus modifying the developmental rate. 4. By an interruption of development during late cleavage stages a considerable percentage of twins and double individuals may be produced. When the eggs of the sea-minnow, Fundulus heteroclitus, are subjected to temperatures of 5° or 6°C. during cleavage stages, development is almost stopped. On returning such eggs to a summer temperature, after several days' sojourn in the refrigerator, there will follow a high mortality, but many specimens will resume development producing a significant percentage of twins and a number of variously deformed conditions along with a good proportion of normally formed young fish. Arresting or stopping development of the same eggs during the same developmental stages by diminishing the available supply of oxygen will be followed by closely similar results. The eggs of the trout are naturally much more inclined to develop into double individuals than are those of Fundulus. When the oxygen supply during early development is not abundant, a great many twin and double trout specimens are frequently found to occur. All of these double conditions result from arrests during very early stages of development, invariably before the process of blastopore formation has in any way begun. No duplicities or twins have been found to occur among the great numbers of fish eggs which have been arrested during postgastrular stages of development. 258 CHARLES R. STOCKARD 5. The bird's egg is usually laid, according to investigations on this subject, after the process of gastrulation has commenced. Yet double chick embryos are not uncommon among the developmental stages observed in the laboratory, although in nature such specimens almost never exist at the time of hatching. The cause for the double chick embiyos is the same, I believe, as that indicated above in the case of the double fish. Although the great majority of hen's eggs are laid and their development stopped by the fall in temperature after gastrulation has begun, still it is recognized by those who have investigated the subject that there is considerable variation in the developmental stages of the eggs at the time of laying, and a minority of eggs are laid before gastrulation has begim. When an egg in this stage is stopped by the fall in temperature following laying, it would be expected from the experience with the fish that just such eggs would frequently give rise to two points of gastrulation and two embryonic fundaments instead of one. The interruption in the process of development at this critical time and the resumption of development at an equally slow rate in all regions of the blastoderm, permits more than one potential embryo-forming region to express itself. The interruption at this particular moment is the very probable cause of twin and double specimens. 6. Polyembryony in the armadillo is in all probability explainable on a similar basis to the cases above. Development begins as in most other mammals in the fallopian tubes and continues until the egg passes down into the uterus as an early blastocyst. Development then stops in the armadillo for a period of several weeks with the blastocyst lying free in the uterus, as Patterson ('13) has reported. The stop here is not due to a temperature change, since none has occurred, but is very probably on account of an exhaustion of the original oxygen supply derived from the ovarian blood. The uterus fails to react immediately to the presence of the blastocyst, implantation is delayed, and no means of obtaining oxygen necessary for continuing development is possible until the egg becomes implanted. After the delayed implantation has taken place, development is slowly resumed in a way which gives rise to multiple embryo formations or budding, as STRUCTURE AND DEVELOPMENTAL RATE 259 has been fully considered above. The 'quiescent period' in the armadillo egg is probably the result of lack of oxygen and thus the cause of polyembryony. Twinning or polyembryony may be considered a typical method of asexual reproduction, and its occurrence in mammals and other vertebrates makes the phenomenon of so-called 'alternation of generations' universal among animals. 7. The degree of duplicity in double individuals depends upon the original distance apart of the embryonic buds on the blastoderm. The relative sizes of the two components in double specimens vary widely. In many double individuals the two components are practically equal, while in others one component is of normal size and the other component in a series of specimens varies from slightly below normal size down to a very small mass. This size difference between components is in no way associated with the degree of duplicity. 8. In double individuals in which the two components are equal in size they are both normal in structure. When the two components of a double specimen are unequal in size, the larger component is almost always normal in structure, and the smaller component is always deformed. The degree of deformity in the smaller component varies directly with the extent of difference in size between the two components. 9. As the large component reaches adult size the lesser component may have become so relatively small as to be represented by a nodular mass on the body of the larger, or it may be lost to sight entirely as a twin inclusion. Such conditions make it evident that.doubleness and twinning are actually more frequent than records would indicate. 10. The types of defects and the degree of deformity exhibited by the smaller component are exactly similar in kind and degree to the deformities found among single individuals. This fact renders the double individual with unequal components a most valuable key to an understanding of the cause of all monstrous development. The two components are from identical germinal origin and are developing in organic connection in exactly the 260 CHARLES R. STOCKARD same environment, yet one is structurally perfect while the other smaller member presents all types of deformities. The difference between the two is in their developmental rate, the larger having a normal rate and the smaller progressing more slowly and in an arrested fashion. The depressed state of the one component is the result of an inhibiting influence exerted by the other. 11. The deformities of the small component in the double individuals and the similar defects induced by stopping the development of single individuals make it evident that all developmental monstrosities are the results of simple arrest. During my experiments with Fundulus eggs it has been possible to induce a single type of defect with a great variety of different experimental treatments. The reverse is also true; all varieties of defects may be induced by subjecting the embryos to one and the same experimental treatment. The primary action of all the treatments is to inhibit the rate of development, and the type of deformity that residts depends simply upon the developmental moment at which the interruption occurs. All monsters are the result of the same cause, and the type of monster depends upon the time at which the cause was in operation. Several developmental moments have been located at which rather definite defects of particular organs may be induced. These are the moments during which the organs are in their most rapidly proliferating condition. Arresting the rate at such a moment gives decidedly injurious results. When an organ is developing at a slow rate the arrest fails to affect it. 12. The development and growth of organs in the single individual are interrelated in a way similar to the interrelations between the components of a double specimen. When one organ or one component has a higher rate than another, it develops at this rate for a limited time and tends to inhibit development on the part of other organs. This is readily demonstrated by the inhibiting effect of the growing shoot over all the potential buds of a plant. When the growing tip is pinched away, the inhibited buds immediately express their capacity to grow. There is much evidence to indicate that a similar interaction exists among the developing parts of an animal embryo. STEUCTURE AND DEVELOPMENTAL RATE 261 13. The initial growth giving origin to an embryonic system, such as the brain and spinal cord, is linear in type, until a definite length is attained when linear growth subsides. This is followed by a series of lateral outgrowths in consecutive fashion. These lateral outgrowths from the central nervous system may be experimentally suppressed by slowing development at definite times, and when all are absent a simple tubular brain is the end result. The same plan of development holds for the foregut and its lateral outgrowths to form the mandibular pouch, etc., and the development of this system may also be modified in a manner similar to that mentioned for the brain. 14. Monstra in defectu and rnonstra in excessu, which have frequently been treated as such distinctly different classes of conditions, are as a matter of fact closely similar. Both classes of anomalies are due to a common cause and may actually both exist in the same specunen. For example, an arrest of development before gastrulation may cause a blastoderm to form two embryonic processes which later develop into a double-headed individual — a typical monstrum in excessu. At a very early stage one of these embryonic processes may become inhibited and later form a cyclopean eye instead of the usual two lateral eyes; this head is then a typical case of monstrum in defectu. The fact that the normal individual stands between these two arbitrary classes of monsters has no other significance than that the monsters themselves are simply modifications of the normal condition resulting from an unusual reduction in the rate of development during certain critical periods. 15. The great importance of developmental rate in influencing the type and quality of structure is not confined solely to embryonic development, but postnatal development, and structures are similarly influenced by the rate at which the processes are accomplished. This phase of the subject is to be presented in a subsequent communication. 16. In view of experimental results, it becomes evident that normal development of the vertebrate embryo depends acutely upon the stability of certain factors in the environment. Changes in the conditions of moisture, temperature, and oxygen supply 262 CHAKLES R. STOCKARD are the most frequent causes of embryonic death as well as monstrous development. The existence of the species may frequently depend upon some means of regulating the developmental environment. Species may be lost during evolution not only on account of failure of their adult structures to fit them for existence, but equally as a result of failure to obtain an environment in which their embryonic development is possible. The highly complex forms, such as birds and mammals, with a long embryonic period have partially succeeded in controlling their developmental environment. But in no case is the regulation constantly perfect and this fact is the underlying cause of frequent malformations and monstrous productions. 17. The double fish specimens with unequal components and the growth reactions between these components are important in connection with certain teratomal conditions in man. If teratoma in man frequently originates as a twin inclusion, we may expect an antagonistic growth reaction to exist between the teratoma and the host. While the host individual is rapidly growing the teratoma will be suppressed and when the host slows its growth the teratoma should tend to grow more rapidly. There should thus be a correlation between the postnatal growth curve and the time of enlargement or recognition of teratomata. Dr. H. E. Himwich has undertaken a survey of this subject which will soon be published. 18. The interaction between the growing organs of a developing individual has been discussed in its relation to regeneration and cancerous growths of old age. In the old individual with but little normal growth still present there can be but slight inhibition to any regenerative process that may be set up following a continued trauma. • STRUCTURE AND DEVELOPMENTAL RATE 263 LITERATURE CITED Alsop, F. M. 1919 Abnormal temperatures on chick embryos. Anat. Rec, vol. 15, no. 6, p. 307. Arxold, L. 1912 Adult human ovaries with follicles containing several oocytes. Anat. Rec, vol. 6, p. 413. AssHETON, R. 1908 A blastodermic vesicle of the sheep of the seventh day, with twin germinal areas. Jour. Anat., 32. liiscHOFF, T. L. W. 1854 Entwicklungsgeschichte des Rehes. Giessen. Child, C. M. 1915 Individuality in organisms. Univ. of Chicago Press. 1916 Experimental control and modification of larval development in the sea-urchin in relation to the axial gradients. Jour. Morph., vol. 28, no. 1. CoN'KLix, E. G. 1905 Mosaic development in ascidian eggs. Jour. Exp. Zool., vol. 2. 1906 Does half an ascidian egg give rise to a whole larva? Roux's Arch., vol. 21, p. 725. 1912 Experimental studies on nuclear and cell division in the eggs of Crepidula. Jour. Acad. Nat. Sci., Philadelphia, ser. 2, vol. 15. Dare.ste, C. 1S91 Mode de formation de la Cyclopie. Annales d'Oculist, vol. 106. 1891 Recherches ■ sur la production artificielle des monstruosites. 2d edition, Paris. Davenport, C. B. 1920 Heredity of twin births. Proc. Soc. Exp. Biol, and Med., vol. 17. Driesch, H. 1892 Entwicklungsmechanische Studien: 1, 11. Zeit. wiss. Zool., Bd. 53, S. 160. 1895 Von der Entwickelung einzelner Ascidienblastomeren. Arch. Entw.-Mech., vol. 1. Donaldson, H. H. 1915 The rat. Memoir, Wistar Institute. Ferret, P., et Weber 1904 Malformations du systeme nerveux central de I'embryon de poulct obtenues expcrimentalement. C. R. Soc. de Biol., vol. 56. Gemmill, J. F. 19^!1 The anatomy of symmetrical double monstrosities in the trout. Proc. Roy. Soc. London, vol. 68, no. 444. 1912 The teratology of fishes. Glasgow (James Maclehose & Sons). Harper, E. H. 1904 The fertilization and early development of the pigeon's egg. Am. Jour. Anat., vol. 3, p. 349. Herlitzka, a. 1897 Sullo sviluppo di embrioni completi da blastomeri isolati di uova de tritone. Arch. Entw.-mech., Bd. 4, S. 624. Hertwig, O. 1892 Urmund und Spina Bifida. Arch. f. mikrosk. Anat., vol. 39. 1895 Beitrage zur experimentellen morphologic und entwicklungsgeschichte. Arch. f. mikrosk. Anat., Bd. 44. 1898 Ueber den Einfluss der Temperatur auf die Entwicklung von Rana fusca and Rana esculenta. Arch. f. mikrosk. x\nat., vol. 51. 1911 Die Radiumkrankheit tierischer Keimzellen. Ein Beitrag zur experimentellen Zeugungs- und Vererbungslehre. Arch. f. mikr. Anat., vol. 77. 264 CHARLES R. STOCKARD Kaestner, S. 1898 Doppelbildungen dei Wirbelthieren. Ein Beitrag zur Casuistik. Arch. f. Anat. u. Physiol., S. 81. 1899 Neuer Beitrag zur Casuistik der Doppelbildungen bei Hiihnerembryonen. Arch. f. Anat. u. Physiol., S. 28. 1907 Doppelbildungen an Vogelkeimscheiben. Fiinfte Mitteilung. Arch. f. Anat. u. Phys. Kellicott, Wm. E. 1916 The effects of low temperature upon the development of Fundulus. Am. Jour. Anat., vol. 20, p. 449. King, H. D. 1903 The influence of temperature on the development of the toad's egg. Biol. Bull., vol. 5. KopscH, Fr. 1895 Ueber eine Doppel-Gastrula bei Lacerta agilis. Kgl. preuss. Akad. Wissensch. 1899 Die Organisation der Hemididymi und Anadidymi der Knochenfische und ihre Bedeutung fiir die Theorien tiber Bildung und Wachstum des Knochenfischembryos. Internat. Monatsschr. f. Anat. u. Phys., Bd. 16. Leplat, G. 1914 Localisation des premieres Ebauches oculaires. Pathog6nie de la Cyclopie. Anat. Anz., Bd. 46. 1919 Action du milieu sur le developpement des larves d'amphibiens. Localisation et difTerenciation des premieres ebauches oculaires chez les vert6br6s. Cyclopie et Anophtalmie. Arch, de Biol., T. 30, p. 231. Leuckart and Schrohe 1891 Die Organisation der Hemididymi und Anadidymi der Knochenfische. Internat. Monatsch. f. Anat. u. Physiol., Bd. 16. Lewis, W. H. 1904 Experimental studies on the development of the eye in Amphibia. Am. Jour. Anat., vol. 3. 1909 The experimental production of cyclopia in the fish embryo (Fundulus heteroclitus). Anat. Rec, vol. 3, p. 175. LiLLiE, Ralph S. 1917 The formation of structures resembling organic growths bj' means of electrolytic local action in metals, and the general physiological significance and control of this type of action. Biol. Bull., vol. 33, p. 135. LoEB, J. 1895 Beitrage zur Entwickelungsmechanik der aus einem Ei entstehenden Doppelbildungen. Arch. Entw.-Mech., Bd. 1. 1915 The blindness of the cave fauna and the artificial production of blind fish embryos by heterogeneous hybridization and by low temperatures. Biol. Bull., vol. 29. 1916 Further experiments on correlation of growth in Bryophyllum calycinum. Botanical Gaz., vol. 62, p. 293. 1918 Chemical basis of correlation. I. Production of equal masses of sister leaves in Bryophyllum calycinum. Botanical Gaz., vol. 65, p. 150. 1918 The law controlling the quantity and the rate of regeneration. Proc. Nat. Acad. Sci., vol. 4, p. 117. 1919 The physiological basis of morphological polarity in regenerations. II. Jour, of Gen. Phys., vol. 1, p. 687. STRUCTURE AND DEVELOPMENTAL RATE 265 Mall, F. P. 1908 A study of the causes underlying the origin of human monsters. Jour. Morph., vol. 19. 1917 Cyclopia in the human embryo. Publication 226 of Carnegie Institution of Washington, p. 5. 1917 On the frequency of localized anomalies in human embryos and infants at birth. Am. Jour. Anat., vol. 22, p. 49. MiTROPHANOw, P. 1895 Teratogenetische Studien. Arch. Entw.-Mech., vol. 1. Morgan, T. H. 1893 E.xperimental studies on teleost eggs. Anat. Anz., Bd. 8, S. 803. 1895 Half-embryos and whole-embryos from one of the first two blastomeres of the frog's eggs. Anat. Anz., Bd. 10, S. 623. Morrill, C. V. 1919 Symmetry reversal and mirror imaging in monstrous trout and a comparison with similar conditions in human double monsters. Anat. Rec, vol. 16, no. 4, p. 265. Newman, H. H. 1917 The biology of twins. Univ. of Chicago Press. 1917 On the production of monsters b}^ hybridization. Biol. Bull., vol. 32, p. 306. Newman, H. H., and Patterson, J. T. 1910 Development of the nine-banded armadillo from the primitive-streak stage to birth; with special reference to the question of specific polyembryony. Jour. Morph., vol. 21, p. 359. Oppermann, K. 1913 Die Entwicklung von Forelleneiern nach Befruchtung mit radiumbestrahlten Samenfaden. Arch. f. mikr. Anat., Bd. 83. Patterson, J. T. 1907 On gastrulation and the origin of the primitive streak in the pigeon's egg. Preliminary notice. Biol. Bull., vol. 13, p. 251. 1909 Gastrulation in the pigeon's egg — a morphological and experimental study. Jour. Morph., vol. 20, p. 65. 1913 Polyembryonic development in Tatusia novemcincta. Jour. Morph., vol. 24, p. 559. ScHULTZE, O. 1-895 Die kiinstliche Erzeugung von Doppelbildungen bei Froschlarven mit Halfe abnormer Gravitationswirkung. Arch. Entw.-mech., Bd. 1. Spemann, H. 1901-1903 Entwicklungsphysiologische Studien am Triton-Ei. I, Arch. Entw.-mech., Bd. 12; II, Arch. Entw.-mech., Bd. 15, 1903; III, Arch. Entw.-mech., Bd. 16, 1903. Stockard, C. R. 1907 .The artificial production of a single median cyclopean eye in the fish embryo by means of sea-water solutions of magnesium chloride. Arch. f. Entw.-mech., Bd. 23. 1909 a The origin of certain types of monsters. Amer. Jour. Obs., vol. 59. 1909 b The development of artificially produced cyclopean fish, 'The magnesium embryo.' Jour. Exp. Zool., vol. 6, p. 285. 1910 a The influence of alcohol and other anaesthetics on embryonic development. Am. Jour. Anat., vol. 10, p. 369. 1910 b The independent origin and development of the crystalline lens. Am. Jour. Anat., vol. 10, p. 393. 1913 a The artificial production of structural arrests and racial degeneration. Proc. N. Y. Path. Soc, vol. 13, N. S. 266 CHARLES R. STOCKARD Stock ARD, C. R. 1913 b An experimental study of the position of the optic anlage in Amblystoma punctatum, with a discussion of certain eye defects. Am. Jour. Anat., vol. 15, p. 253. ~ — 1914 A study of further generations of mammals from ancestors treated with alcohol. Proc. Soc. Exp. Biol, and Med., vol. 11, p. 136. 1919 Developmental rate and the formation of embryonic structures. Proc. Soc. Exp. Biol, and Med., vol. 16, p. 93. Stockard, C. R., and Papanicilaott, G. N. 1918 Further studies on the modification of the germ-cells in mammals: the effect of alcohol on treated guinea-pigs and their descendants. Jour. Exp. Zool., vol. 26, p. 119. Tannretjther, G. W. 1919 Partial and complete duplicity in chick embryos. Anat. Rec, vol. 16, no. 6. Thorndike, E. L. 1905 Measurements of twins. Arch. Phil., Psych, and Sci. Methods, vol. 13, no. 3. Vejdovsky, Fr. 1888-1892 Entwicklungsgeschichtliche Untersuchungen. 2 vols, text and atlas, Prag. von Jhering, H. 1885 Ueber die Fortpflanzung der Giirteltiere. Sitzungsb. d. konigl. Akad. d. Wiss., Bd. 47, S. 105. Wilder, H. H. 1904 Duplicate twins and double monsters. Am. Jour. Anat., vol. 3. 1908 The morphology of cogmobia; speculations concerning the significance of certain types of monsters. Ibid., vol. 8. Wilson, E. B. 1893 Amphioxus and the mosaic theory of development. Jour. Morph., vol. 8, p. 604. 1904 Experimental studies in germinal localization. I. The germregions in the egg of Dentalium. Jour. Exp. Zool., vol. 1, p. 1. Windle, B. C. a. 1895 On double malformations amongst fishes. Proc. Zool. Soc. London, pt. 3. Zoja, R. 1895-1896 SuUo sviluppo dei blastomeri isolati delle uova di alcune Meduse. Arch. Entw.-mech., Bd. 1, u. 2. PLATE 1 explanation of figures A series of young trout that started development with a slightly insufficient supply of oxygen. The series begins with an ordinary single individual and passes through increasing degrees of anterior duplicity, shown in the two upper rows. It then continues with specimens showing step after step of completely formed double bodies and tails and finally ends with perfectly formed identical twins, in which both members of the pair are equally as large and perfect in structure as is the first single individual. The photographs were all made at one magnification and show as nearly as possible the dorsal aspect of each specimen. On careful examination it will be found that in every specimen the two components are practically identical in size, and when the anterior halves are considered all heads are found to be normal in structure. DEVELOPMENTAL RATE AND STRUCTURAL EXPRESSION CHAKLES R. STOCKARD PLATE 1 267 PLATE 2 EXPLANATION OF FIGURES The same series of trout specimens and photographed in exactly the same order as ilhistratcd in plate 1. The individuals are here shown from as nearly as possible the ventral aspects. Selecting any given specimen and comparing its dorsal and ventral surfaces, as shown in plates 1 and 2, it is clearly seen that in all cases the two components are equal in size and both are structurally normal. These are not 'double monsters,' but perfect individuals. The condition of doubleness is unusual, but not deformed or monstrous. The identical twins could not be considered monsters, and they only differ in degree of doubleness from the other members of the series. 268 DEVEI.OPMENTAL RATE AND STRUCTURAL EXPRESSION CHARLES R. STOCKARD 269 PLATE 3 EXPLAXATIOX OF FIGURES Two degrees of duplicity in luiman individuals. The upper photograph illustrates a doubled condition extending superficially only to below the shoulders, but internally the doubleness extends to the sacrum in the skeleton and to the lower ileum in the intestine. The lower photograph shows two complete babies extensively united by their ventral walls. In both of these specimens the components are of equal size and their structures arc normal throughout. In all specimens of human duplicities examined or found recorded in which the components were of etiual size they were both structurally normal. 270 DEVELOPMENTAL RATE AND STRUCTURAL EXPRESSION CHARLES R. STOCKARD PLATE 3 271 J« c ;^ 22 •- - _^ w; o .ti =0 <^ - "7. •- ^ bc c '^ 2 -j:^ g I a « s •? 'g « b£^ 2 a " fe a^ -^ •« .ii ? o 03 t- T^ -c r, o aj •— o o ^ ,„ -^ c O O fcc c o bc 9^ 'Z^ •■.h:■ c " *-■ is « rt o X £ ? > t> .s J; '^ o

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O =3 'r ^ "^ „ .;^. ^ .2 S ^ S >^ 2 - -SJ -^ S p. Ij 'C •- rt r- TZ T .^ .•^>^ ^ ^ /c-\ ivb^^ ^/^. ^ . ^o„ \X3 ^ ^-^^ V /*iB •■v' ^ 8 339 Resumen por el alitor, Chester H. Heuser, The Wistar Institute of Anatomy and Biology y Johns Hopkins Medical School. El establecimiento temprano de la nutrici6n intestinal en el opossum. El sistema digestivo un poco antes e inmedi atamente despues del nacimiento. El periodo de gestacion en el opossum es muy corto ; el intervalo entre la inseminacion y el parto dura tan solo trece dias, y no mas de diez dias pasan entre el principio de la segmentacion del ovulo y el nacimiento. El desarrollo del tubo digestivo es muy rapido durante los ultimos cinco dias de la vida intrauterina. El estomago en el embrion proximo a nacer esta comprimido y su cavidad es pequefia; esta aiin mas reducida en tamano por pliegues o rugosidades de su mucosa. Pronto des pues del nacimiento las rugosidades desaparecen, a causa de la expansion fisiologica del estomago, producida por la leche. En los estados tempranos, que el embrion pasa en la bolsa de la madre, el estomago es una simple camara sacular provista de una pared muy delgada y ligeramente diferenciada. El esofago comunica con el estomago en un punto muy proximo al orificio duodenal. En la mucosa, cerca de los orificios de entrada, existen pliegues poco profundos, pero las glandulas no se han desarrollado todavia en ninguna parte del organo. Las asas primarias del intestino delgado son unas seis en numero, y todas ellas aparecen ya en el embrion proximo a nacer, inmediatamente despues del nacimiento, el estomago, distendido por la leche, desplaza al intestino delgado hacia la derecha,pero la identidad de las asas primarias puede reconocerse todavia. En el embrion proximo a nacer se han desarrollado vellosidades en las tres primeras asas. En los jovenes que habitan la bolsa dichas vellosidades aparecen en todas las regiones del intestino, aunque estan mas diferenciadas en el duodeno. Las tunicas externas del intestino son muy delgadas, tanto en el embrion proxiino a nacer como en el joven situado en la bolsa. En este ultimo especialmente el intestino delgado es notable por la considerable extension de la mucosa. En los extremos distales de las celulas que le revisten existen grandes masas de granulos que se tiiien especialmente. La capa muscular esta indicada solamente por una banda delicada de mioblastos. Los vasos sanguineos del intestino son especialmente visibles a causa de su gran tamano y caracter sinusoidal, estando situados en la inmediata proximidad de la mucosa. Translation by Jose F. Nonidez Cornell Medical College, New York AUTHORS ABSTEACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, NOVEMBER 15 THE EARLY ESTABLISHMENT OF THE INTESTINAL NUTRITION IN THE OPOSSUM— THE DIGESTIVE SYSTEM JUST BEFORE AND SOON AFTER BIRTH CHESTER H. HEUSER The Wistar Instilide of Anatomy and the Johns Hopkins Medical School SIX PLATES (twenty FIGURES) As the intra-uterine period in the opossum is very brief, the intestinal nutrition commences in the newborn animals while they are yet relatively very immature. Ten days after the beginning of segmentation, or thirteen days after insemination, the young opossums appear in the pouch; they become at once attached to the teats, and milk soon enters their stomachs. Five days before birth, however, the digestive tract of the opossum is no further advanced than this system in the three-day chick embryo; the foregut extends forward from the broad connection with the yolk-sac, and the first rudiments of the liver, hindgut, and allantois are just appearing. The transformation brought about during these last five days is remarkable in its rapidity. The stages especially considered in this report are those of the embryo a few hours before birth and the pouch-young about three days old. The external form of the viscera is first examined and the histological structure is then studied. The stomach, small intestine, and large intestine are dealt with in order, special attention being given to the small intestine. METHODS This study was commenced and the material used was obtained while I was a member of the staff of The Wistar Institute. The specimens were collected at Austin, Texas, in cooperation with Dr. Carl G. Hartman, of the University of Texas. Doctor 341 342 CHESTER H. HEUSER Hartman had collected opossum embryos for several years, and as he had worked out methods for recognizing the various stages of pregnancy, we were able to secure a very complete series. The embryos were removed from the excised uterus in Ringer's solution, as described by Hartman ('19).^ With scissors a long incision was first made along the uterine horn, cutting rapidly through the muscular layer but without perforating the mucosa, and the latter was then pulled apart with forceps. In order to avoid injury to the embryonic vesicles, which during the last two or three days before birth are closely pressed against each other and intimately applied to the uterine mucosa, the vesicles were separated from onf another by cutting off the uterine muscle before opening the lumen ; even small pieces of the muscle remaining caused considerable interference by the formation of pockets in the mucosa which surrounded and held portions of the embryonic membranes. This care in separating the vesicles was taken for the reason that I wished to make injections of the bloodvessels in one or more embrA^os of a litter. Such injections could be made very satisfactorily in these embryos with an intact vascular system and a vigorously beating heart. The embryos which were to be sectioned were dropped while alive into Bouin's fluid and left for six to twenty-four hours, depending upon the size of the individual. The Bouin's fluid was replaced with 80 per cent alcohol by adding the latter in small quantities to the fixing fluid — taking several hours for the transfer. The alcohol was later changed several times, and finally lithium carbonate — a few drops of a saturated aqueous solution to the ounce of alcohol — was added to remove the picric acid. As the pouch-young were firmly attached to teats in the pouch, they were removed by exerting a slow continuous pull with blunt forceps pressed against the mouth. The specimens were then placed at once into Bouin's fluid, and after the muscular contractions had ceased, incisions were made through the abdominal wall to insure the rapid entrance of the fixing fluid into the body cavity. Embryos and pouch-young were also fixed and stored in 10 per cent formalin. INTESTINAL NUTRITION IN THE OPOSSUM 343 The whole specimens, which had been fixed in Bouin's fluid, were later run down from 80 per cent alcohol to water — using a fluid-changing device by which one solution was added to the other drop by drop. The apparatus was provided with a glass stirring plunger which was raised and lowered by a cam-shaft,, and an overflow-siphon controlled the volume of the solution surrounding the specimen. After staining in alum-cochineal, the specimens were carried back to 80 per cent alcohol, making use of the same device to insure slow^ interchange of fluids. The abdominal wall was then dissected off so that the external form and position of the viscera could be studied. Next the liver, stomach, and intestines were removed in one piece, and later, in order not to injure the other structures, the liver was dissected off with needles. As a means of recording the appearance of the dissections at the various stages, stereophotographs were made during the progress of the work. The block to be sectioned included the stomach and intestines; it was embedded in paraffin and cut into a series of sections 5/x thick, by Professor Ruber's water-on-the-knife method. The sections were stained with Mallory's connective-tissue stain. The use of the cochineal was advantageous in that the embryos were satisfactorily stained for preliminary dissections before imbedding or for subsequent dissections; the stained embryos could also be photographed well; and in the sections the nuclear detail was better shown than with Mallory alone. The latter stain was very useful for showing differentiation among the various tissues. This method of staining the whole embryos in alum-cochineal and the sections with Mallory's connective-tissue stain has been used on specimens of various ages and has proved to be of value. Several series of sections were prepared and used for reference, but the chief observations in this study were made on an embryo of few hours before birth and on a pouch-young about three days old. THE AMEBICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 344 CHESTER H. HEUSER DESCRIPTION OF THE GASTRO-INTESTINAL TRACT Stomach In the embryo: The stomach in the late embryo is, in general, pyriform in shape, being shghtly compressed from above downward; it lies almost in a transverse zone in the left side of the body. As seen in dissections of the viscera in situ, the stomach is found to be entirely masked by the liver, except on the left where a small portion of the greater curvature projects below the hepatic border (fig. 5) . Upon removing the liver, the stomach may be seen in its characteristic pyriform shape as shown in figures 7 and 9. The greater curvature is about eight times that of the lesser, as determined by measurements of the curves from the esophageal opening to the pylorus. The esophagus enters the stomach at about the middle of the lesser curvature in rather close proximity to the duodenal opening. The pyloric portion is not drawn out into a tubular form as in human embryos (Lewis, '12), and externally at least there is no indication of a pyloric antrum. The blind pouch above the entrance of the esophagus — the fundus (fig. 9) — is relatively long and tapers rapidly upward to its tip. Except that the pyloric portion is not tubular in form, the stomach as a whole has the appearance of the organ in the late rat and early pig embryos. In naming the various subdivisions of the stomach the terms adopted by Lewis ('12) have been used. The external surface of the stomach is smooth, but faintly delineated folds in the mucosa can be seen through the outer layers. These folds of the mucosa, or rugae, are in general arranged longitudinally and extend from the fundus down into the corpus, being more prominent along the greater curvature. They are filled with loose embryonic mesenchyme and extend far out into the lumen so that irregularly shaped projections and compartments are evident in sections (fig. 11). In the pouch-young: The stomach in the pouch-young is no longer compressed as in the embryo, but has become tremendously distended by constant filling with milk. The greatly enlarged organ may be made out in the living animal because of INTESTINAL NUTRITION IN THE OPOSSUM 345 the milk which shimmers through the body-wall as a large white mass. In general, as in the embryo, the stomach lies in the left side of the body with the greater curvature to the left and the lesser curvature toward the right. Measuring the curvatures again from the esophageal opening to the duodenum at its union with the stomach, the ratio is about ten to one — showing an increase in the greater curvature as compared with the ratio of eight to one in the embryo. The esophagus and duodenum still enter the stomach very close together; this striking nearness of these openings into the stomach is a condition similar to that described by Owen ('68) for the ornithorynchus. The inferior border of the pyloric portion bulges below the duodenal opening and above blends imperceptibly with the corpus. The organ has expanded in all directions, but the greatest growth has been in the lower part of the cardiac portion. The fundus does not taper to a narrow tip and is much less conspicuous than in the embryo. Especially as regards this part, the stomach is of the simple carnivorous type seen in cat embryos. By its great expansion the stomach has altered the relations of the other viscera; the small intestine has been crowded to the right side; the lateral margins of the wolffian bodies have been spread far apart; and the liver has been pushed upward and flattened laterally, so that its greatest width is more than twice its vertical thickness. Only a small portion of the stomach now remains capped by the liver, as can be seen by comparison of figures 6, 8, and 10. The rugae present in the gastric mucosa in the embryo have become obliterated in the pouch-young, and except for a series of slight plaited folds between the esophagus and duodenum, the interior of the stomach, like the outside, has a smooth surface. This smoothing of the mucosa may be accounted for by the physiological distension of the organ with milk. The wall along the greater curvature is much thinner than in the region of the plaited folds referred to. The epithelium of the mucosa is made up of cells of a low columnar type with large vesicular nuclei. The cytoplasm is homogeneous and granular, being slightly more condensed along the cuticular border. Just beneath this border 346 CHESTER H. HEUSER terminal bars can be seen and the epithelial cells rest upon a delicate but distinct basement membrane. There is some evidence of a differentiation among the epithelial cells in the pyloric region. Here groups of cells appear slightly more darkly stained and more closely packed together. In each section there are found a few cells which in form appear similar to their neighbors, but are more brightly stained with the acid f uchsin ; they probably represent a phase in cell activity in which the staining reaction has been modified. In the more attenuated portion of the stomach wall along the greater curvature, the distention has been very pronounced and all layers of the wall have been greatly reduced in thickness. The epithelial cells in some cases have less than half the height of those along the lesser curvature. In the thinnest places, the nuclei have been drawn out parallel with the surface of the epithelium; they are also much farther apart in these regions. The muscular layer is everywhere very thin, being only one or two myoblasts in width and the elongated nuclei are few in number. Small intestine A striking feature of the intestine in opossum embryos is the abundance, size, and arrangement of the blood-vessels. In an embryo 8.5 mm. in length — about nine days after the beginning of segmentation and one day before birth — the vessels have a very wide diameter and are also noteworthy for their close approximation to the mucosa, as shown in figure 1 from a transverse section of a loop of intestine. Closely hugging the mucosa, they present the appearance of large sinusoids rather than that of capillaries. The outer borders of the vessels, however, are covered with the mesenchyme, and hence they are not true sinusoids as defined by Professor Minot ('00). In many sections nearly the whole extent of the epithelium of the mucosa is in contact with these large blood-vessels. Four to six folds of the epithelium extend into the lumen, and in each case the crypt of the fold is filled with a large vessel. A reconstruction of a segment of the intestine (fig. 2) indicates a rich plexus of vessels spreading over the outer surface of the mucosa. The crypts of INTESTINAL NUTRITION IN THE OPOSSUM 347 the folds are filled with vessels which are of great size. This condition does not appear in the drawing of the model, but can be seen in segments of it and in sections of the embryo (fig. 1). In one crypt the mucosa is spread out in a thin layer over a large blood sinus. In addition to this great vascularity of the small intestine of the earlier opossum embryos, there are certain other features of interest in the morphology of the intestinal tract not only in the late embryo, but in the pouch-young. In the late embryo: The duodenum extends from the pylorus to the right side of the abdominal cavity, bends dorsally in a right angle and proceeds downward and backward to the midsagittal region, where it turns sharply back on itself and parallels its course to the pylorus. The duodenum may arbitrarily be said to end at this point. Here, at the upper end of limb 4 in figures 3 and 7, the small intestine makes another complete bend and follows the previous limb, lying mesially to it, for half its length, then turns in a right angle to the left side of the body (fig. 3, 5 to 6) and gradually bends upward to the region beneath the corpus of the stomach. Here for a third time it turns sharply back on itself as far as the midventral plane, where three short loops (fig. 3, 8 to 12) are made before it leads into the large intestine. The small coils are surrounded and partially covered by the larger loops. The position of the union of the small with the large intestine is on the left side of the body just below the middle part of the stomach (behind 12 m. fig. 3). The first double loop of the small intestine — duodenum — has a very wide diameter, being enlarged into an antrum pressing against and encroaching upon the stomach. The widest region of the whole intestinal tract is at the middle of the first limb of this loop; the enlargement is a short distance below the opening of the ductus choledochus. Beyond the duodenum the small intestine — from the beginning of limb 5 in figure 3 — gradually diminishes in caliber so that the girth of the smallest coils in the umbilical region is about one-third that of the duodenum in its widest place. The small intestine consists of relatively a few loops, which in most cases have sharp bends. Segments of most 348 CHESTER H. HEUSER of the coils of the mtestine can be seen in a single section, as shown in figure 12, where the great difference in the diameter of the various loops is apparent. Numerous long villi are present in the duodenum and first coils of the small intestine, as shown in figure 12 at 1, 4, 5, '^y 8. In the slender loops 10, 11, and T2 there are no villi, but the mucosa is thrown into four or five folds. The ductus choledochus enters the duodenum in limb 1 eight sections (40m) below the section figured. Limb 5 of section 254 is shown at a higher magnification in figure 13. The low columnar cells of the mucosa rest upon a distinct basement membrane. Definite cell-walls are usually made out and the cuticular border ends abruptly in a narrow dense band, bulging out in the middle part of the cell. Intercellular cement lines are prominent in surface views of the tips, of the cells, and terminal bars are therefore distinct. The nuclei, which are located in the lower part of the cells near the basement membrane, are usually spherical and contain one or two large nucleoli. The relations of vessels and the great difference in thickness of the various layers are also indicated in figure 13. Numerous vessels, as also in the earlier embryo (figs. 1 and 2), appear beneath the mucosa, and the core of each villus is practically filled by a large sinus-like blood-vessel. The outer layers of the intestinal wall are very thin; the muscular coat is especially delicate, consisting of a single, or in places, of a double row of myoblasts. In the pouch-young: After birth, although the intrusive stomach crowds the other viscera out of their former positions, the general arrangement of the coils in the intestines is not fundamentally disturbed. The large loops of the duodenum and the first loops immediately following are similar to their former pattern. This essential similarity in coils of the two stages was strikingly illustrated by a dissection which was made of a formalin-fixed pouch specimen of the same litter as the one shown in figure 6. The coils of the intestine could be spread apart under the binocular microscope so that the various limbs and loops could be compared with those found in the late embryo. As in the latter, the duodenum had by far the largest caliber of any part of the INTESTINAL NUTRITION IN THE OPOSSUM 349 intestine and again the extreme girth was in the first limb at the region of the first right-angle bend. The last three or four coils of the small intestine were distinctly smaller than the others. This great disparity in the caUber of the different parts of the intestine was found to persist in specimens of at least several weeks' age. The villi of the small intestine of the pouch-young are covered by tall columnar cells with large centrally placed nuclei. A delicately striated cuticle rests upon a distinct cement line and terminal bars are very prominent. In the distal ends of the cells are large granular strands and masses which vary considerably in shape and size. The masses are located in a zone which occupies about the middle third of the space between the nucleus and the cuticular border of the cell. The distribution of these granules may be more extensive, but distally there is always a narrow strip of cytoplasm devoid of granules. These cytoplasmic inclusions stain brilliantly with eosin, and with the acid fuchsin of the Mallory stain. Iron hematoxylin brings them out distinctly. After staining with alum-cochineal in toto, the granular masses are faintly visible; following this stain with gentian violet applied to the tissue after sectioning, the masses become intensified but remain much paler than the nucleoli. These granular aggregations, varying in shape and size, appear in nearly all the cells of the villi and in some of the cells of the intervillous spaces. The strands are made up of fine granules; the larger masses are likewise composed of granules or consist of a limiting border of granules enclosing a less densely stained homogeneous interior. The ultimate round granules also vary considerably in size; minute isolated ones may be seen between the larger groups. Some of the cells are filled with a non-staining substance so that they appep-r practically empty; a small amount of protoplasm chngs to the nucleus and a few strands may stretch across to the cell-membrane. Many other cells are partially vacuolated; these occur more abundantly in the first large coils of the intestine. The cells on the left side of the villus shown in figure 18 are of this type. 350 CHESTER H. HEUSER There are no glands present in any part of the intestine, but between the vilh the epitheUal cells are in many places more closely packed, more darkly stained, and of smaller size than those of the villi. A group of these cells is shown in figure 18. In corresponding regions of the two stages, the villi are taller and more highly specialized in the pouch-young (cf. figs. 13 and 17). Of the loops in the older specimen the one at 7-8 in figure 16 has the largest lumen. Here the villi are much lower than in the coils nearer the stomach, but the details of the mucosa and of the other layers are very similar to those found in the duodenum. The smaller coils have increased in size only slightly and the villi are imperfectly formed in some of the limbs and absent in others. The striking thinness of the external layers of the intestine is more marked in the pouch-young than in the embryo. This feature of the pouch-young is well illustrated by the photographs (figs. 17 and 18). The relation of the mucosa and the blood-vessels is again very intimate; the entire core of each villus is occupied by a large sinus-like vessel. Large intestine In the embryo: The large intestine begins at its junction with the small intestine, the point of origin being just below the corpus of the stomach on the left side of the abdominal cavity. From this point it swings gradually toward the midplane of the body and leads a nearly straight course downward tb the cloaca. The diameter of the large intestine is nearly the same throughout its extent, being about equal to that of the most slender of the coils of the small intestine. The caecum is a long slender blind outgrowth from the large intestine immediately below the beginning of this part of the gut. The caecum grows out at a sharp angle and extends ventrally just beneath the stomach, so that the blind end is seen in ventral views of the viscera (fig. 7) . The epithelial tube of the colon is small and laterally constricted with the dorsal edge bent toward the left and the ventral edge toward the right so that in cross-section it appears as a widely opened letter S. Since the epithelial tube is small, the INTESTINAL NUTRITION IN THE OPOSSUM 351 combined width of the external layers is relatively greater than it is in the preceding portions of the digestive tract. The epithelial lining of the colon consists of undifferentiated low columnar cells with large elongated nuclei and homogeneous granular cytoplasm. There are many thin-walled blood-vessels in the loose mesenchyme of the submucosa. The muscular coat is a thin layer made up of about three rows of myoblasts without differentiation into longitudinal and circular coats. The layer of mesenchyme surrounding the muscularis is slightly denser than that constituting the submucosa. In the pouch-young : The large intestine grows a little more rapidly than the other portions of the intestine, but in the pouchyoung studied it is still the least differentiated region of the intestinal tube. The girth is only a little greater than that of the smallest of the coils of the ileum. The caecum remains a long slender lateral outgrowth as in the younger stage. There is in addition a short epithelial diverticulum which has grown out from the large intestine near its union with the small intestine. The significance of this diverticulum is not known. The mucosa has grown so that it is about twice its former width. The tall columnar cells extend from the cuticular border to a distinct basement membrane. The cytoplasm is evenly stained and undifferentiated. The nuclei are confined largely to the lower portion of the cells, but they are not arranged in a single row; with the folding of the epithelial tube the regular distribution of the nuclei is apparently disturbed ; they are found in groups opposite the folds and arranged in a triple layer. The lumen is pentagonal in transverse section. The increased caliber of the large intestine may be accounted for by the increase in size of the lumen and to the growth in thickness of the mucosa; the external layers, however, are relatively thinner than in the embryo. DISCUSSION AND SUMMARY The period of gestation in the opossum is very short; the interval from copulation to parturition is only of thirteen days' duration, and of this period three days have passed before the 352 CHESTER H. HEUSER beginning of segmentation of the ovum. Selenka ('87) calculated that segmentation did not begin until the end of the fifth day, but from much more extensive material Hartman ('19) estimates that the first division of the fertilized ovum occurs two days earlier. During the next three days the embryo develops to a stage with an undifferentiated fore- and hindgut. From this primitive condition the digestive tract, during the last five days before birth, is rapidly transformed to a functioning state. This rapid change from the primitive gut into an alimentary system capable of digesting milk, even at a stage of relatively incomplete differentiation, presents many problems of great interest. The stomach of the late embryo may be described as consisting of a cardiac and a pyloric portion, more or less arbitrarily marked off on the lesser curvature at the angular incisure. The cardiac portion, using the terminology adopted by F. T. Lewis ('12), may be said to include a prominent blind pouch or fundus tapering rapidly to a tip and a large lower part or corpus. The different parts blend imperceptibly with each other along the greater curvature. In the opossum embryo the pars pylorica is not tubular in form as described by Lewis for the human embryo, and as Lewis and I reported ('14 and '15) for the cat, rat, pig, and sheep. In a younger opossum embryo — one measuring 7 mm. in length — the whole digestive tract by reconstruction (Heuser, '18) showed a more elongated portion leading into the duodenum, and the stomach resembled the organ in the 10-mm. human embryo (Lewis, '12, fig. 5). The gastric canal, which Lewis demonstrated to be an epithelial differentiation in human embryos, and as we likewise found to be the case in the other mammals named, has not been definitely made out in the opossum embryos examined; however, it is believed that it would be revealed by wax reconstructions of the gastric epithelium in a proper series of stages. The stomach is small in the late embryo and the lumen is further reduced in size by the presence of folds or rugae in the mucosa. The rugae vary considerably in number and prominence in embryos of this stage, being less conspicuous in two other INTESTINAL NUTRITION IN THE OPOSSUM 353 individuals of the same age. Near the pylorus there are a few shallow folds in the mucosa, indicating a very early stage in the development of pyloric glands, and at the opening of the esophagus, less definitely marked folds are found where the cardiac glands will later develop. The epithelial lining of the stomach, however, is made up of low columnar cells, which are at this stage undifferentiated in all parts of the organ. After birth the distention of the stomach with milk results in the disappearance of the rugae which were present in the embryo. In the living animal the position and large size of the stomach are very evident on account of the milk which can be seen through the transparent body-wall. The greatest enlargement has occurred in the portion of the corpus along the greater curvature and in the upper part of the pyloric region. The fundus ends bluntly and is not prominent as in the embryo. Bensley ('02), in his comprehensive paper, described the stomach of the adult opossum as a simple structure, resembling closely in shape and arrangement of parts those of insectivores and carnivores. He showed in a diagram (fig. 10 A) that the fundic gland zone occupied the greater portion of the mucous surface, with the remainder covered by pyloric glands, and stated also that the cardiac gland zone occupied an extremely narrow zone at the termination of the esophagus. Except that the glands have not yet developed, the arrangement of parts in the early pouch-young is similar to that described for the adult. However, the epithelial cells are yet of a simple type, and although certain cells in the pyloric region are slightly more darkly stained and more closely packed together, there is no differentiation among them. A few slight plaited folds occur near the pylorus and also around the entrance of the esophagus. Glands, however, are nowhere present nor can any of the elements be regarded as cells associated with the formation of acid as found in the functioning stomach of higher mammals. . The coils of small intestine in the late embryo are placed medially in the abdominal cavity, lying between the liver and the wolffian bodies. The first loops extend toward the right side, partially surrounding the remaining smaller coils. Mall 354 CHESTER H. HEUSER ('98) showed that the primary coils of the intestine in the human embryo could be recognized very early and that even the various loops of the adult intestine, as well as their position, could be made out in embryos of five weeks. The first three of the primary loops, I believe, can be identified in the 7-mm. opossum embryo; the smaller distal coils, however, are not as yet indicated. Villi have developed in the first three loops of the small intestine of the late opossum embryo. It was not determined whether the villi originate singly as thickenings of the epithelium, as Johnson ('10) has described for the human embryo, or whether they are preceded by longitudinal folds. Four to six low folds are present in the mucosa of the last slender coils and there are a few diverticula of the mucosa similar to those found by Lewis and Thyng ('08) in various mammals. One diverticulum of this nature is shown in limb 12 of figure 12. The epithelial lining is made up of undifferentiated cells of a low columnar type, filled with homogeneous cytoplasm and with nuclei proximally placed near the basement membrane. The entire width of the intestinal wall, exclusive of the mucosa, is remarkably thin; in some places the whole wall beneath the basement membrane is equal to about one-third the height of the epithelial cells above it. The muscular coat particularly is very delicate; it consists of a fine strand of mj'oblasts only one or two in thickness. In the pouch-young the small intestine has been crowded to the right by the enlarged stomach. The primary coils, however, can still be recognized; the numerals on figures 3 and 4 have been placed on comparable limbs of the coils. Villi are present in all parts of the small intestine, although they are older and more advanced in the duodenum. The external coats are yet very thin and the intestine is noteworthy for the very large surface of the mucosa. The muscular layer is only faintly indicated by a.few circular myoblasts, so that peristaltic contractions must be very feeble. The intestinal blood-vessels are especially noticeable on account of their large size and sinusoidal character, being closely applied to the mucosa. The cytoplasm of the cells covering the villi is coarsely granular and the large vesicular nuclei are placed centrally in the cell. INTESTINAL NUTRITION IN THE OPOSSUM, 355 In many places between the villi there are patches of epithelial cells which are smaller and more darkly stained than those covering the villi. In the distal ends of the cells covering the villi one sees very prominent masses which can usually be resolved into granules with the high power. These granules stain brilliantly with eosin and with acid fuchsin. The chief interest in them lies in speculation as to whether they are products of secretion or of absorption; no data, other than that afforded by histological examination, have been obtained. The large intestine had changed to a much smaller degree relatively in the transformation from the late embryo to the pouch-young. Although the external caliber of the colon has increased considerably in the interval of growth, there is but little advance in the epithelial or mesoblastic differentiation. In the pouch-young the epithelial lining-cells have doubled in height; the lumen is considerably increased in size and the mesenchyme is markedly condensed. These morphological changes are, however, much less pronounced than are the differentiations which have occurred in the upper portions of the small intestine. The process of differentiation apparently proceeds from above downward, in the direction of the physiological passage of food-stuffs. The relatively slight dilation of the colon and the histological findings indicate that the residue of the foodstuffs reaching the colon in the opossum pouch-young is relatively small; practically all of the milk must be disposed of in the upper portions of the alimentary canal. In summary it may be said that the changes in the gastrointestinal canal from the late embryo to the early pouch-young, in which the digestion of milk must occur, are marked. Certain of the changes, as the enormous dilatation of the stomach, must be largely due to mechanical causes; other alterations, as in the formation of villi and the differentiation of the epithelial cells in the upper parts of the small intestine, must be accounted for by the natural processes of development of the embryo, aided or hastened possibly by the early functional requirements of the organs. 356 CHESTER H. HEUSER LITERATURE CITED Bensley, R. R. 1902. The cardiac glands of mammals. Am. Jour. Anat., vol. 2, pp. 105-156. Hartman, C. G. 1919 Studies in the development of the opossum (Didelphys virginiana L.). III. Description of new material on maturation, cleavage, and entoderm formation. IV. The bilaminar blastocyst. Jour. Morph., vol. 32, pp. 1-142. Heuser, C. H. 1914 The form of the stomach in mammalian embryos. Proc. Am. Assoc, of Anatomists. Anat. Rec, vol. 8, p. 130. 1919 The anatomy of the 7-mm. opossum embryo. Proc. Am. Assoc. of Anatomists. Anat. Rec, vol. 16, p. 150. JoHNSox, F. P. 1910 The development of the mucous membrane of the oesophagus, stomach and small intestine in the human embryo. Am. Jour. Anat., vol. 10, pp. 521-575. Lewis, F. T. 1912 The form of the stomach in human embryos, with notes upon the nomenclature of the stomach. Am. Jour. Anat., vol. 13, pp. 477-503. 1915 The comparative embryology of the mammalian stomach. Proc. Am. Assoc, of Anatomists. Anat. Rec, vol. 9, pp. 102-103. Lewis, F. T., and Thyng, F. W. 1908 The regular occurrence of intestinal diverticula in embryos of the pig, rabbit and man. Am. Jour. Anat., vol. 7, pp. 505-519. Mall, F. P. 1898 Development of the human intestine and its position in the adult. Bull, of the Johns Hopkins Hospital, vol. 9, pp. 197-208. MiKOT, C. S. 1900 On a hitherto unrecognized form of blood circulation without capillaries in the organs of vertebrata. Proc. of the Boston Society of Natural History, vol. 29, pp. 185-215. Owen, R. 1868 On the anatomy of vertebrates, vol. 3. London. Slenka, E. 1887 Studien iiber Entwickelungsgeschichte der Thiere, viertes Heft, Das Opossum (Didelphys virginiana). Wiesbaden. PLATES 357 PLATE 1 EXPLANATION OF FIGURES 1 Transverse section of a loop of the small intestine in an 8.5-mm. opossum embryo. Wistar series 16177, section 555. The section shows the relation of the large sinus-like blood-vessels to the intestinal mucosa. A diverticulum of the epithelium is seen in the lower right portion of the section. X 125. 2 Wax-plate reconstruction of a segment of the small intestine in an 8.5-mm. opossum embryo. Wistar series 16177, sections 553-576. The model shows a freely anastomosing plexus of blood-vessels closely pressed upon the mucosa. X 125. 3 Outline drawing of the viscera in an opossum embryo a few hours before birth to show the positions of the sections shown in figures 11, 12, and 13. From embryo no. 154. X 18. 4 Outline drawing of the viscera in a young opossum about three days old to show the positions of the sections shown in figures 14, 15, and 16. From pouchyoung no. 156. X 18. 358 INTESTINAL NUTRITION IN THE OPOSSUM CHESTER H. HEUSER PLATE 1 Fia.15 Figr16 359 THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 PLATE 2 EXPLANATIOX OF FIGURES 5 Dissection of an 11.75-mni. opossvnn embryo to show the positions and rekxtions of the viscera in the body. The stomach and intestines of this embrj'o — one a few hours before ]>irth — are shown in figures 3, 7, and 9. These organs were then embedded and sectioned. Embrvo no. 1.54. X 12. 360 INTESTINAL NUTRITION IN THE OPOSSUM CHESTER H. HEUSBR PLATE 2 5 361 platp: 3 EXPLANATION OF FIGURE 6 Dissection of an 11.75-min. pouch-young of the opossum. This specimen is about three days old. The greatly distended stomach has crowded the intestines toward the right. The stomach and intestines are shown again in figures 4, 8, and 10. Photographs of sections of the same organs are shown in figures 14 to 20. Pourh-voung no. 150. X 12. 362 INTESTINAL NUTRITION IN THE OPOSSUM CHESTER H. HEUSER PLATE 3 363 PLATE 4 EXPLANATION OF FIGURES 7 Dissection of the stomach and intestines from an 11.75-mm. opossum embrj'o. Ventral view of the organs from the specimen shown in figure 5. From embryo no. 154. X IS. '8 Dissection of the stoniuch and intestines from an 11.75-mm. pouch-young of the opossum. Ventral view of the organs from the sjiecimen shown in figure 6. From pouch-young no. 156. X IS. 9 Same organs as shown in figure 7 oriented to show the form of the stomach. From embiyo no. 154. X IS. 10 Same organs as shown in figure S oriented to show the form of the stomach. From pouch-young no. 156. X IS. 364 INTESTINAL NUTRITION IN THE OPOSSUM CHESTER H. HEUSER PLATE 4 Caecum E50phaqus^ Fundus Rectum Vit.Vein-^^ 10 365 PLATE o EXPLANATION OF FIGURES 11 Photograph of a section of the stomach of the kite opossum embryo. The position of this section and those shown in figures 12 and 13 are indicated in figure 3. From section 83 of embryo no. 154. X 25. 12 Photograph of section 2;.3. From embryo no. 154. X 25. 13 Photograph of limb 5 in section 254 of the small intestine. From embryo no. 154. X lot). 14 Photograph of section 128 of the early pouch-young. The positions of the sections in the series are indicated in figure 4. From pouch-young no. 156. X20. 15 Photograph of section 195. From pouch-young no. 156. X 20. 366 INTESTINAL NUTRITION IN THE OPOSSUM CHESTER H. UEUSER PLATE 5 i'\ .;-■%-/% Reciu 12 '^ \.. 14 ^ . -.^ 9 - . . 8 ■ 15 367 PLATE 6 EXPLANATION OF FIGURES Photographs of sections passing through the stomach and intestines of a pouoh3^oung of the opossum. From pouch-young no. 1.56. 16 Section 295. X 20. 17 Limb 5 of the small intestine in section 195 (fig. 15). X 100. IS From limb 4 of the small intestine in section 295 (fig. 16). Some of the cells of the villi are largel.y filled with a non-staining substance. A group of darkly stained and smaller cells is also shown between the villi. X 400. 19 Limb 7 of the small intestine in section 295 (fig. 16). The prominent granular masses in the epithelial cells are here shown in a tangential section of the villi. X 400. 20 Villus from limb 1 of the small intestine in section 128 (fig. 14). X 400. 368 INTESTINAL NUTRITION IN THE OPOSSUM CHESTER H. HEUSER PLATE ^^■f ^ ^^v.^ '-^/n 5^^ iite. t 16 % 5 11 9 !8 m

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■^ 17 ^^^^^^^^^A^-v.-' •«* ~_<L«»-i* -:^S.*^^ ,^ 59 € 20 369 Resumen por el autor, John Lewis Bremer, Escuela Medica Harvard, Boston. Ramas recurrentes del nervio abductor en embriones humanos. En la mayor parte de los embriones humanos, el nervio abductor esta provisto de una o mas ramas recurrentes. En los que no poseen esta rama, su lugar esta ocupado por una raiz anterior del hipogloso. Las fibras acompanan al vago o glosofaringeo, entran en los miotomos espinales mas craniales o se dirigen hacia el dorso. Pueden degenerar en los embriones de 15 mm.; las mas antiguas se encontraron en un embrion de 31 mm. La degeneracion comienza en el extremo nuclear de las fibras y va acompanada de fagocitosis. La atraccion que produce a estas ramas parece estar circunscrita a los miotomos postoticos mas anteriores, y la que da lugar al recorrido de la porcion principal del abducens, a los musculos oculares pre6ticos. La fuerza relativa de ambas varia aparentemente, en parte a causa de su distancia con relacion a las fibras del abducens en el momento de su emergencia del cerebro. Las diversas posiciones de las raices del abductor y su orden de emergencia en las diversas clases de los vertebrados, y las posiciones relativas de las dos masas musculares, son objeto de discusion en el presente trabajo, para explicar la falta habitual de las ramas recurrentes en las formas inferiores, excepto como raras anomalias. La ausencia completa de ambos nervios abductores, excepto una rama recurrente a cada lado, se describe en un embri6n humano de 18 mm. El autor atribuye esta anomalia a la condicion retardada del ojo y sus musculos, que de este modo no ejercen la fuerza necesaria para cambiar la direccion de las fibras emergentes del abductor en su direccion craneal ordinaria. Translation by Jose F. Nonidez Cornell Medical College, New York AUTHOR 8 ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, NOVEMBER 15 RECURRENT BRANCHES OF THE ABDUCENS NERVE IN HUMAN EMBRYOS JOHN LEWIS BREMER Harvard Medical School, Boston, Massachusetts ONE DIAGRAM AND FOUR FIGURES Recurrent branches of the abducens have been known since 1898, and then- significance commented on by Neal, Piatt, Dohrn, Belogolowy, and others. In an earher paper I showed them, along with other unusual roots and branches of the abducens and hypoglossal nerves. Only recently, however, have I realized that in human embryos the recurrent branches of the abducens are to be considered normal, instead of infrequent variations, and that in man they attain their greatest frequency, size, and duration. In many classes of vertebrates the gap along the floor of the hind-brain between the most anterior roots of the adult hypoglossal nerve and the most posterior roots of the abducens is more or less filled in the embryo by numerous ventral rootlets, some running to join the hypoglossal, caudal to the vagus and accessory nerves, some passing between the vagus and the glossopharyngeal, below the upper ganglia, others taking a more dorsal course, like the dorsal rami of spinal nerves, to end in the dorsal head musculature. Many of these rootlets are abortive, and end before their definite direction can be more than imagined. They may, if they persist long enough, cause probably transient foramina in the cartilaginous base of the skull, in line with the hypoglossal foramina, or they may pass out by the jugular foramen. They may be connected by longitudinal strands, at a short distance from their point of origin from the brain, thus forming a series of loops often continuing the direction of the abducens caudally toward the hypoglossal. Such a continuation of the abducens caudally, or a separate caudally running root 371 THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 372 JOHN LEWIS BREMER from the . immediate vicinity of the permanent abducens roots, is a true recurrent branch of the abducens, as opposed to the other scattered rootlets more immediately connected with the hypoglossal nerve, which might be called anterior hypoglossal roots. The significance of these transient roots as a whole is the indication that the vagus, glossopharyngeal, and facial nerves were originally provided with ventral roots in addition to their dorsal and lateral ones, and that probably the abducens was diverted to its present disconnected position by the migration of the more ventral pre-otic musculature to the vicinity of the eyeball. The recurrent branches of the abducens should be correlated either with the migration forward of a postotic myotome to form the external rectus muscle, or, as Neal suggests, with the loss of the postotic myotome and the 'piracy' of the nerve in acquiring attachment to pre-otic muscles. If we examine the descriptions and drawings of those authors who have mentioned these recurrent branches, mostly in connection with studies on the derivation of the eye muscles, the impression is left that they are occasional variations, small and of significance only in the comparative morphology of the vertebrate head. NeaV describing Acanthias, says "that not all of the nerve fibrils extend anteriorly toward the third somite (van Wijhe's), but that in later stages of development, e.g., in embryos with 70-80 somites, a nerve fibril is seen to pass from the posterior root of the nerve in a posterior direction toward the myotome of the sixth somite." Miss Platt^ speaks very briefly of the abducens, also in Acanthias, as being "at first distributed not only to the walls of the third head cavity, but also to the general mesoderm posterior to that cavity." Thus two investigators find these recurrent branches in the same class of vertebrates at different embryonic ages, the one 'at first,' the other 'in later stages.' Scammon, in the Normentafeln of Acanthias, does not mention these branches, and in the material used by Scammon in this laboratory, I have found them in only one or two embryos, ^ Piatt, 1891.2, p. 101. (Full data for references in foot notes are given in bibliography.) 2 Neal, 1898, p. 232. RECURRENT BRANCHES OF ABDUCENS NERVE 373 and then only as two or three short nerve fibers, soon lost and with no connection with the muscle. Dohrn in an early paper describes the abducens in Torpedo: it is further noteworthy that the root fibers at their first appearance are directed analwards, like the motor spinal nerves, and that only after a certain course in this direction the separate root bundles run together into a common nerve stem, directed forward." This is obviously not meant to imply a recurrent branch of the abducens in the strict sense; but later Dohrn* did find such branches in Heptanchus cinereus. On the left side of an embryo of 23 mm. he observed and drew carefully a large branch running dorsalward and connecting with the myotome called by him 'u.' He apparently did not know of Miss Piatt's work, and only later of Neal's, to which he refers in a foot-note. The branch was represented on the right side by a much smaller nerve, reaching only half as far. He considered it so little likely that such a picture should be pure chance, that it became of great importance to search in the other embryos to see whether in them also a ramus recurrens of the abducens could be found, and in fact succeeded in finding it in a 14-mm. embryo on both sides, though in a much reduced form, as a short wavy nerve of only one fiber; and in an embryo of 20 mm. on the left side only, also as a short branch. Thus after careful search he found it in three out of some dozen Heptanchus embryos studied, and in all but one case "in a much reduced form." Belogolowy,^ studying the cranial nerves of the chick, finds frequently three groups of rootlets for the abducens, the first pointing forward, the second indifferent, and the third pointing mostly backward; they become, as his figures show, connected with each other and with the most anterior hypoglossal roots by slender bundles of fibers, making a continuous nerve parallel with the floor of the medulla and prolonging the abducens caudally to the hypoglossal nerve. This arrangement is found in chicks of between three and five days' incubation, and is termi 3 Dohrn, 1890-2, p. 343. 4 Dohrn, 1901, p. 28. ^ Belogolowy, 1910, p. 271. 374 JOHN LEWIS BREMER nated by the loss of the ventral roots between the two definitive nerves, so that the nerve origins become separated by a widening gap. None of these transient rootlets or connections grows toward the vagus or glossopharyngeal nerves, and there is no evidence that any fibers have an independent growth caudally from the permanent abducens roots. Belogolowy examined also pig embryos for comparison of the head nerves with those of chicks, and figures in an embryo of 3 mm. frontoparietal measurement (about 8 to 9 mm. greatest length) short rootlets continuing the ventral series between the abducens and hypoglossal, but without joining either of these nerves. These I have also found in the majority of the 125 class specimens of 10- to 12-mm. pig embryos cut in transverse section (though in this plane they are not conspicuous) and in some of the younger specimens in the collection. None of these is of more than a few fibers, and, as Belogolowy noticed, none remains for longer than the growth period of 5 or 6 mm. In sheep and rabbit I find even these rootlets much less frequently, so that a recurrent branch of the abducens must be very rare in these embryos. In many of the rabbit embryos of thirteen or fourteen days the anterior XII roots run between the vagus and the glossopharyngeal nerves, but these seem to disappear whoUy after this time. They have no connection with the abducens, all of whose fibers point immediately forward. In Lacerta muralis, between 5 and 7 mm. in length, and in Chrysemys marginata, from 7 to 9 mm., the abducens is provided with a few posterior rootlets, joined in loops, behind the permanent roots. In this respect these two species of reptile resemble the birds (chick), but the rootlets do not extend as far caudally in the reptiles, and there is never, apparently, a continuous connection between the abducens and the hypoglossal. I have found no remains of these rootlets in older embryos. From this brief resume of the findings in Acanthias, Heptanchus, chick, reptile, pig, sheep, and rabbit, it is clear that transient nerve roots arising from the ventral surface of the hind brain between the permanent abducens and hypoglossal roots are to be expected in a certain, usually small, percentage of these RECURRENT BRANCHES OF ABDUCENS NERVE 375 embryos. They are commonly small enough to escape notice unless under special scrutiny. In man they are more constant, larger, and apparently of longer duration (found in relatively older embryos) than in any other of these groups. Of the twenty-four human embryos in this collection between the first appearance of the abducens nerve and the crown-rump length of 18 mm. only two are entirely lacking in this respect; three others have the recurrent branch on one side only. The two embryos of this group, one of 12 mm. (H. E. C, no. 816), the other of 16 mm. (H. E. C, no, 1128), which lack the recurrent braneh, show instead long anterior hypoglossal roots running in front of the vagus nerve, between it and the glossopharyngeal. Between 18 mm. and 31 mm. there are again twenty-four embryos, of which twelve are provided with the recurrent branch on one or both sides. Beyond this age I have not found it present. As there is evidence that these branches may degenerate and disappear as early as the 15-mm. stage, the first group of twenty-four embryos would seem to be a fairer index of the usual occurrence of this feature, and one might state therefore that the recurrent branch of the abducens is present in over 90 per cent of human embryos; in other words, that their absence is an anomaly, their presence, up to the 18-mm. stage, to be normally expected. In spite of this fact, these branches have been noticed only three times, to my knowledge, in papers on the description of individual human embryos. Phisalix, in 1887, writing of the cranial nerves of a human embryo of thirty-two days, 10 mm., seeks to prove that the cranial nerves are similar to the spinal, and after mentioning the dorsal and lateral roots of the vagus and glossopharyngeal nerves continues:^ "Beside these intrabulbar motor roots I have discovered others which differ in no way from the anterior spinal roots, neither in their origin nor in their connections. Thus for each of these nerves there is a very slender bundle of motor fibers which arises from the bulb, at its base near the median line, and which runs to join the nerve at the distal end of the superior ganglion." No drawing accom • Phisalix, 1887.2, p. 243. 376 JOHN LEWIS BREMER panies this description, but it is evident that either anterior hypoglossal or posterior abducens roots are indicated. Thyng^ speaks of the presence in a 17.8-mm. embryo of a caudal aberrant root on each side running from the region of the vagus and glossopharyngeal nerves to join the abducens. This embryo is in this laboratory, and represents one of the better examples of recurrent branches — the root to the abducens being actually a branch from this nerve, ending below the upper gangUon of the vagus. Barniville^ describes a posterior root or branch of the abducens in a human embryo of 8.5 mm., which, joining a branch from the most anterior hypoglossal nerve, turns dorsally, like a dorsal ramus of one of the spinal nerves, and extends to the level of the ganglionic commissure of the vagus, where it comes very close to the brain wall. Barniville is in doubt whether it actually has a connection here or not, and therefore in doubt whether to call the fibers sensory roots of the hypoglossal and abducens, or dorsal rami of these nerves. He refers to my earlier figures, where similar dorsal rami are shown. Streeter, in his article on the cranial nerves in the Keibel-Mall Textbook of Embryology, mentions the aberrant roots of the abducens and hypoglossal nerves as possible results of a caudal extension of the abducens nucleus or cephalic extension of the hypoglossal nucleus, but gives no hint of their frequency, and does not mention or figure the recurrent branches. Elze, in 1907, describes two roots of the abducens in an embryo of 7 mm., one arising far caudally; but this is not a recurrent branch, as the posterior fibers pass forward from the brain to join the anterior root on the way to the eye muscles. Several reasons may be considered for the failure of other investigators to notice these recurrent branches of the abducens in man: the usually transverse plane of section places the abducens roots at a disadvantage, cutting them across where they are smallest at their origin from the medulla floor and where small branches of the vessels entering the medulla are very numerous and in close relationship with the nerve roots, thus ^ Thyng, 1914, p. 56. 8 Barniville, 1914. p. 11. RECURRENT BRANCHES OF ABDUCENS NERVE 377 making their presence difficult to detect; in fact, the roots may run in the walls of these vessels for a considerable distance. The not uncommon shrinkage of the brain in preservation maybreak the small abducens roots and make their connections uncertain. Another consideration is that in the usual reconstruction of the cranial nerves as seen from the side the abducens is partially hidden by the large ganglion of the trigeminal and by the otocyst and its attendant ganglia, and therefore perhaps not so much an object of study as the other nerves. Finally, it may be possible that the human embryos of this collection are too few to justify any statements as to the frequency of the presence of these recurrent branches, and that with a wider study a much smaller percentage would be obtained. Considerable variation in the exact origin and course of the recurrent branches is found, but it is possible to designate certain types into which all may be grouped. In their method of origin from the medulla floor they may arise with the main abducens roots, some of the fibers from the same roots pointing caudally, some curving sharply forward, so that there is a crossing of the individual bundles in the same roots; or the recurrent nerves may arise by one or more separate roots, closely caudal to the last abducens root. The usual course is ventral and lateral toward the jugular foramen, with the vagus and glossopharyngeal nerves, but some pass laterally, to the dDrsal musculature of the neck, while others turn dorsally, as in the cases described by Barniville and by me formerly, to end beside the more dorsal part of the hind brain. It is quite common to find the recurrent branch doubled on one or both sides of an embryo, as in figure 1, and, as is shown there, the two branches may be joined at some distance from the brain wall. In size there is again great variation; some recurrent nerves are of a few fibers only, others as large or larger than the abducens itself; frequently the large branches end in terminal expansions. In length they also vary, some running only a few micra, others extending past the vagus nerve far ventrally or far laterally to the musculature, as has been noted. 378 JOHN LEWIS BREMER The fate of these recurrent branches is undoubtedly degeneration and complete disappearance, as no record has ever been made, to my knowledge, of their presence after the 31-mm. stage. The course of this degeneration can be followed in my specimens with a fair degree of certainty. At first the nerve fibers of the branch, especially at their tips, become wavy and shghtly separated from one another, thus causing the terminal expansion mentioned above. Then follows a reduction in the number of fibers in the proximal portion of the branch, near the nerve roots. All gradations of this peculiar feature can be seen in different embryos, from a scarcely noticeable change of size in the two ends' of the branch to a complete severance of the distal end from its origin, so that the nerve fibers, still readily recognizable, lie in the mesenchyma entirely separated from the medulla and from the main abducens nerve. Whether or not this is preceded by the degeneration of the nerve cells in the abducens nucleus, I have no means of judging, as the material available is not stained to show the details of nerve-cell degeneration. No obvious differences in the cells of the nucleus are noticeable in any of the specimens. Similar free fibers have been noticed and figured before by me and by Thyng^ in relation to the degenerating anterior hypoglossal roots, but the result was not so striking in that case, as the nerve bundles were distally connected, at least physically, with a live nerve; the degenerating branches of the abducens nerve, with no connection at either end, seem much more surprising. The final dissolution or absorption of the disconnected nerve fibers is apparently accompUshed by the action of phagocytes. The process, as shown in figure 4, is indicated by the presence within the nerve bundle of phagocytic cells in various stages of engorgement, as evidenced by vacuoles in the cell protoplasm; they are apparently recruited from the surrounding mesenchyma, and pass out into it again after engorgement, or perhaps they are cells of the perineurium in a new role. The cell reaction is slight, as only a few small phagocytes are ever found, showing that the process is slow and gradual; but these few cells are easily recognized, as

  • See figure 1, x, and also Bremer, 1908, figures 1 and 2, x. Thyng, 1914, plate 3.

RECURRENT BRANCHES OF ABDUCENS NERVE 379 they are the only cells among the nerve fibers which throughout the abducens are free from nuclei, except for the perineurium. The end result is the total disappearance of the recurrent branch. In one embryo I have seen a small group of phagocytes, present in only three consecutive sections, clustered about two or three scarcely recognizable nerve fibers. Only its position, along the Diagram A The floor of the medulla is seen in profile from the right side, with the proximal ends of the glossopharyngeal, vagus, and hypoglossal nerves. The main abducens points to the right, the recurrent branch to the left. In nos. 1, 2, and 3 the different courses of the recurrent branches are shown; to the occipital somitic muscles, to the branchial muscles, accompanying either the vagus or glossopharyngeal nerves, and to the postotic dorsal musculature, respectively. No. 4 gives a variation showing a separate root for the recurrent branch. No. 5 shows a degenerating recurrent branch so short that its destination is not certain, and already disconnected from its nucleus in the medulla. 380 JOHN LEWIS BREMER usual course of the recurrent branch, made me reasonably certain that this group of cells represented the last stage in the process of dissolution of this nerve. Usually the recurrent branches either degenerate relatively early, before the precartilage of the base of the skull is laid down, or if they persist pass toward or through the jugular foramen with the vagus and glossopharyngeal nerves. On the left side, however, of one embryo of 25 mm. (H. E. C, no. 2042) the long well-marked recurrent branch, larger than the main abducens nerve, passes through a separate foramen, walled off from the jugular foramen by a substantial plate of precartilage, and containing in addition to the recurrent branch of the abducens a meningeal branch from the trunk of the vagus, and a small vein, probably the inferior petrosal sinus. These two structures commonly accompany long recurrent branches of this type, but the partition of precartilage across the jugular foramen I have seen in no other embryo. It probably marks off the anterior compartment of the jugular foramen mentioned in some textbooks of anatomy as containing the inferior petrosal sinus, but in other embryos of this age, and even considerably older, the cartilaginous plate does not exist, nor is it present on the right side of this embryo, where the recurrent branch is much shorter. The possibility is strongly suggested that this early subdivision of the jugular foramen is caused by the presence of the recurrent branch, and a bony canal in this position might even be looked for as an anomaly. In attempting to explain the fact that the recurrent branches of the abducens nerve attain in man their greatest duration and size and frequency, obviously phylogenetic considerations are of no assistance. Nor can Ave turn with any hope of enhghtenment to the history of the head somites, for they are not recognized generally in man, in spite of a paper by Zimmerman, who reported finding them in an embryo of 3.5 mm. We are forced to an examination of the nerves themselves at their earher stages, and of any adjacent structures which might influence them, in the search for a possible explanation. RECURRENT BRANCHES OF ABDUCENS NERVE 381 The earliest abducens root fibers in man leave the medulla wall at right angles to its surface, and continue growing in this direction for a considerable distance. In an embryo of 7.5 mm. (H. E. C, no. 256) and in another of 9.6 mm. (H. E. C, no. 1001) the fibers point straight away from the brain wall. In one of 6 mm. (H. E. C, no. 2094) the very few fibers as yet present curve shghtly at their distal ends, and point some craniaUy, some caudally, in about equal numbers. In an embryo of 4.6 mm. (H. E. C, no. 374) a single rootlet divides, sending a recurrent branch caudally and the main nerve cranially, both short, but of about equal size and length. Another rootlet is just emerging from the brain on one side a short distance cranially. Incidentally it will be noted that the total length of these embryos is not a reliable measure of their development at this time, the smallest of this group being really the oldest in general configuration as well as in the growth of the abducens nerve. The first rootlet, then, seems to be attracted both forward and backward with equal force, and the individual fibers seem to hesitate, and often interdigitate in their final course. I have already noted that Dohrn found these rootlets in a similar condition in Torpedo, but after a short caudal growth they usually all turned forward. In pig, sheep, and rabbit, on the other hand, the first abducens roots turn cranially almost from the start. Apparently, then, there is in man some force acting on the earliest growing fibers to turn some of them caudally, a force which is lacking or of insufficient strength at the crucial moment in other mammals. Another point to be taken into consideration is the relative time of the emergence of the numerous abducens rootlets. Belogolowyi" makes the observation that in chicks the anterior spinal nerves appear earliest, and the motor nerves of the brain later, but that the order of development of this forward group is subject to great variation. Of the abducens roots particularly he makes this generalization, 11 that in birds they develop one after the other from behind forward, while in mammals the reverse is »» Belogolowy, 1910, p. 270. " Belogolowy, 1910, p. 322. 382 JOHN LEWIS BREMER true, the growth of the abducens rootlets spreading backward toward the hypoglossal nerve. This generalization is, I think, too broad, and was probably made after the study of only the two classes of embryo mentioned in his paper, namely, the chick and the pig. It also might properly be amended to include the other ventral cranial nerve, the oculomotor, arising still farther forward. With this in view, I have examined embryos of sheep, pig, and rabbit, and find in each that, as Belogolowy states, the abducens roots emerge serially from before backward, and that in each the development of the oculomotor nerve precedes that of the abducens. In man, on the other hand, if one may judge by the four embryos available at this critical stage, the abducens is the earlier of the two nerves, and the growth wave spreads forward. The oculomotor nerve seems to grow much faster and reaches the eye muscles sooner than the abducens, which would lead to a false idea of their relative priority. Thus the first rootlets of the abducens are probably the caudal ones, nearer and therefore more suceptible to any postotic attraction; while in the other mammals studied the earliest rootlets are cranial and therefore removed from this attraction and brought nearer to a pre-otic influence. Both influences act apparently even as far back as the first human roots, vying with each other and causing the divergence of the individual fibers. The attractions suggested are, of course, the masses of developing muscle, the pre-otic eye muscles rapidly developing, and the postotic premuscle mass either degenerating or migrating forward, according to the two prevalent theories of the loss of the postotic myotome. In the human embryo of 6 mm. mentioned above, in which the abducens fibers are short and turn, some cranially, some caudally, and in which the oculomotor nerve is as yet absent, the occipital muscles are already indicated by a continuous band of elongated cells in the somites extending as far forward as the vagus ganglion. Ventrolateral to this ganglion and again ventrolateral to the glossopharyngeal, facial and trigeminal gangha, there are condensations of mesenchyma, but without elongated cells. After a gap there is again a mesenchymal condensation dorsal and caudal to the eye, in which elon RECURRENT BRANCHES OF ABDUCENS NERVE 383 gated cells are found. In the 9.6-mm. and 7.5-mm. embryos of about the same age, the eye-muscle mass is not so clearly defined, but the somitic muscle is continuous up to a point just caudal to the vagus ganglion. In a sheep embryo of 7.2 mm. (H. E. C, no. 1226), on the other hand, the oculomotor nerve is large and can be traced to a well-defined eye-muscle mass, while the somitic muscle ends some distance caudal to the vagus; the abducens is just beginning, and turns almost immediately forward. The same is true in rabbit embryos of 5 mm. (twelve days), as regards the oculomotor and abducens nerves and the disposition and condition of the muscle masses. A difference exists, however, between sheep and rabbit in the extent of the hypoglossal roots, which are confined to a caudal position in sheep, but reach far forward in rabbit, in these early stages, leaving only a small gap between them and the abducens roots. The corollary of this appears in the frequent occurrence in the latter embryos of anterior hypoglossal roots passing in front of the vagus nerve. In the rabbit the earliest roots of the abducens are almost pre-otic and actually nearer to the eye-muscle mass than to the somitic muscle. In the pig the muscles have about the same disposition as in man, but the oculomotor nerve arises first and the eye-muscle mass may be considered to predominate and attract the anterior abducens roots, which arise next in order; the later, more posterior abducens roots, however, point frequently toward the occipital muscles, to which they are nearest, giving the small short-lived recurrent branches, which are always separate roots, not interlaced with those of the abducens proper. In the chick the order of emergence of the abducens rootlets seems to progress usually from behind forward, as Belogolowy said, and they are at first a continuation of the anterior hypoglossal roots. This would lead one to expect recurrent branches, as the order is similar to that found in man, as opposed to sheep and rabbit. The oculomotor nerve is, however, well developed before the abducens appears, probably in conjunction with the large and precocious eye, which is accompanied by precocious eye muscles. The external rectus is already recognizable as a 384 JOHN LEWIS BREMER separate muscle in chicks of little over forty-eight hours' incubation, and in an embryo of about three days, 6.2 mm. (H. E. C, no. 511), is well developed and with clear-cut edges, though the abducens fibers have not yet emerged from the brain. The large eye has also altered the position of this muscle, the caudal end of which lies far caudal to the anterior edge of the trigeminal ganglion, much nearer the otocyst than in mammals. The occipital muscles in this embryo are also readily recognizable, and end with a sharp edge medial to the vagus ganglion. The abducens roots, as they emerge, would perhaps feel the attraction of both muscles, the anterior group of them turning forward, the posterior group backward, and the intermediate group indifferent, as described by Belogolowy. These groups, however, include all the rootlets' back to the hypoglossal nerve, whereas the anterior alone remain permanently as the abducens nerve, and since these have been influenced by the proximity of the external rectus muscle, no recurrent branches in a strict sense are found. Belogolowy figures fibers continuing the abducens caudally, to be sure, but a study of their course and shape shows that they are the remains of more caudal rootlets which were attracted forward to join the abducens by loops, and which now are degenerating, by loss of their connection with the brain, as in the case of the degenerating hypoglossal roots already referred to. In other words, they are the remains of cranially directed fibers from caudal rootlets not caudally directed recurrent fibers. The disposition of nerve roots and muscle masses in Lacerta and Chrysemys is similar to that just described in chicks. The permanent, cranial abducens rootlets are much nearer to the large, external rectus muscle than to even the most cranial of the occipital muscles, and the caudally disposed abducens rootlets degenerate so rapidly that no recurrent branch becomes established. In Acanthias new elements seem to influence the course of the abducens fibers. The lack of a neck bend would remove the occipital muscles relatively farther caudally, but the more complete development of the postotic head cavities would probably more than overbalance this; and the position of the nerve roots RECURRENT BRANCHES OF ABDUCENS NERVE 385 themselves tends further to approximate the postotic muscles and nerve roots. Belogolowy/- as noted by Neal, found that in Acanthias the roots of the abducens arise from neuromeres next posterior to those from which they originate in chicks, thus subjecting the caudal roots to a stronger postotic attraction, represented by the occasional recurrent branch. The same is very probably true in Heptanchus, but no material is available to me for examination. The presence or absence of a recurrent branch would depend upon a delicate balance between the time of emergence of .the fibers from the brain and the degeneration of the postotic somite, or perhaps its ability to produce muscle fibers. It will be seen, then, that the presence of recurrent branches of the abducens is correlated with the proximity of postotic premuscle masses to the emerging roots, supplying apparently an attraction in opposition to that of the eye-muscle group. Probably the degree of differentiation of the various muscles concerned plays a large role in the force of these attractions, independent of their actual proximity, but our lack of knowledge of the minute details of early muscle differentiation, or of possible difference between muscles developed from definite somites and those, like the eye muscles and the branchial musculature, arising from mesenchymal condensations, does not permit us to be critical in this respect. The three types of distribution of the recurrent branches, to the lateral head musculature, to the branchial muscles accompanying the vagus nerve, and to the dorsal side of the head, show further that the postotic influence may be from different sources. The first two types have a common course as far as the line of the vagus and hypoglossal junction, and the majority of recurrent branches follow this course, but end before reaching the vagus, so that it is impossible to tell whether they would have continued laterally or curved to accompany the vagus or hypoglossal nerves. In the first case the muscle attraction would have been the most anterior somite, in the latter the mesodermal condensation to form the branchial muscles. The third type represents those recurrent branches 12 Belogolowy, 1910, p. 279. 386 JOHN LEWIS BREMER which turn doi sally, along the wall of the hind brain. Here we must suppose, if the musculature plays any part in determining their course, that certain cells of the somites migrate dorsally, as in the body somites, attracting the nerve fibers with them as dorsal rami. The same possibility is invoked by Neal and others to explain the course of the trochlear nerve, and it may be of interest to record that in one embryo of 16 mm. (H. E. C, no. 2095) the trochlear itself, though otherwise normal, has dorsal rami in the form of short branches which pass dorsally from each nerve just lateral to the chiasma. The lateral muscles have retained their position, the ventral or branchial and the dorsal muscles have migrated, carrying their nerve branches with them. In connection with this discussion of the probable causes of the development of the recurrent branches of the abducens, it is of interest to describe a human embryo of 18 mm. which shows a total absence of this nerve to the external rectus muscle. The embryo, which was cut in the sagittal plane, was found too poorly preserved for the regular Embryological Collection, and so kept in a supplementary group, and numbered 393. In spite of the poor preservation, however, all nerves can be readily followed, and such structures as muscles and organs easily recognized ; the brain and allied structures have, as usual, suffered the most damage. Complete absence of the abducens nerve on one or both sides is recorded as a rare anomaly in text-books of anatomy, but I know of no mention of its occurrence in embryonic stages. The external rectus muscle is in these cases usually supplied by a branch from the oculomotor nerve, and that is true in this instance. No other anomalies, except those noted in connection with this nerve, were found on casual examination. On the left side the abducens is entirely absent; no trace of fibers arising from the medulla in the usual ventral position can be found between the roots of the oculomotor and those of the hypoglossal nerve. A peculiarity in the lateral nerve roots is the presence of a fairly large bundle which emerges between and in line with the lateral roots of the trigeminal and facial RECURRENT BRANCHES OF ABDUCENS NERVE 387 nerves and runs medially and ventrally to join the tympanic branch from the petrosal ganglion of the glossopharyngeal nerve, median to the gasserian ganglion, to form with it the small superficial petrosal nerve. It is to be considered, therefore, merely as the motor facial component to this nerve, which arises more cranially than usual and takes a separate course, at a considerable distance from the geniculate ganglion. It passes through a separate foramen in the precartilage of the base of the scull. On the right side of this embryo there is again no forwardrunning abducens nerve; the external rectus muscle is supplied by a branch of the oculomotor. But in the usual position of the abducens roots fibers arise which turn ventrally, slightly caudally, and laterally to join the motor division of the facial nerve, distal to the geniculate ganglion. Thus a ventral root from a position slightly cranial unites with a lateral root just beyond the ganglion of the accompanying dorsal fibers, and the relations of a complete nerve are closely approximated. Applying to this case the ideas gathered from the study of the recurrent branches of the abducens, I examined the eye muscles of this embryo, and also the various parts of the eye itself. The external rectus muscle was clearly differentiated on both sides, the eyes were apparently normal, though the retina and lens w^ere in a poor state of preservation. But in comparison with other embryos of the same size and even smaller, it was found that the eye and all its appurtenances were definitely retarded. The retina was thinner and less differentiated, the lens showed shorter fibers and was still widely vesicular, the eyelids ^vere less developed and the anterior chamber was not even suggested, though already present in other embryos of 18 mm. As a whole, the eye of this embryo resembled most closely that of a 14.5 mm. embryo. With this retardation of the eyeball, the eye muscles were found to be much smaller than usual, though clearly differentiated. The probable history of the abducens nerves in this embryo can, then, be sketched as follows: all of the emerging fibers were submitted to the caudal attraction of the postotic musculature THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 2 388 JOHN LEWIS BREMER or of the branchial muscles, and, because of the probable late development of the eye and its accompanying structures, were relieved from the usual counter-attraction of the eye muscles. Even the most anterior rootlets thus turned caudally, not, to be sure, to the occipital muscles, but to the ventral condensation for the muscles supplied by the facial nerve. A little later, after the abducens fibers had all emerged and taken a caudal course, the eye muscles developed and attracted the oculomotor nerve, which, since the external rectus muscle was unprovided by its usual nerve, sent an unusual branch to this muscle also. Of the recurrent branches of the abducens we can imagine that only the most anterior on one side has persisted, all the others undergoing the degeneration commonly found before this age. Probably the one remaining would soon have disappeared; in fact, its caliber at its origin is already much less than at its union with the facial nerve, and in the brain the nucleus of the abducens, apparently entirely absent on the opposite side, is very doubtfully represented by a slight grouping of cells. It seems probable, then, that the absence of the abducens nerve in a human subject is due to some very unusual retardation of the development of the eye, which causes the abducens fibers, released at the crucial moment from any anterior attraction, to turn caudally, thus leaving the external rectus muscle free to be annexed by some other nerve. With the degeneration of the recurrent abducens fibers, as is always to be expected, the nerve in these cases is completely lost, and no trace of its original emergence from the brain is carried into adult life. SUMMARY Recurrent branches of the abducens nerve consist of fibers whose growth is in a caudal or ventrocaudal or dorsocaudal direction. They are present infrequently in Acanthias, Heptanchus, and pig, absent in chick, lizard, sheep, and rabbit. They are most frequent in man, where they appear in about 90 per cent of embryos up to 18 mm., and less frequently up to 31 mm., since they msiy begin to degenerate at 15 mm. Degeneration RECURRENT BRANCHES OF ABDUCENS NERVE 389 is shown by the separation of the fibers from the brain (probably after loss of the nerve cells in the abducens nucleus) and their digestion by phagocytes. The distribution, of those fibers long enough to make their destination sure, is to the anterior spinal muscles, the branchial musculature, or the dorsal muscles of the head. They may arise from the same roots as the abducens proper, or by separate but closely adjacent rootlets. They are absent in embryos whose eye muscles predominate over the occipital muscles, either by proximity or precocity, but the position of the first abducens roots on the floor of the medulla is also a factor, as the more caudally these arise, the more often are recurrent branches to be expected. Among the mammals studied, man alone shows the abducens roots emerging in order from behind forward, with the oculomotor appearing later than the abducens. In sheep, pig, and rabbit the growth wave is reversed, beginning with the oculomotor and extending caudally. In man, then, the earliest abducens roots are caudal, nearer to the occipital muscles, in the other mammals they are cranial, approaching the eye-muscle mass. In birds and reptiles the eye and its muscles are not only precocious, but the muscles are actually displaced caudally by the large eyeball, so that their influence on the emerging abducens fibers extends far caudally and only the fibers close to the hypoglossal roots turn caudally. These are soon lost, and the permanent abducens shows no recurrent branches. In fishes the abducens roots arise from more caudal neuromeres, and occasionally some fibers turn caudally, but only as a rare anomaly. 390 JOHN LEWIS BREMER LITERATURE CITED Barniville, H. L. 1914 The morphology and histology of a human embrj^o of 8.5 mm. Jour. Anat. and Phys., vol. 49. Belogolowy, J. 1910 Zur Entwickelung der Kopfnerven bei Vogel. Bull. Sec. Imp. Moscou, Hft. 3, 4. Bremer, J. L. 1908 Aberrant roots and branches of the abducent and hypoglossal nerves. Jour. Comp. Neur., vol. 18, no. 6. DoHRN A. 1890.2 Studien u. s. w. 15, Mitt. Zool. Stat. Neapel, Bd. 9. 1901 Studien u. s. w. 18-21, Mitt. Zool. Stat. Neapel, Bd. 15. Elze, C. 1907 Beschreibung eines menschlichen Embryo von zirca 7 mm. Anat. Hefte, Heft 106. Neal, H. V. 1898 The segmentation of the nervous system in Squalus acan thias. Bull. Mus. Comp. Zool. Harvard, vol. 31, no. 7. 1914 The morphology of the eye muscle nerves. Jour. Morph., vol. 25, no. 1. Platt, J. B. 1891 Morphology of the vertebrate head. Jour. Morph., vol. 5, no. 1. Phisalix, C. 1887 Sur les nerfs cranien d'un embryo humain de trente-deux jours. Comp. rend. acad. sc. Paris, T. 104, no. 4. Scammon, R. E. 1911 Normal plates on the development of Squalus acanthias (F. Keibel). Streeter, G. L. 1912 Development of the nervous system, in the Keibel Mall Human Embrj'ology. Thyng, F. W. 1914 The anatomy of a 17.8 mm. human embryo. Am. Jour. Anat., vol. 17, no. 1. PLATES 391 •~^ - aj o) Ki X. <o ^ X o (U to to & O o m a) tc ^ 1* «3 CO 3 c 03 bC .s "-3 to o o s CO O to CO O 'bi) .2 bO c3 > to 3d s s ^^ o to S '5 _C T3 c3 >5 r-F-f ■ w o QJ C > to 0? o wi fl +i a s o o c3 .a ^ 3 >% -£:: (N c3 bC -(-5 PL, , bO 4:; -3 o -c r « ^ o S « c 't^ -ti c -c -3 rt fl O ^ O p Jh cj S ^ s ^ <B CO _ Si O --H to" CJ 'i? -d c3 S 3 a " l-H O a " 2 II. I 2.H r^ CO to ^ » tH C3 .-^ o q; '" j- bC o ^ M 9s 03 C3 O O ^ " 'I o & (O " 'O .'T: bC £5 j^^ -. ^ ^ ,. „ -^ bC '=^ :S ^ fl ^ Ji -a 3 S u X 03 -IJ bC «^ .S =3 CO o O — ' ^^ ^ «*-■ & (U Pi w •z W O & Q K « w -fl S f^ «  o «  w to K ^ O z z O w » 393 Ah 2 ii.-'-S ^ « o o 3 oj o o o o 5 ^ >>.ii -5 -c _, o 2 c cj P rt o — > S! ^ ^ C C »2 3 - q; jE, rt .^ > ^ _2 c ^ c 5 ji; o c ^ ~ ;i t; o «  -^ Ci O ,i^ '^ « kT J-i 02 fl X X a o c ^ & CD H t«  _o _c '^ o o o O '-1J o CO c,-a o CO CO -^ 02 o c -f^ o ^ «  rC to .2 «4-l o ^^ (M ji; bC o 5 o «  c»r CO 5 O C d o o Sd tC o _c o a 7^, • *N a (in o H > CO o a c ? C; s CO tl^ -c 3 rt o O c ■» f- 3 n ■^1 3 "S — CO bb O 394 C B o 395 PLATE 3 EXPLANATION OF FIGUKE 4 Detail under high power of the tip of the lower recvirrent branch of the abdu^ cens shown in figure 1. The nerve fibers, surrounded by a perineurium, have been invaded by phagocytes, which become vacuolated and apparently cause the dissolution of the fibers, as evidenced by the vacant spaces within the nerve sheath. Some of the engorged phagocytes can be seen among the cells of the perineurium. (H. E. C, no. 2095, sect. 269.) X 600. 396 RECURRENT BRANCHES OF ABDUCENS NERVE JOHN LEWIS BREMER PLATE 3 g .^^ ^^'M J rjm .^^ % 's,^ ® s® >■ «  ^■, v ,<9 397 Resumen por el autor, Tokuyasu Kudo, Universidad de Minnesota. Estudios sobre los efectos de la sed. I. Los efectos de la sed sobre el peso de los diversos organos y sistemas de la rata albina adulta. En los experimentos en que se someti6 a los animales a una sed aguda, los cuales duraron desde seis hasta diez y seis dias, la cantidad de agua susministrada con los alimentos era muy escasa y provoco una perdida media de 36.1 por ciento del peso total de las ocho ratas objeto del experimento. Algunas de las glandulas perdieron mas agua que el cuerpo en total, menos agua la musculatura y el tegumento, mientras que el cambio fue aun menor en el esqueleto, sistema nervioso y en algunas glandulas. En los experimentos de sed cronica, que duraron de cuarenta y siete a cincuenta y cinco dias, la cantidad de agua susministrada no era tan pequena como en los mencionados mas arriba, pero a causa de la duracion del experimento la perdida media total de agua fue mayor, alcanzando el 52.4 por ciento en las siete ratas empleadas, al cabo de siete semanas. Las perdidas de peso en los diferentes organos y sistemas fueron semejantes a las que siguen despues de una sed aguda. L'n animal, despues de una inanicion total de once dias, presento perdidas de peso semejantes a las que aparecen despues de la sed cronica. Estos resultados son semejantes a los publicados por otros observadores, que ban estudiado los efectos de la inanicion total o parcial. Es dudoso, sin embargo, en que grado las perdidas que ha encontrado el autor son los efectos especificos de la sed, o en que grado dependen del regimen alimenticio defectuoso a que se someti6 a los animales. Translation by Jose F. Nonidez Cornell Medical College, New York AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, NOVEMBER 15 STUDIES ON THE EFFECTS OF THIRST I. EFFECTS OF THIRST ON THE WEIGHTS OF THE VARIOUS ORGANS AND SYSTEMS OF ADULT ALBINO RATS TOKUYASU KUDO The Institute of Anatomy, University of Minnesota, Minneapolis THREE TABLES CONTENTS Material and methods 402 General observations 408 Body weight 409 Lengths of body and tail 410 Weights of organs and systems 411 Integument, skeleton, musculature, viscera and 'remainder,' brain, spinal cord, sciatic nerves, eyeballs, heart and aorta, spleen, lungs, parotid and submaxillary glands, liver, pancreas, stomach and intestines, kidneys and bladder, testes, epididymis, thyroid gland, thymus, suprarenal glands, hypophysis. Discussion 420 Summary 430 The importance of water for the Hving organism is well recognized, but the effects of thirst (water inanition) have been studied only to a relatively slight extent. It has long been known that a diet deficient in water causes a loss in body weight in various species of animals, and this principle has often been followed in the treatment of human obesity. As to the effects upon the individual organs, however, but few observations have been published. The present investigation was undertaken to determine the changes in the weight of the body and of the various organs in adult albino rats subjected to a relatively dry diet for various periods. The work was done in the Institute of Anatomy at the University of Minnesota. This opportunity is taken to express my indebtedness to Dr. CM. Jackson, Director of the Department, for valuable aid and direction. 399 400 TOKUYASU KUDO MATERIAL AND METHODS The albino rat (]Mus norvegicus albinus) was chosen for use in this experiment. As the rat normally requires but little water in its diet, it seems (like the pigeon) to be especially suitable for thirst experiments. The material used in the present experiments included twentyfive adult albino rats from the colony in the Institute of Anatomy. Three died during the tests, and are excluded because the coagulated blood from postmortem congestion might affect the weights of the organs. Two additional females (one pregnant) are also excluded from the tables for lack of adequate controls. The remaining twenty rats (table 1), which were killed and autopsied, are all males except one. Twelve of these were from six litters of known age (167 to 259 days). The other eight were stock rats of unknown age. Four of the rats were used for normal controls. They include the one female, which, however, is excluded in the case of organs (suprarenals, hypophysis, and gonads) that show a sex difference in weight. The rats had all been fed chiefly upon maize, and Graham bread soaked with whole milk, with occasional fresh vegetables. They were usually placed upon a maintenance diet of whole milk and dry biscuit for some days before beginning the experiment. The biscuit used in the experiments was the 'Old Grist Mill' brand ('dog-bread') manufactured by Potter and Wrightington, Charlestown, Massachusetts, according to Castle's formula, and having approximately the following composition: per cent Moisture at 100°C 4.63 Phosphates as tricalcium 6 . 73 Crude fiber 0.58 Proteids 15.40 Carbohydrates, starch, sugar, etc 65.57 Ether extract (fat, etc.) 7. 10 Estimated calories per gram 4.6 The test rats were weighed daily and individual records kept. The temperature of the animal room was fairly constant at about 77° F. (range 65° to 80°). QHOO ^VNIJS ,HaaNivrv3H, dnoao IVHaOSIA aHaiviQosQw (Z) Hi'iivaj aNV (Z) snaanjaH MOX -aqass 9aoN -lovnixavo NOiaaaas snoiNaiMvon ixawaoaiNi Sh 5 ^ ^; ^ 2; <! 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This allowed the feces and urine to drop through. Both feces and food were carefully weighed. Acute thirst. Nine rats (eight included in table 1) were used for the acute-thirst experiment. They were allowed dry food ad libitum. The greatest difficulty in thirst experiments is to induce the animals to eat dry food. If they refuse to eat, the result is total inanition, instead of water inanition. The occurrence of partial inanition (due to deficiency in vitamines or other necessary dietetic elements) must also be avoided so far as possible. Various methods of feeding in the acute-thirst experiments (table 1) were used as follows : Group 1. Two rats (K 4.1, K 3.4) were fed with maize (Indian corn) only, either whole grains or crushed, without water or milk. They consumed an average of about 1 gram daily. The feces averaged 2 to 2.7 grams daily. Group 2. Two rats (Sr, 8, Si. 1) were fed with dry biscuit ad. libitum and 3 grams of milk daily. Each rat daily ate 2.5 grams of biscuit and passed 2 grams of feces (average) . Group 3. Two rats (K 3.3, K 3.2) were fed with dry biscuit and 5 grams of milk daily. They daily ate 2.9 grams of dry biscuit and passed 0.8 gram of feces. Group 4. In this group, two rats (K 7.1 and Si. 3) in the earlier days of the test were given (in addition to dry biscuit or maize and biscuit) relatively greater amounts of milk (15 to 20 grams), which was gradually reduced in the later days to 2 to 2.5 grams. By this method the rat ate relatively more food and continued active for a much longer period (killed after sixteen to seventeen days). The daily average food intake for rat K 7.1 was 1.9 grams of biscuit and 6.3 grams of milk. The feces averaged 2.7 grams daily. For the other rat (Si. 3) the corresponding daily averages were 2 grams of biscuit and maize; 5.1 grams of milk; 0.4 gram of feces. In addition to the groups above mentioned, a male rat (Si. 2) was kept alive without food or water (D ; total inanition) for eleven days. This rat will be compared with those in the thirst series. EFFECTS OF THIRST — ALBINO RATS 405 Chronic thirst. In the chronic-thirst experiments, seven rats were used. Six (table 1) were fed upon dry biscuit ad Hbitum with milk gradually decreased in amount, so as to reduce the body weight about 1 per cent (of the initial weight) daily. They were killed after an average of 50.6 (range 47 to 55) days (table 1) . These rats daily ate an average of 6.9 grams of dry biscuit and 9 grams of milk. The feces averaged 1.1 grams daily. One rat (Sr. 3) ate a daily average of 6.7 grams of maize and 6.5 grams of whole milk. Another rat (Si. 4) was fed with dry biscuit and 6 grams of milk daily. This rat took an average of 6.3 grams of biscuit and passed an average of 1 gram of feces daily. In the rats of the acute-thirst series, it was observed that those getting 5 grams of milk a day (that is, only a little less than in the chronic series above) weakened rapidly and could live only about ten days. It therefore appears that, under the conditions stated, the water contained in about 6 grams of milk represents a critical limit, below which the rats can live but a few days. In order to determine the amount of dry biscuit or corn required for maintenance, when water is allowed ad libitum, supplementary experiments were made upon adult rats. It was found that under these conditions about 13 to 15 grams of dry biscuit or maize are required to maintain approximately constant body weight for short periods. Jackson ('15) found that in adult rats with water ad libitum a diet of Graham bread soaked in whole milk and amounting to 10 per cent of the body weight daily is not quite sufficient for maintenance. It is therefore evident that in the present experiments the food-intake was not sufficient for maintenance, so that to some extent the effects of general inanition are added to those of thirst. At the end of both acute and chronic thirst experiments, the rats were killed by chloroform and the various organs and parts carefully dissected and weighed. The technique followed is that described by Donaldson ('15), with a few modifications. The submaxillary glands and thyroid gland were removed first. The blood is so thick during thirst that slight pressure upon the body is necessary to obtain any appreciable flow even from the larger vessels. The organs and parts upon removal were placed in a closed jar upon glass plates resting on moist filter-paper. 406 TOKUYASU KUDO The organs, after weighing, were dried to constant weight in an oven at about 95°C., in order to determine their water content (not included in this paper). Percentage losses in the various organs were calculated as follows. The average weight (table 2) for each organ or part in the test rats was compared with the corresponding average for the controls. The data in the column 'Difference' express the apparent percentage change ( + or — ) as the result of the experiment. In calculating these percentage changes the slight differences in average initial body weights between the controls and the test rats are ignored. For various reasons, the data obtained do not justify any final conclusions as to the effects of thirst. The number of animals used (especially for controls) is comparatively small, and in some cases there are marked individual variations (due partly to unavoidable variations in technique), as shown in table 1. Moreover, it is obviously difficult to separate the effects of thirst from the accompanying inanition due to inadequate food-intake. By comparison of the results M'ith those obtained by inanition with water or b}^ total inanition, however, it is possible to reach some general conclusions of interest and importance. GENERAL OBSERVATIONS The symptoms noted in the albino rats during the thirst experiments are briefly as follows. The skin becomes roughened and the hairs are easily detached. (Likewise noted in dogs on a dry diet by Bowin, '80.) Dryness and desquamation were observed in the skin of the plantar surfaces. The fecal material is usually hard, but sometimes diarrheal in character. The urine is scanty. Opacity of the lens apparently caused visual disturbance in some cases. The conjunctiva was observed in some cases to be congested, and in one case hemorrhagic. Hemorrhage from the nose was noted in several cases. Paralysis of the right posterior extremity occurred in one case. During the period of twelve to twenty-four hours before death the rats will take neither food nor milk and are exhausted, apathetic, and convulsive. Some rats apparently become mentally deranged and will bite, although EFFECTS OF THIRST — ALBINO RATS 407 previously they were very tame. Mental disturbance has also been noted in men during extreme thirst (Tiedemann, '36; Zabaver, '04). The rat (Si. 2) subjected to total inanition ate about half of its tail during the experiment. In the chronic-thirst series, the rats always had the penis extruded, possibly from attempts to get the urine. The general conditions observed at autopsy are as follows. There is extreme general emaciation. The skin and muscles appear dry and are difficult to separate. The blood is very thick. The fat has almost disappeared from the- subcutaneous and muscular tissues, but persists in small amount in the orbit. The adipose tissue of the interscapular region is reduced to a small, brownish-red mass. A small amount of serous fluid is found in the peritoneal, pleural, and pericardial cavities. The appearances of the brain, spinal cord, lungs, and abdominal viscera will be mentioned later in discussing the individual organs. BODY WEIGHT As shown in tables 1 and 2, the average initial gross body weight of eight rats in the acute-thirst series is 218.8 grams and in the three controls, 218.3 grams. The loss in body weight in the acute-thirst series averages 36.1 per cent, the loss in individual cases ranging from 31.8 per cent to 45.9 per cent. This represents nearly the maximum possible loss during the acute-thirst experiments, as the rats were killed only in very advanced stages of inanition. Three other rats of 152, 162, and 208 grams that were fed small amounts of milk (average 3, 2, and 2.8 grams daily) died with losses in body weight of 33.8, 31.6, and 35.7 per cent, respectively. These rats have been excluded from the tables, as previously explained. In the rat (Si. 2) on total inanition, the body weight decreased from 216 grams to 114 grams, a loss of 47.2 per cent in eleven days, at the end of which time the rat was killed. These losses in total and acute water inanition are greater than those obtained by Jackson ('15) in acute inanition with water. In a series of fifteen albino rats, he obtained an average loss of 33.1 per cent (range 25 to 40 per cent) in an average of nine (six to twelve) days. 408 TOKUYASU KUDO In my chronic- thirst series the loss in body weight is remarkably greater than in acute thirst. The average body weight for the seven test rats (tables 1 and 2) shows a loss of 52.4 per cent (range, 50.6 to 60.2 per cent). The loss in body weight in this chronic-thirst series is also markedly greater than that obtained in albino rats in chronic inanition with water by Jackson ('15), which averaged 36.1 per cent (extreme 37.5 per cent) in five weeks. In a dog, given water only, Kumagawa ('98) observed a loss of 56 per cent in the body weight in ninety-eight days. As the result of a dry diet, Bowin ('80) obtained a loss of 50 per cent in the body weight of dogs and rabbits; Scheffer ('52) a loss of 45.8 per cent in pigeons; and Pernice and Scagliosi ('95) a loss up to 41.2 per cent in chickens. In rabbits, Skoritschenko ('83) noted very irregular losses in body and organ weights. In the acute thirst of experimental diarrhea, however, Tobler ('10) produced the death of dogs in three and one-half to seven days, with loss of only 22 to 30 per cent in body weight. In the present experiments there is no constant relation between the percentage loss in body weight and either the initial body weight or the length of the thirst period. However, in each case the loss in body weight during acute thirst is greater during the first half than during the second half of the experiment. As might be expected, the loss during thirst is invariably the greatest on the first day, as found by numerous investigators in various animals and types of inanition. This is probably due chiefly to the reduction in contents of the stomach and intestines. LENGTHS OF BODY AND TAIL The body length is measured from the tip of the nose to the anus and the tail length from the anus to the tip of the tail. The measurements were taken immediately after death, the body and tail being extended by very slight tension. The body length (table 1) in the acute-thirst series shows an average loss of 11.4 per cent and in the chronic-thirst series a loss of 14.7 per cent, in comparison with the controls. In the rat (Si. 2) on total inanition the corresponding apparent decrease is 12.5 per cent. This decrease in the trunk length, as noted by EFFECTS OF THIRST — ALBINO RATS 409 Jackson ('15) in rats on water only, is probably due to shrinkage of the intervertebral disks. In the tail the shrinkage is evident, though less marked than in the body; thus the tail becomes relatively longer in the test rats. WEIGHTS OF ORGANS AND SYSTEMS Integument As shown in table 2, the integument (which includes the skin appendages, external ear, hair, and claws) has in the acute-thirst series apparently decreased in absolute weight from an average of 41.9 grams to 28.5 grams, which is a loss of 31.9 per cent in average weight (table 2). In the chronic-thirst series, the corresponding decrease is from 40.2 grams to 21.3 grams, a loss of 47 per cent. In the rat (Si. 2) on total inanition (table 1) the apparent loss in weight of the integument amounts to 42.5 per cent. Since these losses are only slightly less than those of the whole body, there is but little change in the relative weight of the integument. Jackson ('15) also found that in rats after inanition with water the loss of weight in the integument is nearly proportional to that of the entire body. (Scheffer, '52, and Falck and Scheffer, '54, in the dog on dry diet, found the loss in the body weight (20.7 per cent) somewhat less than that in the integument (25 to 28 per cent.) Skeleton The skeleton (tables 1 and 2) was prepared in two ways. According to Jackson ('15), the bones, together with the cartilages, periosteum, and ligaments, constitute the 'ligamentous skeleton.' The bones and cartilages, after removal of the periosteum and ligaments by immersion for about one hour in 1 per cent of solution of 'gold dust' (a soap mixture) at 90°C., constitute the 'cartilaginous skeleton.' The humerus and femur were also weighed fresh, after removal of the periosteum and ligaments, to avoid possible effects of heating in the soap solution. As shown in table 2, the ligamentous skeleton of the acutethirst series shows a loss of 4.3 per cent in average weight, and no TOKUYASU KUDO (lurinii tlu' ('lii<)ni(^-thirst series a loss of 10.3 per cent. Since the corresponding loss in the body weight is much greater, the skeleton increased correspondingly in relative weight during the experiments. The cartilaginous skeleton similarly shows a loss in average weight of 11.8 per cent in the acute- thirst series and 5 per cent in the chronic-thirst series. (The apparent loss of 34.1 per cent in the skeleton of rat Si. 2, on total inanition (table 1), is due to an error in technique.) The data for the humerus (2) and femur (2) show a loss in average weight of 21.1 per cent in the acute-thirst series and 12.1 per cent in the chronic-thirst series. This indicates that the loss in these large bones is relatively greater than in the remainder of the skeleton. Jackson ('15) found that in albino rats during acute and chronic inanition (with water) there is apparently little or no loss of weight in the skeleton, and cited numerous data showing similar results by other investigators in various species. Falck and Scheffer ('54), in a dog on dry diet, found a loss of 20.7 per cent in body weight and a loss of 5.3 per cent in the ligamentous skeleton. In the acute thirst, caused by experimental diarrhea in dogs, Tobler ('10) found a loss of 4 to 15 per cent in the weight of the (cartilaginous?) skeleton. Thus it is evident that during inanition, either with or without water, the skeleton decreases but slightly in weight. Musculature The musculature in the acute-thirst series shows a loss in average weight of 33.1 per cent and in the chronic-thirst series a loss of 61.2 per cent (table 2). The corresponding percentage losses in body weight and in musculature found by previous observers as a result of thirst are as follows: in the pigeon (Schuchardt, '47), body —43.9, musculature -37; in the dog (Falck and Scheffer, '54), body -20.7; musculature —29.3. During inanition with water, Jackson ('15) observed the following percentage losses in body weight and weight of muscula EFFECTS OF THIRST — ALBINO RATS 411 ture, respectively: acute inanition, body -33.9, musculature -30.9; chronic inanition, body -36.1, musculature -40.8. Results of other investigators cited by Jackson support the conclusion that in general the loss of weight in the musculature during inanition is roughly proportional to the loss in body weight, with a tendency to relatively greater loss during chronic inanition. This is in general agreement with the results of thirst, as above given. Viscera and 'remainder' In the visceral group (tables 1 and 2) have been included the brain, spinal cord, hypophysis, eyeballs, parotid, and submaxillary glands, as well as the thoracic and abdominal viscera. The visceral group in the acute-thirst series shows a loss of 30.6 per cent in average weight and in the chronic-thirst series 42.2 per cent. This is relatively somewhat less than the corresponding loss in body weight. Thus the relative (percentage) weight of the visceral group is not much changed, although (as will appear later) there is great variation in this respect among the individual organs. The 'remainder' includes that which remains after dissecting from the rat body the skin, skeleton, musculature, and the visceral group mentioned above. It does not include the escaped blood and other fluids. It includes the dissectable fat tissue, mesentery, some nerves and vessels, a few small unweighed organs, and lymphatic glands. The latter were apparently enlarged in some of the test rats. The 'remainder' shows a loss of 72.7 per cent in average weight in the acute-thirst series and of 88.8 per cent in the chronic series. In the rat (Si. 2) on total inanition (table 1) the corresponding loss in the 'remainder' amounts to 77.8 per cent. This loss is undoubtedly due chiefly to the loss of fat, which has almost disappeared in the test rats. 412 TOKUYASU KUDO Brain The brain in some cases appeared markedly congested at autopsy. In the acute-thirst series the brain apparently remains nearly constant in average weight with an insignificant increase of 0.12 per cent (table 2). In the chronic-thirst series the average brain weight lost 4.2 per cent. The ininvidual variations are small (table 1). In the rat (Si. 2) on total inanition the corresponding apparent loss in the weight of the brain is 7.6 per cent. The mean brain weight for the controls was about 7 per cent below the table value for rats of this length (Donaldson, '15). The data available in the literatm'e concerning the effects of thirst on brain weight are somewhat contradictory. Falck and Scheffer ('54) in a dog losing 20.7 per cent in body weight, noted an apparent gain of 7.2 per cent in brain weight. Bowin ('80) in rabbits on dry food with 50 per cent loss in body weight, noted an absolute decrease (but relative increase; amounts not stated) in the brain weight. Tobler ('10), in three dogs after experimental diarrhea with loss of 22 to 30 per cent in body weight, observed an apparent gain of 4.2 to 13.7 per cent in brain weight. The increase in weight might perhaps be due partly to the congestion or to indi^ddual differences between the test animals and the controls. As to the effects of ordinary inanition, it has long been known that of all of the organs, the central nervous system is one of the most resistant to changes in weight. Jackson ('15) found an apparent loss of only 5 or 6 per cent in the brain of rats subjected to acute or chronic inanition (with water) and cites from the literature numerous data indicating little or no change in brain weight in other animals. On the whole, it appears evident that, during thirst, both acute and chronic, as well as during ordinary acute and chronic inanition with or without water, there is but little change (usually a slight decrease) in the weight of the brain. McCarrison ('19), as an effect of vitamine-deficient diets, finds an apparent slight decrease in the brain weight of pigeons, but an increase (average 1/7) in monkeys. EFFECTS OF THIRST — ALBINO RATS 413 Spinal cord The spinal cord, like the brain, sometimes appeared definitely congested at autopsy. In the acute-thirst series the spinal cord shows an apparent increase of 1.8 per cent in average weight, and in the chronic-thirst series a loss of 6.7 per cent (table 2). It is evident that in the spinal cord, as in the brain, there is no appreciable change in weight during acute thirst and a slight loss during chronic thirst. The apparent loss of 16.7 per cent in the spinal cord of the rat (Si. 2) on total inanition (table 1) is probably due to an individual variation. Falck and Scheffer ('54) observed an apparent loss of 7.1 per cent in the weight of the spinal cord of a dog kept on dry diet with a loss of 20.7 per cent in body weight. Likewise in albino rats after inanition with water, Jackson ('15) noted no significant change in the weight of the spinal cord during acute inanition and a loss of but 4 per cent during chronic inanition. He cited observations of several previous investigators who obtained similar results in various animals during inanition with or without water. Sciatic nerves In order to study the effects of thirst upon the weight of the peripheral nerves, sciatic nerves (nn. ischiadici) on both sides were cut proximally at the exit from the pelvic cavity in the thigh and distally at the middle of the thigh. As seen in table 1, the data show considerable individual variation, so no stress can be laid upon differences between the results. In the acute-thirst series (table 2) the nerves show an apparent loss of 21.3 per cent in average weight, and in the chronic-thirst series a loss of 22.1 per cent. This would indicate that during thirst the loss in weight is relatively greater in the peripheral than in the central nervous system. No observations by others upon this point have been found in the literature, but Doctor Hatai has called my attention to the fact that there is a large amount of fat usually present in the normal sciatic nerve. 414 TOKUYASU KUDO Eyeballs The occurrence of conjunctival congestion (or hemorrhage) and the opacity of the lens were mentioned under 'General observations.' Conjunctival congestion during extreme human thirst was noted by Tiedemann ('36) and conjunctival infection was reported in the dog by Pernice and Scagliosi ('95). Opacity of the lens in dehydrated frogs was noted in some cases by Durig Coi). As shown in table 2, the eyeballs in my acute-thirst series lose 10.2 per cent in average weight, and in the chronic-thirst series 13.3 per cent. In the rat (Si. 2), on total inanition, the apparent loss of weight in the eyeballs is 13 per cent. In a dog on dry diet with loss of 20.7 per cent in body weight, Falck and Scheffer ('54), on the contrary, found an apparent gain of 19.7 per cent (probably due to an individual variation) in the weight of the eyeballs. During inanition with water in the albino rat, no significant change in the weight of the eyeballs was found by Jackson ('15), who cites a few similar observations by previous authors. Bitsch ('95) found the absolute weight of the eyeballs in the dog usually increased during inanition, and suggested that it might be due to edema. Possibly the lack of available water during thirst may explain the decrease in the weight of the eyeballs in the present experiments, but a further investigation on this point is required before a final conclusion can be reached. Heart and aorta Heart. The heart in the acute-thirst series (table 2) shows a loss of 30.6 per cent in average weight, and in the chronic-thirst series a loss of 46.3 per cent. In the rat (Si. 2) , after total inanition (table 1), the corresponding apparent loss is 42.6 per cent. Since the heart during thirst loses somewhat less in weight than does the body as a whole, it increases accordingly in relative (percentage) weight. Few data in the literature are found concerning the weight of the heart during thirst. Falck and Scheffer ('54) noted in the dog a loss of 4 per cent in the heart and aorta, with a loss of 20.7 EFFECTS OF THIRST — ALBINO RATS 415 per cent in body weight. Bowin ('80) also noted in rabbits an absolute decrease (but relative increase) of weight in the heart during thirst, Tobler ('10) obtained variable results (+4.1 to — 21.5 per cent) in the weight of heart and lungs together in dogs subjected to experimental diarrhea with loss of 22.4 to 29.8 per cent in body weight. After inanition of albino rats with water, Jackson ('15) likewise noted that the loss of weight in the heart is somewhat less than that in the body as a whole. He cites data from several previous observers showing similar results in various animals. An atrophy of the heart in pigeons and monkeys on vitaminedeficient diets has recently been observed by McCarrison ('19). Aorta. The aorta was cut anteriorly at its junction with the heart and posteriorly at its bifurcation. All the branches of the aorta were clipped close to the vessel, and the surrounding fat tissue and lymphatic nodules were removed. The blood was removed from the aorta, and the vessel was weighed in two controls, two test rats in acute-thirst, and seven in chronic-thirst experiments (table 1). The aorta in the acute-thirst rats apparently decreased in average weight 42.5 per cent and in the chronic-thirst 44.6 per cent. While the number of observations (in the controls and the chronic tests) is too small to be very significant, the results indicate that the loss in the weight of the peripheral arteries may be fully as great as that of the heart. Spleen The spleen at autopsy usually appeared small and atrophic. The capsule is sometimes thickened. Upon section, the pulp appeared relatively pale brownish, and the trabeculae more prominent than in the normal organ. The spleen in the acutethirst series (table 2) shows a loss of 66 per cent in average weight and in the chronic-thirst series a loss of 73.3 per cent. In the rat (Si. 2), after total inanition, the corresponding apparent loss in the weight of the spleen is 62.9 per cent. As seen from table 1, the weight of the spleen, even in normal rats, is exceed 416 TOKUYASU KUDO ingly variable. And yet the atrophy in both acute and chronic tests is so profound that the results are unquestionable. Of previous investigators, only Falck and Scheffer ('54) record an apparent increase of 9 per cent in the weight of the spleen in a dog on dry diet (loss in body weight, 20.7 per cent). Bowin ('80) found a decrease in both absolute and relative weight of the spleen in dogs and rabbits with loss of about 50 per cent in body weight. In three dogs, after experimental diarrhea (with loss of 22.4 to 29.8 per cent in body weight), Tobler ('10) found a loss of 32.5 to 75.9 per cent in the weight of the spleen. In albino rats, after inanition with water, Jackson ('15) noted an average loss of 51 per cent in the spleen in the acute series (body weight, —33.9 per cent) and of 29 per cent in the chronic series, (body weight, —31.1 per cent). He cites similar results by numerous observers in man and various animals subjected to inanition with or without water. It thus appears that profound atrophy of the spleen is not characteristic of thirst, but usually appears also in inanition with or without water. As an effect of vitamine-deficient diets, a marked atrophy of the spleen has been observed by McCarrison ('19) in pigeons, and a less marked atrophy in monkeys. Lungs The characteristic pathological changes due to lung infection were rarely observed in my test rats, although slight infection is probably responsible in part for the abnormally high weight in the controls (table 1). The average weights (table 2) show an apparent loss of 44 per cent in the weight of the lungs in the acute-thirst series, and of 51.5 per cent in the chronic-thirst series. In the rat (Si. 2) after total inanition the corresponding apparent loss is 52.7 per cent. If, however, the normal weight of the lungs given by Donaldson ('15) be taken for comparison, the loss of weight in the lungs of the test rats would be only about 16 and 22 per cent, respectively, in the acute- and chronicthirst series. Of previous observers on the effects of thirst, Falck and Scheffer ('54) noted an apparent loss of 26.9 per cent in the weight of EFFECTS OF THIRST — ALBINO RATS 417 lungs, trachea, and pharynx, in a dog on dry diet with loss of 20.7 per cent in body weight. Bowin ('80) concluded that the changes in the weight of the lungs in dogs and rabbits on dry diet are the same as during total inanition. In rats after inanition with water, Jackson ('15) found an apparent loss of 30.9 per cent in the lungs of the acute series and 40 per cent in the chronic series, which was roughly proportional to the loss in body weight. He cites previous observations by others indicating that the loss in weight of the lungs during inanition (with or without water) is usually less than that of the body. McCarrison ('19) has likewise noted a decrease in the weight of the lungs in monkeys on vitamine-deficient diets. Parotid and submaxillary glands The parotid glands are easily removed and separated from the adjacent lymphatic nodules and facial nerve. The submaxillary glands were similarly isolated. The adjacent lymphatic glands sometimes appeared enlarged in the test rats, in contrast with the salivary glands. The weights of the submaxillary glands in the controls were a little heavier than those given by Hatai ('18). The parotid glands in the acute-thirst series show a loss of 57.6 per cent in average weight (table 2) and in the chronicthirst series a loss of 69.7 per cent. In the rat (Si. 2) on total inanition (table 1) the apparent loss in the weight of the parotid glands is 67.7 per cent. The submaxillary glands similarly lose 47.1 per cent in average weight in the acute-thirst series, and 64.5 per cent in the chronic-thirst series. In the rat (Si. 2), after total inanition, the apparent loss in weight of the submaxillary glands is 63.3 per cent. Thus the salivary glands during thirst and after total inanition evidently undergo a profound atrophy, decreasing in weight relatively more than the body as a whole. Falck and Scheffer ('54) similarly found a loss of 33.7 per cent in the weight of the salivary glands in a dog on dry diet with loss of 20.7 per cent in body weight. McCarrison ('19) finds an atrophy of the submaxillary glands in monkeys on vitamine-deficient diets. 418 TOKUYASU KUDO Liver At autopsy, the liver usually appears somewhat hyperemic^ the capsule being normal. In the acute-thirst series (table 2) the liver shows a loss of 37 per cent in average weight and in the chronic-thirst series a loss of 55.3 per cent. In the rat (Si. 2) after total inanition (table 1) the apparent loss in weight of the liver is 53 per cent. Thus it is evident that the liver during thirst has lost weight in nearly the same proportion as the whole body, but relatively more in the chronic-thirst series and during total inanition. Of other investigators on the effects of thirst, Falck and Scheffer ('54) found a loss of 25.3 per cent in the liver weight (body weight, — 20.7 per cent). In dogs and rabbits with loss of 50 per cent in body weight, Bowin ('80) found the loss in liver weight similar to that during total inanition. Finally, in three dogs after experimental diarrhea with loss in body weight of 22.7 to 29.8 per cent, Tobler ('10) noted a loss of 17.9 to 34.8 per cent in the weight of the liver. In albino rats subjected to inanition with water, Jackson ('15) obtained a loss of 58 per cent in the acute tests (body weight, — 33.9 per cent) and of 43 per cent in the chronic tests (body weight, —36.1 per cent). He cites numerous data from previous observers indicating that in man and other species the liver invariably decreases markedly in weight during inanition (with or without water) . The loss is nearly always relatively greater than that of the body as a whole. Lasarew ('95) found that the loss in the weight of the liver in the guinea-pig is relatively greater during the earlier stages of inanition. McCarrison ('19) finds an atrophy of the liver of pigeons and monkeys on vitamine-deficient diets. Pancreas The pancreas, especially in the case of the thirst experiments, appears atrophic and is rather difficult to dissect out from the surrounding adipose tissue, omentum, and lymphatic glands. The pancreas in the acute- thirst series shows a loss of 53.1 per cent in average weight (table 2), and in the chronic-thirst series EFFECTS OF THIRST — ALBINO RATS 419 a loss of 52.7 per cent. In the rat (Si. 2), after total inanition, the corresponding apparent loss in weight of the pancreas (table 1) is 58.6 per cent. Thus the relative loss appears to be similar to that in the combined salivary glands, though somewhat less during chronic inanition. In a dog on dry diet with loss of 20.7 per cent in body weight, Falck and Scheffer ('54) found a loss of 36.1 per cent in the weight of the pancreas. Similarly, Lukjanow ('89) observed a loss of 54.4 per cent in the weight of the pancreas (body weight, —34 per cent) in pigeons after total inanition. These results are in fair agreement with my observations on rats. Atrophy of the pancreas was also observed by McCarrison ('19) in pigeons and monkeys on vitamine-deficient diets. Stomach and intestines In two cases of acute thirst ulceration and hemorrhage of the stomach were observed in the mucosa near the cardia. The digestive tube, from the level of the diaphragm to the anus, was removed and separated from the pancreas, mesentery, etc., and weighed with and without contents. The stomach and intestines, including their contents in acute thirst, show an apparent loss of 36.4 per cent in average weight, and in chronic thirst a loss of 28.3 per cent (table 2). In the rat (Si. 2) after total inanition (table 1) the apparent loss is greater, amounting to 52.1 per cent. The stomach and intestines, without contents, in the acutethirst series show a loss of 29.1 per cent in average weight, and in the chronic-thirst series a loss of 31.8 per cent (table 2). In the rat (Si. 2) after total inanition (table 1) the corresponding apparent loss is 20.3 per cent. In all cases the loss is relatively less in the stomach and intestines than in the body as a whole. In a dog on dry diet with loss of 20.7 per cent in body weight, Falck and Scheffer ('54) noted an apparent loss of 7.6 per cent in the weight of the stomach and of 17.9 per cent in that of the intestines. In three dogs after experimental diarrhea with loss of 22 to 30 per cent in body weight, Tobler ('10) found apparent losses of 2.5 to 34.5 per cent in the weight of intestinal tract. 420 TOKUYASU KUDO Following the inanition of albino rats with water, Jackson ('15) found a loss of 57 per cent in the gastro-intestinal tract of both the acute- and chronic-inanition series (bod}^ weight loss 33.9 and 36.1 per cent). He cites previous observations by Sedlmair ('99), indicating in the cat during inanition a loss in the intestinal tract proportional to that in bod}^ weight, and a relatively smaller loss observed by Voit ('66). McCarrison ('19) finds marked inflammatory changes with degenerative atrophy of the gastric and intestinal mucosa in pigeons, guinea-pigs, and monkeys on vitamine-deficient diets. Kidneys and bladder Kidneys. At autopsy the kidneys usually appear hyperemic. In a few cases the surface was roughened. The capsule is easily removed. The kidneys in the acute-thirst series (table 2) show a loss of 23.8 per cent in average weight and in the chronic-thirst series a loss of 31.4 per cent. In the rat (Si. 2) after total inanition (table 1) the apparent loss in the weight of the kidneys is 30.5 per cent. In all of these it appears that the kidneys lose in weight relatively less than the body as a whole, thus gaining in relative (percentage) weight. In thirst experiments on the dog, Falck and Scheffer ('54) likewise found the loss in the kidnej^s and ureters (—8.7 per cent) less than in the whole body ( —20.7 per cent). Tobler ('10) found in three dogs after experimental diarrhea a loss of 5.64, to 30.1 per cent in kidney weight (body weight loss 22 to 30 per cent) . Durig ('01), however, in dehydrated frogs found a relative increase in the w^eight of the kidneys. In rats after inanition with water, Jackson ('15) found a loss of 25.5 per cent in the acute series (body weight, —33.9 per cent) and of 26.8 per cent in the chronic series (body weight, —36.1 per cent). He cites results from previous observers likewise indicating for other animals (in most cases) a loss in kidney weight relatively less than in body weight in inanition with or without w^ater. McCarrison ('19) likewise finds an atrophy of the kidneys in pigeons and monkeys on vitamine-free diets. EFFECTS OF THIRST — ALBINO RATS 421 Bladder. The urinary bladder was isolated from the surrounding adipose tissue and peritoneum. At autopsy the bladder is always empty and contracted; its mucosa appeared hyperemic. Unfortunately, the weights were recorded in only a few cases (table 1). The bladder in the test animals (table 2) shows in average weight an apparent loss of 39.7 per cent in the acutethirst series and of 46.9 per cent in the chronic-thirst series. Although the observations are too few for definite conclusions, it appears that the loss of weight in the bladder is relatively greater than in the kidney, and is more nearly proportional to that of the body as a whole. Falck and Scheffer ('54) in a dog on a dry diet found an apparent loss of 34.9 per cent in the weight of the bladder (body weight, — 20.4 per cent) . Testes The testes during thirst, especially in the chronic-thirst series, appear atrophic and often become softer than in the controls. The test rats show no spermatozoa in the ductus deferens or seminal vesicles. In the acute- thirst series the testes apparently lose only 15.1 per cent in average weight, but in the chronic-thirst series the loss is 59.9 per cent (table 2). In the rat (Si. 2) after total inanition (table 1) the corresponding apparent loss is 36.9 per cent. In a dog on dry diet, Falck and Scheffer noted an apparent loss of 23.5 per cent in the weight of the testes and penis (body weight, —20.7 per cent). In albino rats after inanition with water, Jackson ('15) found the loss in weight of the testes is, relatively, slightly less than that of the body as a whole during acute inanition, and slightly greater than that during chronic inanition. In the cat a relative decrease in the weight of the testis during inanition was observed by Voit ('66). McCarrison ('19) finds a marked atrophy of the testis in pigeons on vitamine-free diets and a less marked atrophy in monkeys. 422 TOKUYASU KUDO Epididymis The epididymis in the acute-thirst series (table 2) shows a loss of 30 per cent in average weight and in the chronic-thirst series a loss of 64.8 per cent. In the rat (Si. 2) after total inanition (table 1), the corresponding apparent loss in weight of the epididymis is 55.8 per cent. In all cases, therefore, the loss appears slightly greater (relatively) in the epididymis than in the testis. Jackson ('15) concluded that in the albino rat after inanition with water the loss in weight of the epididymis is relatively not very different from that in the testis. Thyroid gland The thyroid gland in the acute-thirst series undergoes a loss of 23.9 per cent in average weight, and in the chronic-thirst series a loss of 33.1 per cent. In the rat (Si. 2), after total inanition (table 1), the apparent loss in weight of the thyroid is 41.7 per cent. In a dog on a dry diet, Falck and Scheffer ('54) noted an apparent loss of 30 per cent in the weight of the thyroid (body weight, —20.7 per cent). In acute inanition with water, Jackson ('15) found in the thyroid of the albino rat a loss in weight proportional to that of the whole body (about 34 per cent) , and during chronic inanition with water a loss of 21.8 per cent (body weight, —36.1 per cent). The extent of the loss in weight of the thyroid during inanition appears quite variable, due in part to the difficulty in dissecting out the gland uniformly. McCarrison ('19) noted a slight atrophy of the thyroid in pigeons and monkeys on vitamine-free diets. Thymus In the acute-thirst series the thymus (table 2) shows an apparent loss of 78.1 per cent in average weight and in the chronicthirst series a loss of 90 per cent. There is some uncertainty about these conclusions, however, as the age of the rats is unknown in some cases. It has been shown (Jackson, '13; Hatai, '14) that the thymus in the rat normally undergoes an age involu EFFECTS OF THIRST — ALBINO RATS 423 tion, and the smaller weight of the thymus in the test rats may be partly due to their greater age. The weight of the thymus in the test rats is far below the normal for the corresponding age, however. In a dog on dry diet, Falck and Scheffer ('54) observed an apparent loss of 63.1 per cent (body weight, -20.7 per cent). Hammar ('06) has shown the general occurrence of hunger involution of the thymus during inanition, which has been thoroughly investigated in the rabbit by Jonson ('09). McCarrison ('19) has likewise noted a very great atrophy of the thymus in pigeons and monkeys on vitamine-deficient diets. Suprarenal glands Since the suprarenal glands normally show a sexual difference in weight from the age of about six weeks, the sexes must be considered separately. In the following discussion, the data concern only the males. In the acute-thirst series the suprarenal glands show a loss of 21.3 per cent in average weight (table 2), while in the chronicthirst series there is an apparent loss of 27.1 per cent. In the latter case, the initial body weight of the (three) male controls is about 3 per cent greater than that of the test rats, so there is definite loss in the weight of the suprarenals during the thirst experiments. In the rat (Si. 2) after total inanition (table 1), the apparent decrease in weight of the suprarenals amounts to 16.6 per cent. No data were found in the literature concerning the weight of the suprarenal glands during thirst. During inanition of albino rats with water, Jackson ('15) found in the acute-inanition series an apparent increase of 1.5 per cent in weight and in the chronic-inanition series a decrease of 8.9 per cent (loss in body weight, 33.9 and 36.1 per cent, respectively). It therefore appears that during inanition with water the suprarenal glands are more resistant to changes in weight than they are during thirst and total inanition. In pigeons, guinea-pigs, and rabbits on vitamine-deficient diets, McCarrison ('19) finds a definite increase 424 TOKUYASU KUDO in the weight of the suprarenals. In scorbutic guinea-pigs he finds the weight, even doubled, but associated with degenerative changes. Hypophysis In the case of the hypophysis, as with the suprarenal glands, there is normally a sexual difference in weight (Hatai, '13). Males only are considered in the following discussion. The hypophysis in the acute-thirst series shows an apparent loss of 1.7 per cent (in an average weight) and in the chronicthirst series a gain of 1.7 per cent. These differences are too small to be considered significant. The apparent increase of 8.3 per cent in the weight of the hypophysis in rat Si. 2 (table 1) on total inanition is perhaps due to an individual variation. No data have been found in the literature concerning the changes of weight in the hypophysis during thirst. In albino rats after inanition, with water, Jackson ('15) found the weight of the hypophysis apparently decreased relatively slightly less than the whole body. It would therefore appear that during inanition with water there is a marked loss in the weight of the hypophysis, whereas during the thirst experiments and total inanition it remains more nearly constant. McCarrison ('19) finds as result of vitamine-deficient diets the hypophysis (glandular lobe) slightly increased in pigeons, and usually so in monkeys. DISCUSSION The changes in the absolute weights of the various systems and organs as a result of thirst in the adult albino rats are summarized in table 2. While (as previously stated) no great emphasis can be laid upon the exact values, it is apparent that, with reference to the relative loss in weight during the thirst experiments, the various organs and systems may be divided into three groups. In the first group, the loss of the organs in weight is relatively greater than that for the whole body. In the second, the loss is less than that for the entire body — but more than half as great — and finally in the third group it is less than half that for EFFECTS OF THIRST — ALBINO RATS 425 TABLE 2 Average loeights for controls and test rats in the thirst experiments, with percentage difference. Data from table 1 ACUTE thirst: 6 TO 16 DAYS CHRONIC thirst: 47 to 55 d.4.ys Average weight in Average weight in Difference Controls (3 males) Test rats (8 males) Controls (3 m., 1 f.) Test rats (7 males) grams grams per cent grams grams per cent Initial gross body weight . . 218.3 218.8 +0.2 211.8 211.3 -0.2 Final gross body weight . . . 218.3 139.6 -36.1 211.8 100.9 -52.4 Integument 41.93 20.24 28.54 19.36 -31.9 -4.3 40.15 20.07 21.27 18.00 -47.0 Ligamentous skeleton -10.3 Cartilaginous skeleton 16.34 14.41 -11.8 15.22 14.46 -5.0 Humerus (2) and femur (2) . 2.08 1.64 -21.1 1.95 1.62 -12.1 Musculature 88.7 26.2 59.3 18.2 -33.1 -30.6 86.6 25.4 33.6 14.6 -61.2 Visceral group -42.2 'Remainder' 15.15 1.766 0.520 4.14 1.768 0.529 -12. n +0.12 + 1.8 15.72 1.742 0.546 1.76 1.668 0.509 -88.8 Brain -4.2 Spinal cord -6.7 Sciatic nerves 0.055 0.303 0.848 0.129 0.043 0.272 0.589 0.074 -21.3 -10.2 -30.6 -42.5 0.052 0.303 0.819 0.129 0.040 0.263 0.440 0.072 -22.1 Eyeballs -13.3 Heart -46.3 Aortal -44.6 Spleen 0.638 1.94 0.217 1.C9 -66.0 -44.0 0.627 1.94 0.167 0.94 -73.3 Lungs -51.5 Parotid glands 0.160 0.489 0.068 0.258 -57.6 -47.1 0.154 0.462 0.047 0.164 -69.7 Submaxillary glands -64.5 Liver 7.21 4.54 -37.0 7.40 3.31 -55.3 Pancreas 0.93 13.78 0.43 8.77 -53.1 -36.4 0.84 13.23 0.40 9.49 -52.7 Stomach-intestines (filled). -28.3 Stomach-intestines (empty) 6.15 4.36 -29.1 6.01 4.10 -31.8 Kidnevs 1.87 0.109 1.43 0.065 -23.8 -39.7 1.89 0.109 1.29 0.058 -31.4 Bladderi -46.9 Testes^ 2.33 0.806 1.98 0.565 -15.1 -30.0 2.33 0.806 0.93 0.283 -59.9 Epididymides^ -64.8 Thyroid 0.0180 0.0137 -23.9 0.0178 0.0119 -33.1 Thymus 0.187 0.041 -78.1 0.145 0.015 -90.0 Suprarenals' 0.0409 0.0322 -21.3 0.0409 0.0298 -27.1 HvDonhvsis' 0.0060 0.0059 -1.7 0.0060 0.0061 +1.7 1 In these cases the number of observations does not include all the members of the group, as explained in the text (table 1). 426 TOKUYASU KUDO the entire body. In three organs in this last group there is apparently a slight gain. To make the comparison of the results in this manner, the data have been arranged as in table 3. In addition to the organs listed in table 3, the few observations on the bladder and aorta (table 2) during thirst, both acute and chronic, indicate that these organs suffer a marked loss in weight. If we compare the changes in weight of the organs or systems during acute thirst with those during chronic thirst, it appears that they are, on the whole, very similar, making due allowance for the greater loss of body weight in the chronic-thirst series. In a few organs, however, there is a considerable difference. For example, the testis, epididymis, and musculature suffer relatively a much heavier loss in weight in the chronic-thirst series than in the acute-thirst series. On the whole, however, it is evident that the results in the two series are remarkably similar to each other, and also (especially the chronic series) to those obtained in the rat after total inanition. A further study of the data for the acute-thirst series (tables 2 and 3) shows that the large losses in weight are suffered by 'the remainder' (composed mainly of fat, lymphatic glands, etc.), the integmiient (carrying fat), the visceral group (highly cellular), and the musculature, parts which taken together represent about 80 per cent of the gross weight of the control rats. It is evident from this that variations in the loss of other parts or organs will modify but slightly the total losses observed. Comparing my results on thirst with the changes in weight of organs and systems of the albino rat found by Jackson ('15) during inanition with water, there appears a surprising degree of similarity. There are, of course, some organs which are more or less different. For instance, the thyroid gland, suprarenals, and intestinal tract apparently suffer a greater loss during thirst than during inanition with water; but it is somewhat doubtful whether the difference is due entirely to the difference in diet. The changes in the chronic series, either on a dry diet or with water, might possibly be due in part to lack of vitamines in the diet; but this could hardly be the case in the acute series, where the EFFECTS OF THIRST — ALBINO RATS 427 TABLE 3 Based on table 2 and on table 1 — for complete inanition. The percentage losses are given in three groups and in a regularhj descending order for the series subjected to acute thirst.' In the series for chronic thirst, and in the one rat after complete inanition, the losses are entered in the same order as in the acute-thirst series. In the last two series the values do not decrease regidarly, but the arrangement facilitates comparison betiveen the several series ACUTE THIRST CHRONIC THIRST COMPLETE INANITION Organs showing a loss in weight greater than that of the entire body Loss of weight for entire body .... Organ or part Thymus Remainder Spleen Parotid glands Pancreas Submaxillary glands Lungs Liver Stomach and intestines (filled) per cent 36.1 -78.1 -72.7 -66. n -57.6 -53.1 -47.1 -44.0 -37.0 -36.4 per cent 52.4 -90.0 -88.8 -73.3 -69.7 -52.7 -64.5 -51.5 -55.3 -28.3 per cent 47.2 -77. S -62.9 -67.6 -58.6 -63.3 -52.7 -53.0 -52.4 Organs showing a percentage loss in weight less than that for the entire bodybut more than half as great Organ or part Musculature Integument Heart Visceral group Epididymides Stomach and intestines (empf>vO. Thyroid Kidneys Suprarenals Humerus Femur -61.2 -47.0 -46.3 -42.2 -64.8 -31.8 -33.1 -31.4 -27.1 -12.1 -39.2 -42.5 -42.6 -38.7 -55.8 -20.3 -41.7 -30.5 -16.6 Organs showing a percentage loss in weight less than half that for the entire body Organ or part Testes Cartilaginous skeleton Eyeballs Ligamentous skeleton. Hypophysis. Brain Spinal cord , -36.9 -34.1 -13.0 +8.3 -7.6 -16.7 428 TOKUYASU KUDO resulting changes in organ weights are in general very similar, although the experiments lasted but a few days. The extensive investigations of McCarrison ('19), however, have shown that in animals (pigeon, guinea-pig, monkey) on vitamine-deficient diets the various organs present atrophic changes, which resemble and are in some way related to those occurring in ordinary inanition. The evident fact that the changes in organ weights in the present thirst experiments are in general very similar to those found during total inanition and in partial inanition of various kinds, may perhaps be explained in one of two ways. Either the effects of thirst closely resemble those of general inanition (perhaps in some way interfering with the normal process of metabolism) or they are masked and overshadowed to a large extent by the inanition which necessarily accompanies the thirst experiments, due to the inadequate food-intake. Further research will be necessary before this problem can be solved. SUMMARY The more important results of the present investigation may be suiruiiarized briefly as follows: In the acute-thirst experiments, lasting from six to sixteen days, the amount of water in the diet was greatly reduced in eight test rats, resulting in a loss of 36.1 per cent in the average gross body weight in a few days. The corresponding percentage changes in the organs are most readily seen in table 3. In the chronic-thirst experiments, lasting from forty-seven to fifty-five days, the amount of water in the diet was not so greatly reduced, resulting in a more gradual but greater loss of body weight, the loss in average weight of seven test rats being 52.4 per cent in about seven weeks. The corresponding percentage changes in the various parts are most readily seen in table 3. In a single rat, after total inanition, the loss in body weight was 47.2 per cent in eleven days. The apparent loss in the individual parts is very similar to that during thirst (especially the chronic) series (table 3) , making due allowance for the differ EFFECTS OF THIRST — ALBINO HATS 429 ence in the loss in body weight and the probabihty of individual variation in a single specimen. The striking similarity in the changes in organ weights produced in the thirst and total inanition experiments is confirmed by the data from previous investigators as to the effects of total and partial inanition, with or without water, in the rat and other animals. It is yet uncertain whether this is because the primary effect of thirst is the same as that of other forms of inanition or whether the thirst effects are largely obscured by the accompanying inanition due to inadequate food intake. LITERATURE CITED BiTSCH 1895 Pathologisch-anatomische Veranderungen der Netzhaut des Hundes beim Hungern. Dissertation, St. Petersburg. (Cited by Miihlmann, '99.), BowiN, M. 1880 Beitrage zur Frage iiber die Trockenernahrung. Dissertation, St. Petersburg. (Cited by Miihlmann, '99.) Donaldson, Henry H. 1915 The rat. Data and reference tables. Memoirs of the Wistar Institute of Anatomy and Biology, no. 6. Philadelphia. DuRiG, A. 1901 Wassergehalt und Organfunktion. Erste Mitth. Arch. f. die gesamte Physiol., Bd. 85, S. 401-504. Falck, p., tjnd Scheffer, T. 1854 Untersuchungen iiber den Wassergehalt der Organe durstender und nicht durstender Hunde. Arch, f . physiol. Heilkunde, Bd. 13, S. 508-522. (Cited by Tobler, '10.) Hammar, J. A. 1906 Ueber Gewicht, Involution und Persistenz der Thymus im Postfetalleben des Menschen. Arch. f. Anat. u. Phys., Anat. Abt. Hatai, S. 1914 On the weight of the thymus gland of the albino rat (Mus norvegicus albinus) according to age. Am. Jour. Anat., vol. 16, pp. 251-257. 1918 On the weight of the epididymis, pancreas, stomach and of the submaxillary glands of the albino rat (Mus norvegicus albinus) according to body-weight. Am. Jour. Anat., vol. 24, no. 1. Jackson, C. M. 1913 Postnatal growth and variability of the body and of the various organs in the albino rat. Am. Jour. Anat., vol. 15, pp. 1-68. 1915 Effects of acute and chronic inanition upon the relative weights of the various organs and systems of adult albino rats. Am. Jour. Anat., vol. 18, pp. 75-116. KuMAGAWA, M., UND MiURA, R. 1898 Zur Frage der Zuckerbildung aus Fett im Thierkorper. Arch. f. Physiol., S. 431-450. Landauer, a. 1895 Ueber den Einfluss des Wassers auf den Organismus. Ung. Arch. f. Med., Bd. 3, S. 136-188. (Cited by Rosenstern, '11.) Lasarew, N. 1895 Zur Lehre von der Veranderung des Gewichts und der zelli gen Elemente einiger Organe und Gewebe in verschiedenen Perioden des vollstandigen Hungerns. Dissertation, Warschau. (Cited by Miihlmann, '99.) 430 TOKUYASU KUDO Ltjkjanow, S. M. 1889 Ueber den Gehalt der Organe und Gewebe an Wasser und festen Bestandtheilen bei hungernden und durstenden Tauben . im Vergleich mit dem beziiglichen Gehalt bei normalen Tauben. Zeitschr. f. physiol. Chemie, Bd. 13, S. 339-351. ^NIcCarrison, R. 1919 The pathogenesis of deficiency disease. Parts I to IX. Indian Jour. Med. Research, vol. 6, nos. 3, 4; vol. 7, nos. 1, 2. (Abstr, in Brit. Med. Jour., (1919) Feb. 15, p. 177; July 12, p. 36; Aug. 16, p. 200.) MfHLMANN, M. 1899 Ru:sische Literatur iiber die Pathologie des Hungerns. Centralbl. f. allgem. Path, und pathol. Anat., Bd. 10, S. 160-220. NoTHWAXG, J. (F.) 1891 Die Folgen der Wasserentziehung. Arch. f. Hyg., Bd. 16, S. 122; Bd. IS, S. 83. Also, Dissertation, Marburg. (Cited by Tobler, '10.) Pernice, B., und Scagliosi, G. 1895 Ueber die Wirkung der Wasserentziehung auf Thiere. (Virchow's) Arch. f. path. Anat., Bd. 139, S. 155-184. RosENSTERN, J. 1911 Ueber Inanition im Sauglingsalter. Ergebn d. inneren' Med. u. Kinderheilk., Bd. 7, S. 332^04. ScHEFFER, Th. 1852 De animalium, aqua iis adempta, nutritione. Diss. Marburg. (Cited by Tobler, '10.) ScHUCHARDT, B. 1847 Quaedam de effectu, quem privatio singularum partium, etc. Dissertation, Marburg. (Cited by Tobler, '10.) Sedlmair, a. C. 1899 Ueber die Abnahme der Organe, insbesondere der Kno chen, beim Hunger. Zeitschr. f. Biol., Bd. 37 (N. F. Bd. 19), S. 25-58. Skoritschenko 1883 Untersuchungen iiber einige Factoren des Hungerns. Protokolle der Conferenzsitzungen der Kais. Mil. Med. Akad., S. 175 233. (Cited by Miihlmann, '99.) TiEDEMAXN, F. 1836 Physiologie des Menschen, Bd. 3. Tobler, L. 1910 ZurKenntnissdesChemismus akuter Gewichtsstiirze. Bezie hungen zwischen Wasser und Salzen im Organismus. Arch. f. e.xper. Pathol., Bd. 62, S. 431-468. VoiT, Carl 1866 Ueber die Verschiedenheiten der Eiweisszersetzung beim Hungern. Zeitschr. f. Biol., Bd. 2, S. 308-365. Zabaver, Dora 1904 La mort par la soif. Th. Med. Lyon. THE AMERICAN JOURNAL OP ANATOMY, VOL. 28, NO. 3, MARCH, 1921 Resumen por el autor, Warren H. Lewis. Johns Hopkins Medical School. Los efectos del permanganato potasico sobre las celulas mesenquimatosas de cultivos de tejidos. El permanganato potasico produce una serie definida de cambios sobre las celulas mesenquunatosas, cuya rapidez depende de la concentracion de la solucion. Con una soluci6n de 1 por 40,000 u otra de 1 por 80,000 los cambios son bastante lentos para poder seguirse, y la muerte de las celulas tiene lugar al cabo de una media hora. El niicleo es el primero afectado y al cabo de unos cuantos minutos presenta cambios de coagulaci6n a los cuales sigue una contraccion del material cromatico en una masa densa, picnotica y fuertemente coloreable, y la expulsi6n del jugo nuclear en forma de vacuolas claras. Poco despues del cambio nuclear comienzan a disociarse los filamentos mitocondriales, convirtiendose en bastoncitos y granulos, los cuales se transforman finalmente en vesiculas redondeadas. Probablemente se hacen mas fluidas y se hinchan y puesto que se suponen constituidas en gran parte por fosfolipinas, estos cambios indican que ha habido o^idaci^; pues cuando se oxidan las fosfolipinas adquieren agua. Las mitocondrias tefiidas previamente con verde janus o negro janus num. 2, pierden su color. Los granules degenerativos y las vacuolas pierden tambien color cuando se tinen previamente con rojo neutro, y los granos incoloros desaparecen generalmente. La perdida de estos colores es una indicacion de la muerte de la celula. A menudo se presenta tambien una condensacion de algunas partes del citoplasma en una masa que se transforma en finamente granulosa y fuertemente coloreable despues de la fijacion. La centrosfera no parece ser afectada. Existe cierto paralelismo entre estos cambios y los que tienen lugar durante la mitosis. Translation by Jose F. Nonidez Cornell IMedical College, New York AUTHOR S ABSTRACT OP THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JANUARY 17 THE EFFECT OF POTASSIUM PERMANGANATE ON THE MESENCHYME CELLS OF TISSUE CULTURES WARREN H. LEWIS Carnegie Laboratory of Embryology, Johns Hopkins Medical School, Baltimore, Maryland SIXTEEN FIGURES (ONE PLATE) INTRODUCTION Potassium permanganate (KMn04) is usually looked upon as a strong oxidizing agent and, when in contact with protoplasm, is supposed to give up nascent oxygen, 2 KMn04 + H2O = 2 KOH + 2 MnO + 50. The experiments herein described were begun with the idea that an excessive supply of nascent oxygen might produce visible characteristic changes in the cells. One cannot be sure that any particular series of changes is peculiar to the especial agent employed, unless there have been similar experiments with many other substances which can be used for comparison. Potassium permanganate does, however, cause a sequence of changes which lead to the death of the cell. These changes are not peculiar to potassium permanganate since dead cells in cultures that are not treated with any agent sometimes show a somewhat similar condition. In normal cultures, however, the changes leading to this specific death condition cover hours or days, while with potassium permanganate a few minutes suffice to produce them. The nucleus is the particular cell organ one would first examine, since it is supposed to be especially concerned either with oxidations and reductions or with syntheses.^ The normal resting nucleus can usually be located without difficulty. It is 1 Lynch, V, 1919 The function of the nucleus of the living cell. Amer. Jour. Phys., vol. 48. 431 432 WARREN H. LEWIS markedly homogeneous and transparent, having a refractive index near to that of the cytoplasm, and often it can be identified only by the arrangement of the mitochondria and the granules about it and of the nucleolus within. No nuclear membrane is distinguishable, although the border of the nucleus in the resting cell is clear and sharp. The nucleoli are, as a rule, quite distinct, irregular in outline, and somewhat ragged. They vary in size and usually each nucleus contains one or two large ones and sometimes one or more small ones in addition. In the normal living mesenchyme cell during prophase the chromosomes become visible and the nuclear outline is lost, but reappears again after division during the reconstruction period. No fibers can be seen in the clear nuclear spindle. The substance of the spindle seems to come from the nucleus itself by what appears to be a coagulation-like process resulting in the segregation of the chromatin material into the more highly refractive chromosomes and their separation from the clear nuclear sap. This nuclear sap disappears after division and the endosplasm then closely surrounds the small mass of chromosomes. During the reconstruction period the nucleus, or rather the chromosomes, which constitute the early daughter nuclei, swell slowly, apparently by the imbibition of fluid, lose their highly refractive condition, and become so closely packed together that the nucleus appears almost homogeneous and the individual chromosomes are no longer distinguishable. It is generally believed, however, that they retain their individuality. Attention should be called to the important supposition that the chromosomes take up water or fluid during the reconstruction period and lose fluid during the prophase stage of mitosis, because one of the most marked effects of potassium permanganate was to bring about shrinkage of the nucleus with expulsion of fluid vacuoles. The cytoplasm appears to be more complex than the nucleus and less homogeneous. The cytoplasm proper is usually regarded as a homogeneous semifluid or semisolid substance, in which are imbedded certain bodies or inclusions, namely, the mitochondria and the granules. The cytoplasm, when examined under the microscope in the living mesenchyme cells of our cultures, is MESENCHYME CELLS OF TISSUE CULTURES 433 not, however, a homogeneous substance. The cells are flattened out against the under-surface of the coversUp and present a much more favorable condition for the observation of their internal structure than the thicker, more irregular shapes in the embryo. Furthermore, the living cells flatten (each into a comparatively thin plate, while functioning and readjust their internal architecture in such a manner as to exaggerate more or less their structure. It is usually possible to distinguish a clear homogeneous ectoplasm and a slightly darker, less homogeneous endoplasm which seems to have a very finely powdered texture. It is thus shghtly less transparent than the ectoplasm. There is no indication of a cell membrane distinct from the ectoplasm. Each cell contains a centriole or centrosome located close to one side or one end of the nucleus. I am not certain that I have ever seen the centriole in the living cell, so that the proof of its existence in the form of a small granule in these living cells is dependent upon the condition found in fixed material. We cannot be sure, however, that the granule which we call the centriole in the fixed cell is not a coagulation product. It, however, undoubtedly represents a differentiation of the cytoplasm in this region which is much emphasized in degenerating cells. Its position can usually be determined by the general configuration of the cell contents, especially if there is a slight amount of degeneration with a consequent accumulation of granules about it. From the behavior of the centriole (and centrosphere) in cell division and cell degeneration, it is generally believed that it constitutes an important cell organ or that certain activities are in some way centered in or about it. The mitochondria are variable bodies, usually long and threadHke and often branched in the more normal cells. In the older cultures and in dividing cells they tend to break up into rods and granules, and in extreme degeneration they may round up into vesicles of various sizes. In the more normal cells they do not appear to have any definite arrangement, but in degenerating cells they tend to accumulate about the centriole or centrosphere, more or less radially in one type or stage of degeneration 434 WAEREN H. LEWIS and concentrically in another type. The mitochondria are supposed to be a combination of phospholipin and albumin."^ Many histogenetic functions have been ascribed to them, such as the formation of neurofibrils, myofibrils, collogenic fibrils, etc., there is, however, reason to doubt even the existence of such fibrils in living cells, and more doubt still as to whether the mitochondria take any especial part in their formation. The mitochondria are probably concerned with functions more general in nature, such as respiration, storage of food stuffs or waste products, etc. We are not sure that they are to be considered as part of the living cytoplasm; they may be merely cytoplasmic inclusions, dependent for their existence on the activities of the protoplasm. In cultures over twenty-four hours old practically all of the cells contain granules that take up the neutral red dye with great avidity and tend to accumulate about the centriole and centrosphere. Vacuoles which likewise take up the neutral red dye are found in the cells, especially in the older cultures; they also tend to accumulate about the centriole and centrosphere. Since they occur in degenerating cells, we have called them degeneration granules and vacuoles. It is not uncommon to find slender channels, much the same in size and shape as mitochondria, connected with the vacuoles. These channels are very unstable — they come and go and often anatomose. They take up the neutral red, methylene blue (Ehr.), and brilliant cresyl blue in exactly the same manner as do the vacuoles, and are therefore to be regarded as merely extensions of them. I have looked upon the granules and vacuoles as indicating degenerative changes^ in the cell, in that they probably represent accumulated waste products which the cell is unable to get rid of in the normal manner. The vacuoles may indicate also that there has been a certain amount of autolysis of the cjrtoplasm. ^ Cowdry, E. V. 1918 The mitochondrial constituents of protoplasm. Contributions to Embryology, VIII, Carnegie Institution of Washington, Pub. no. 271. ^ Lewis, W. H. 1919 Degeneration granules and vacuoles in the fibroblasts of chick embryos cultivated in vitro. Johns Hopkins Hosp. Bui., vol. 30. MESENCHYME CELLS OF TISSUE CULTURES 435 It is not possible, from our knowledge, to form a definite picture of the part played by the various visible structures in the metabolism of the simplest cell. We know in a general way that cells take in oxygen, water, salts, and food substances; that these are built up into its own substance or transformed or stored, and in the process carbon dioxide and other waste products* are formed which ultimately find their way to the exterior of the cell. MATERIALS AND METHODS The experiments were made on cultures of mesenchyme cells from various regions of chick embryos, six to nine days old. The cultures were grown in either Locke's solution plus 0.5 per cent dextrose, or in Locke-Lewis solution (the above solution plus 10 to 20 per cent of chicken bouillon). The potassium permanganate was dissolved either in normal Locke's solution or in Locke's solution without the sodium bicarbonate. At first definite percentages of potassium permanganate were used and the culture drop was usually entirely washed away with the solution in strengths varying from 1 to 20,000 to 1 to 80,000. The rapidity of action depended on the strength of the solution and how completely the old culture drop had been replaced by the new. If, for example, a very small drop of a 1 to 40,000 solution was added without washing away the old drop, the new drop was so diluted that its action was much retarded. Most of the cultures were stained with neutral red or with neutral red and janus black no. 2 before the potassium permanganate was added. One can follow the changes in the cell with greater ease after the neutral red is deposited in the granules and vacuoles and the janus black no. 2 in the mitochondria. Since these structures in dead cells do not retain the dyes in the dilute solutions as here used, we had a good criterion for cell death in the loss of color. Dead cells do, however, take up these dyes diffusely, if the solutions are strong enough. Some of the cultures were fixed at varying periods after the application of the potassium permanganate and stained in the usual manner with iron haematoxylin. 436 WARREN H. LEWIS SEQUENCE AND RAPIDITY OF CHANGES The nucleus appeared to be the first structure to show changes, but it was often difficult to tell whether they preceded those in the mitrochondria, especially the mitochondria in the thinner, more peripheral parts of the cell. The changes in the nucleus and mitochondria proceeded, in part at least, concurreijtly. The granules and vacuoles were usually not affected until sometime later. No especial changes were observed in the cytoplasm or in the centriole or centrosphere. The fixed material usually showed, however, that important changes had taken place in the cytoplasm. The stronger the solution of potassium permanganate, the more rapidly did the changes leading to the death of the cell take place, and it was only with the weaker solution (1 to 20,000 to 1 to 80,000) that the changes were slow enough to follow. With 1 to 40,000 or 1 to 80,000 these changes were prolonged over a period of half an hour before the cell was entirely dead. In strong solutions death occurred in less than one minute, even at room temperature. I have recently observed the same series of changes at ordinary room temperature. Most of the observations, however, were made in a warm box. The rapidity of action was dependent also on the thickness of the cells, the process being much slower in thick cells than in thin ones. In general the cells at the periphery were flatter and thinner than those near the explant ; the peripheral cells were the first to show any change and the first to die. A single preparation with extensive growth sometimes exhibited the whole series of changes at a single instant; some of the cells at the periphery were dead, while some of those near the explant appeared normal. The final end was the death of the cell. Different regions or parts of the cell probably cease to function at different times. The loss of color from the mitochondria and from the granules and the changes such as were seen in the nucleus after the addition of potassium permanganate are all indications that the cell is dying. MESENCHYME CELLS OF TISSUE CULTUEES 437 CHANGES IN THE NUCLEUS Normally, the outline of the nucleus can usually be determined by the structures within the surrounding cytoplasm. Within a minute or more after the application of the potassium permanganate the nucleus gradually grew brighter, more opaque, and somewhat mottled, and became the most striking feature of the cell. Its outline became sharp and clear and it appeared to have an enclosing membrane. Accompanying these changes in the nucleus there was often a slight alteration in form, in that it tended to become more spherical as though taking up fluid. Finally, vacuoles of clear colorless fluid were exuded from the periphery of the nucleus. As these vacuoles increased in size they pushed away the surrounding cytoplasm on the one side and indented the nucleus on the other, so that the latter, as it condensed, became more or less crenated or pycnotic. These pycnotic nuclei stood out very prominently in the cell and in fixed specimens stained deeply with iron haematoxylin, like the chromatin. Nuclei, which were completely surrounded by vacuoles did not, as a rule, appear as irregular as those with only two or three vacuoles. Not all of the larger nuclei, however, showed this vacuolar formation and shrinkage, since a few in every culture retained more or less their usual size and shape (fig. 16). The position of origin, the size and the number of the vacuoles varied from one or two or several (figs. 8, 9, 10, 2) to many (figs. 13, 14, 15). It is possible that the variations in the number and size of the vacuoles or the total amount of fluid exuded and in the correlated size of the shrunken nucleus, depended, in part at least, on the condition of the nuclei (with reference to age) as regards the relative amount of chromatin substance and nuclear sap. Young nuclei of daughter cells have very little nuclear sap and a comparatively large amount of chromatin. The old resting nuclei have a greatly increased actual amount of nuclear sap and also an increase in the amount of chromatin, but the proportion of chromatin to nuclear sap is much less than in the young cells. In most fixed cultures nuclei vary in size and depth of stain. The smallest and most deeply stained nuclei are the youngest, namely, those of the daughter 438 WAEEEN H. LEWIS cells soon after division.^ Parallel with the increase in the size of the cell and of its nucleus, there is a diminution in the depth of stain retained by the nucleus. This is very marked if one compares the nucleus of a young daughter cell with that of an old cell just before mitotic changes again take place. Immediately after cell division the nuclei are small and deeply staining, because the chromosomes contain but little fluid and the chromatin material which takes up the stain is dense. As the nuclei enlarge through the imbibition of fluid by the chromosomes, they become less deeply stainable, because the chromatin material becomes less dense. This process of swelling of the chromosomes goes on until a certain metabolic balance is reached and either a more or less stable condition results or there is another complete upset and fluid is again squeezed out of the chromosomes, leaving them floating in the nuclear sap. During the resting period there is also a supposed gradual increase in the amount of chromatin material, as well as an increase in the size of the nucleus through the imbibition of fluid. If, then, we bear in mind these differences in the normal nuclei, we can explain the differences in the number and size of the nuclear vacuoles and the correlated shrinkage of the nuclei to pycnotic forms in these experiments. In some manner the potassium permanganate caused a contraction of the chromatin material and a squeezing out of the clear nuclear sap in the form of vacuoles. The younger nuclei have less nuclear sap and less chromatin than the older nuclei and were probably the ones which gave rise to the fewest and smallest vacuoles and to the smallest residual chromatin mass (figs. 4, 5, 6, 7, 8, 10). During metaphase, anaphase, and telaphase there are no indications of the formation of these nuclear vacuoles (figs. 1, 2, 3). During the reconstruction period, however, when the chromosomes are supposed to be absorbing fluid and the nucleus increases in size, there was an increase in the amount of vacuolar fluid and in the residual chromatin mass. The older nuclei give rise to a still larger total vacuolar volume and to a larger chromatin residue.

  • Lewis, M. R. and W. H. 1915 Mitochondria and other cytoplasmic structures in tissue cultures. Amer. Jour. Anat., vol. 17, p. 368.

MESENCHYME CELLS OF TISSUE CULTURES 439 The question as to just how the potassium permanganate brings about these changes in the nucleus is a difficult one and not to be answered easily. In the first place, the changes are not specific for potassium permanganate, as they are sometimes seen in cells that die in untouched cultures. In the latter condition they take place much more slowly; they cannot be followed from minute to minute, but consume hours or even days. The most obvious explanation is one of increased permeability, associated with an increased oxidation of the nuclear material and breaking down of some of it, accompanied by a coagulation and clumping of the chromatin material. This may account for the mottled appearance and increased visibility of the nucleus and the final aggregation of the chromatin into a compact mass. Hogue'^ found that when cells were treated with hypertonic solutions they took up fluid and that the nucleus frequently gave rise to a vacuole as an outlet for the extra amount of liquid absorbed. No accompanying shrinkage of the chromatin material was noted, however,- such as occurs in the potassium-permanganate experiments. CHANGES IN THE MITOCHONDRIA The mitochondria usually began to show changes after the nucleus had been affected, at about the time that the nuclear vacuoles appeared. Before the potassium permanganate was added, the mitochondria were in the form of threads, rods, and granules. A cell might contain any one or all of these forms. Potassium permanganate caused the mitochondria to gradually assume a spherical form, and the size of the spheres varied according to the length of the mitochondrium from which each one arose. The small circles in figures 1 to 16 represent spherical mitochondria. The threads gave rise to large vesicles, the short rods to small ones, and the granules to still smaller ones. The long threads sometimes broke up into short threads before they rounded up. Cells which contained both long and short mitochondria showed both large and small vesicles. Ones with long

  • Hogue, Mary J. 1919 The effect of hypotonic and hypertonic solutions on

fibroblasts of the embryonic chick heart in vitro. Jour. Exper. Med., vol. 30. 440 WARREN H. LEWIS mitochondria contained mostly large vesicles; cells with rods and granules, small vesicles. As the mitochondria changed into vesicles they lost most of the blue color if previously stained with janus black no. 2. At the same time they appeared to become somewhat darker or more opaque than normal, and were then more easily seen, especially when they became spherical. The vesicles in certain foci appeared as dark rings. In changing from threads and rods to vesicles the mitochondria often became at first irregularly varicose with swollen places here and there. Two or more swollen places often developed on the longer threads and rods and remained connected for a time by narrow segments; these later expanded, forming oval vesicles which changed to spherical ones. The threads and rods shortened quite perceptibly as they expanded. The factors which cause the rounding up of the mitochondria can only be surmised. It seems evident that they take up fluid and increase in volume. The potassium permanganate causes, perhaps, an increase in the permeability of the cytoplasm, so that not only the cytoplasm itself, but also the mitochondria take up water, since both swell. If, as is commonly supposed, mitochondria are composed of phospholipins, a simple explanation of their swelUng can be offered, since the affinity for water is increased when phosphohpins are oxidized.^ It seems probable that there is also an increase in the surface tension of the mitochondrial substance which is normally rather low. The transformation of mitochondrial threads and rods into vesicles by the action of potassium permanganate is not a specific reaction to this substance, as it can be brought about by other reagents and is not infrequent in the later stages of cell degeneration in cultures that have not been disturbed. CHANGES IN THE CYTOPLASM AND CENTROSPHERE There was very little alteration to be seen in the cytoplasm of the living cell. More or less swelling of the cells occurred, sometimes blebs appeared or the cells rounded up somewhat. There was often more or less liquefaction and Brownian motion ^ Mathews, A, P. 1915 Physiological chemistry, p. 98. MESENCHYME CELLS OF TISSUE CULTUEES . 441 of some of the fine granules and fat globules, if the latter were present. The cytoplasm, in the later stages, sometimes became more or less vacuolated. These vacuoles were of a different type from those that take the neutral red. They were difficult to see and did not take up the neutral red. In fixed material many of the cells showed a peculiar, finely granular, deeply staining area in the cytoplasm (figs. 1, 4, 6, 9, 11, 12, 14), seeming to indicate that the cytoplasm, probably endoplasm, had become denser in certain areas and on fixation had coagulated into a finely granular mass. These areas did not appear to have any definite location. In some cells they were near the nucleus, in others some distance away; in some instances on the same side of the nucleus as the centrosphere, in others on the opposite side; they occurred in cells with or without centrospheres, in young daughter cells, in older cells, and even in dividing cells. They were present in some cells with nuclear shrinkage, in others they were absent. The condition of the fixed cytoplasm of these areas reminds one very much of that usually seen in dividing cells. No changes were observed in the centrosphere. CHANGES IN THE GRANULES AND VACUOLES The granules and vacuoles that had previously taken the neutral red stain retained their usual condition until after the nuclear and mitochondrial alterations had taken place, then there was either a gradual loss or a rather sudden disappearance of the red color. In the former case the vacuoles and granules seemed to otherwise remain unaltered and could be seen unstained. With the disappearance of the red color from the vacuoles and granules the cells lost the last vestige of life. The vacuoles and granules of dead cells never took up the neutral red, although the cytoplasm sometimes became diffusely stained if the neutral red was sufficiently concentrated. In the living cell the dye was evidently held in some peculiar chemical or physical combination in the granules and vacuoles and accumulated there until the saturation point was reached, 442 WARREN H. LEWIS or until the dye was exhausted from the solution. With the death changes in the cell some alteration apparently took place in the granules and vacuoles and they were unable to retain the dye. Potassium permanganate does not decolorize the neutral red in vitro in Locke's solution. DISCUSSION Certain reactions are produced by potassium permanganate that seem to have some bearing upon mitosis. The segregation of the chromatin material from the nuclear sap takes place in both mitosis and in the potassium-permanganate experiments, but in the latter the chromatin is clumped. The reduction of the mitochondria to rods and granules occurs in mitosis and also in our experiments. The condensation of the cytoplasm is another peculiar phenomenon common to both mitosis and to the experiments. These reactions suggest that some of the factors involved in mitosis and in potassium-permanganate experiments are alike. Can it be that an increased supply of oxygen is one of the common factors? The evidence is too slight to go into a prolonged discussion. The fact that the centrosphere, when present, does not seem to be affected is perhaps more important than appears on first thought. If the centriole and its centrosphere represent the most active or dynamic center of the cell, as is sometimes supposed, we should expect it to be involved. In studying cell degeneration^ one is much impressed by the picture of a gradually enlarging centrosphere surrounded by the accumulating granules and vacuoles and the mitochondria. In a former paper^ I expressed the opinion that this seemed to indicate that the centriole or centrosphere was the dynamic center of the cell, as expressed by Boveri years ago. There is, however, another possibility that has gradually been forcing itself into consideration, namely, that the centrosphere may have just the opposite significance and is rather to be looked upon as a degenerating ' Lewis, W. H., 1920 Giant centrospheres in degenerating cells of tissue cultures. Jour. Exper. Med., vol. 31. MESENCHYME CELLS OF TISSUE CULTURES 443 area. If the latter is true, we should not expect an increased supply of oxygen to have as much effect upon it as it would if it were a very active part of the cell and using a relatively large amount of oxygen. SUMMARY Potassium permanganate produced a definite sequence of changes in the mesenchyme cells, their rapidity depending upon the strength of the solution. With a 1 to 40,000 or a 1 to 80,000 solution the changes were slow enough to be followed and death of the cells occurred in about one-half hour. The nucleus was first affected and in a few minutes showed coagulation changes that were followed by a contraction of the chromatin material into a dense, pycnotic, deeply stainable mass and the expulsion of the nuclear sap in the form of clear fluid vacuoles. Shortly after the nuclear changes began the mitochondrial threads broke up more or less into rods and granules and these rounded up into vesicles. They probably became more fluid and swollen and, since they are supposed to consist largely of phospholipins, this indicated that oxidation had occurred as phospholipins when oxidized take up water. The mitochondria if previously stained with janus green or janus black no. 2 lost color. The degeneration granules and vacuoles also lost color if previously stained with neutral red and the colorless granules usually disappeared. The loss of these colors is an indication of cell death. A condensation of some portion of the cytoplasm into a mass which became finely granular and deeply stainable after fixation often occurred. The centrosphere did not seem to be affected. There is a certain parallelism between these changes and those which occur in mitosis. PLATE 1 EXPLANATION OF FIGURES The sixteen figures are all of mesenchyme cells from the same culture. The explant from the leg of a seven-day chick embryo, gave rise to an average outgrowth in Locke's solution plus 5 per cent dextrose. After forty-eight hours the culture was washed with a solution of neutral red and janus black no. 2. The mesenchyme cells contained rather few red granules. The mitochondria were in the form of threads and rods. The centrospheres varied from small to fairly large ones. After a brief examination, the culture was washed with one drop of a 1 /40,000 solution of potassium permanganate and the usual changes were observed in the nucleus and in the mitochondria. Sixteen minutes after the application of the potassium permanganate the specimen was fixed in Zenker's fluid without acetic acid, and stained with iron haematoxylin and eosin. The figures were drawn with a camera lucida at a magnification of*r250 diameters with onehalf reduction in printing. Many of the fine processes of the cells were not carried out to full length. 1 Metaphase, mitochondrial vesicles (small circles), dense cytoplasmic mass. X 625. 2 Anaphase. 3 Telophase, mitochondrial vesicles in dense cytoplasm. 4 Daughter cells with dense cytoplasm, small mitochondrial vesicles, one nuclear vacuole. 5 Daughter cells, mitochondrial vesicles, centrospheres, nuclei each with one nuclear vacuole. 6 Older daughter cells with mitochondrial vesicles, each cell with a small dense cytoplasmic area and centrosphere and each nucleus with halo of small nuclear vacuoles about a small dense chromatin mass. 7 Mesenchyme cell, small dense cytoplasmic area probably about centriole, mitochondrial vesicles, nuclear vacuoles about pycnotic dense nucleus. 8 Cell with mitochondrial vesicles, nuclear vacuoles and pycnotic nucleus, but no dense cytoplasmic area or centrosphere. 9 Cell with dense cytoplasmic area, large centrosphere, mitochondrial vesicles, and nuclear vacuoles. 10 Cell with mitochondrial vesicles, large centrosphere, nuclear vacuoles and pycnotic nucleus. 11 Elongated mesenchyme cell with small area of dense cytoplasm, mitochondrial vesicles, and dense nucleus. 12 Cell with area dense cytoplasm and centrosphere in the same end of the cell; mitochondrial vesicles, nuclear vacuoles, and dense pycnotic nucleus. 13 Cell with mitochondrial vesicles and a corona of nuclear vacuoles about pycnotic nucleus. 14 Cell with large dense cytoplasmic area, mitochondrial vesicles, and corona of nuclear vacuoles about pycnotic nucleus. 15 Binucleate cell with one large centrosphere, mitochondrial vesicles of various sizes, corona of nuclear vacuoles about dense nuclei. 16 A much flattened mesenchyme cell from periphery with mitochondrial vesicles. No nuclear vacuoles were formed. 444 MESENCHYME CELLS OF TISSUE CULTURES WAEREN H. LEWIS PLATE 1 445 THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 3 Resumen por el autor, George A. Baitsell. Yale University, New Haven, Estudio del desarroUo del tejido conectivo en los Anfibios. El esbozo primitivo del tejido conectivo de los embriones de rana es un material amorfo y gelatinoso, el cual a causa de su escasa avidez hacia los colorantes durante las primeras fases del desarroUo, durante las cuales se tine ligeramente por los diversos metodos empleados, es muy dificil de poner en evidencia. Esta substancia fundamental puede observarse alrededor del notocordio a raiz de la formacion de este ultimo y un poco antes de aparecer la yema caudal del embrion. Un poco mas tarde este material, que entrara a formar parte del tejido conectivo, rodea la medula espinal y una capa formada por el se extiende ventralmente a lo largo de la pared del cuerpo, y al cabo de cierto tiempo termina rodeando por completo la cavidad del cuerpo. La formacion de esta substancia alrededor del notocordio tiene lugar antes de haber aparecido un sincicio de celulas mesenquimatosas en esta regi6n. ' Es evidente, por lo tanto, que esta substancia fundamiental primitiva del tejido conectivo se ha formado por secrecion intercelular de las celulas embrionarias y no a consecuencia de una fusion sincicial del protoplasma. Las fibras del tejido conectivo comienzan a aparecer en la substancia fundamental a raiz de formarse esta. Pueden constituir la substancia fundamental en regiones desprovistas de celulas, de modo que es evidente que no se han originado por una accion intracelular. En algunos casos puede comprobarse que las fibras se originan por una transformacion gradual de la substancia fundamental, primero en una delicada estructura reticular y despues en las largas fibras tipicas del tejido conectivo. En sus rasgos morfologicos por lo menos, este proceso coincide con el observado previamente por el autor en la transformacion del coagulo sanguineo. Translation by JosS F. Nonidez Cornell Medical College, New York author's abstract op this paper issued by the bibliographic service, februart 28 A STUDY OF THE DEVELOPMENT OF CONNECTIVE TISSUE IN THE AMPHIBIA GEORGE A. BAITSELL Osborn Zoological Laboratory, Yale University, New Haven, Conn. SIX FIGURES (four PLATES) INTRODUCTION The studies in which the author has been engaged for several years have shown, in brief, that the plasma clot obtained from frog's blood is of such a nature that, when influenced by the proper factors, it is possible to radically transform its structure. The reactions of the clot were studied in tissue cultures ('15) and in wound healing ('16). In both these cases it was possible to demonstrate clearly that the clot could be transformed into a fibrous tissue which was apparently identical in structure, save for the absence of connective tissue cells, with regular connective tissue. In wounds made in frog skin, the experiments further showed that the fibrous tissue which developed from the fibrin clot functioned, at least temporarily, as a normal connective tissue and there was no evidence to indicate that it would ever be replaced. In the final paper of the series ('17) — in which a study of the normal clotting and the transformation of the clot under the influence of certain mechanical factors was studied with the aid of a microscope equipped for dark field illumination^ — it was possible to demonstrate that the transformation of the clot w^as brought about by a fusion and consolidation of the fine elements of which it is composed. This process resulted in the formation of long fibers which united to form wavy, fibrous bundles. These bundles of fibers anastomosed with other bundles, ramified in various directions throughout the clot and the result was the formation of a fibrous material which closely resembled regular connective tissue. 447 448 GEORGE A. BAITSELL The above results led naturally to the question as to whether a similar process normally takes place in the histogenesis of the connective tissues in amphibian embryos and thus to the present study in which this problem is considered. It is worthy of note that, notwithstanding the great amount of work that has been done upon the development of connective tissue, no general agreement has been reached. At present there are two main theories held regarding connective tissue formation — the intracellular theory and the intercellular theory — and, in general, the results of the various investigators support one or the other of these. In order to get the matter clearly in mind it may be well to set forth a statement of the opposing views, which are as follows : According to the intercellular theory there is early formed in development an amorphous, gelatinous, non-living ground substance. It is generally held that this is formed as a secretion of the mesenchyme cells. Later, fibers are formed in this ground substance by a deposit or secretion which is given off by the cells of the tissue. It is held that the cells have no morphological connection whatever with the fibers which form in the ground substance. The intercellular theory substantially as stated above was the original theorj^ held for connective tissue formation and was first stated by Henle. With various minor modifications, it has been supported by v. Kolhker, Alerkel, Ranvier, v. Ebner, Renaut, Schafer, Laguesse, and others. The material used in the researches of these investigators was obtained from many different sources and included both embryonic and adult tissue. The intercellular theory has been, perhaps, most strongly supported by Merkel. In his last paper ('08) he gives a splendid review of the question of connective tissue development as well as the results of his own studies. His conclusions are that the primitive origin of all connective tissues lies in an amorphous gelatinous substance which is secreted by the syncytium of the mesenchyme cells. This secretion constitutes the ground substance and in some cases it is formed sparingly and in other cases very abundantly (e.g., Amphibia, umbilical cord). The fibers. CONNECTIVE TISSUE IN AMPHIBIA 449 according to Merkel, are really strands of the amorphous ground substance and they increase in size through an assimilation of the surrounding ground substance. The cells play no direct part in the formation of the fibers but serve only to produce the ground substance by secretion. Merkel also believes that the formation of the connective fibers is due in every case to the influence of mechanical factors acting on the ground substance; e.g., where there is a decided stretching, as in a tendon, parallel fibers are formed from the ground substance. According to the intracellular theory, the ground substance which is early formed is regarded as living material, although it is intercellular in position. The fibers which form in this ground substance are considered by some investigators to be strands of cell cytoplasm w^hich have been given off from the periphery of the connective tissue cells. Others beUeve that a transformation of the entire cell into fibers takes place. In either case, in the first stages of fiber development, all the fibers are regarded as. being merely modified strands of cell cytoplasm. The fibers when once formed by the cells may, and often do, become separated from the cells but nevertheless it is held that they are able to grow both in thickness and in length by intussusception due to the assimilation of food material. The living intercellular material is also able to grow in the same manner and it increases greatly in amount in proportion to the cells present. The intracellular view has been modified by the Hansen-Mall theory, which is widely held at present. In accordance with this theory, the ground substance arises as a result of a sjmcytium of the mesenchyme cells and is therefore regarded as being a common living exoplasm of the mesenchyme cells. In this exoplasm, each of the nuclei of the mesenchyme cells are surrounded by a minute mass of cytoplasm which constitutes the endoplasm of the cells. According to this theor}^ the fibers arise in the common exoplasm either in the regions in immediate contact with the cells or in the regions of the exoplasm which may be far removed from cells. 450 GEORGE A. BAITSELL The above view regarding connective tissue formation is clearlyset forth by jMall ('02) who says:^ In very early embrj'os the mesenchj-me is composed of individual cells which increase rapidly in protoplasm and then unite to form a dense syncytium. The protoplasm of the syncytium grows more rapidly than the nuclei divide, so that in a short time we have an extensive syncytium with a relatively small number of nuclei. In its form the syncytium appears as large bands of protoplasm with spaces between them filled at times with cells and at other times with fluid. The second condition we have in the umbilical cord of young human embryos. About this time the protoplasm of the syncytium differentiates into a fibrillar part, which forms the main portion of the syncytium — the exoplasm — and a granular part, which surrounds the nucleus — the endoplasm. The view of Mall, as stated above, is in quite close agreement with that of Hansen ('99 and '05) which is based upon his work on the development of cartilage. He believes that,^ in many cases, it is not possible to distinguish between protoplasm and ground substance and also that^ cells of cartilage are to be considered as an endoplasm and the ground substance as a common ectoplasm which is more or less independent of the endoplasm. In the concluding section of this paper will be found an analysis of these two theories and a discussion of the relations shown to them by the results obtained from the present work. It may, however, be stated at this time that the results reported in the present paper^ strongly support the intercellular theory of connective tissue development, and furthermore they show that the process of connective tissue formation closely resembles, morphologically at least, the formation of fibrous tissue by transformation of a plasma clot. 1 Mall, 1902, p. 331. 2 Hansen, 1S99, p. 434. 3 Hansen, 1905, p. 747. ^ A report of this work was given at the meeting of the National Academy of Sciences held in New Haven, Nov., 1919, and an abstract of the work is published in the Proceedings of the National Academy, 1920, vi, 77. CONNECTIVE TISSUE IN AMPHIBIA 451 MATERIAL AND METHODS In the present work, embryonic amphibian material has been used. It was obtained from a number of species, chiefly Rana sylvatica, R. palustris, R. catesbiana and Amblystoma punctatum. From each of these species, five series of nine or ten embryos, each ranging from the medullary plate stage by regular gradations to well developed free swimming larvae were obtained for preservation. In all about 200 embryos were selected for permanent preservation. In addition to this, through the kindness of Professor R. G. Harrison, access was had to several series of transverse, sagittal and frontal serial sections of R. palustris, cut 7.5/x and stained in haematoxylin and Congo red. Figures 1 and 2 are taken from this series. The following preserving fluids were used: a) Zenker with 5 per cent glacial acetic acid; b) Zenker without acetic acid; c) saturated solution of mercuric chloride with 5 per cent glacial acetic acid; d) saturated solution of mercuric chloride without acetic acid; e) 80 per cent alcohol. Several series of embryos were preserved in these fixing fluids. They were later imbedded in paraffin, and transverse, sagittal and frontal serial sections were made ranging from 7 to 10 micra in thickness. The following stains have been used: a) Delafield's haematoxylin; h) iron haematoxylin; c) picro-fuchsin connective tissue stain; d) Mallory's anihn blue connective tissue stain, and e) Mall's modification of the Mallory stain. With the large amount of material that was available, preserved by various methods, and with the various methods of staining which were used, it was possible to test a large nmuber of combinations and to determine the combination which was best adapted for the present work. Considerable time was spent in making this comparative study of the preserving fluids and stains and their reactions with the tissues, and it is believed that the combination, consisting of Zenker with acetic for the killing fluid and Mallory's aniline blue connective tissue stain which was finally adopted and used for most of the material is the best possible one for the problem in hand. 452 GEORGE A. BAITSELL With regard to this combination, it may be well to say in the first place that, of all the preserving fluids tried, Zenker with acetic gave the best results. The only objection to this fluid is that it has a tendenc}^ to harden the yolk material in the embryos somewhat more than some of the other fluids and this makes the sectioning of the earlier stages difficult. However, the difference here is not great and the superior preservation in the Zenker fluid warrants its use. As to the staining, it was found that the JNIallor}^ connective tissue stain was superior to any of the other stains tried for the work in hand. A comparative study was made of the reactions of this stain as originalh" given and the same stain as modified by Mall ('01-'02). The results obtained show very clearly that the modified Mallory does not give such a clear differentiation between cytoplasm and ground substance as does the original Mallory stain. Mall says,^ "My best specimens were obtained by staining the sections with ^Vlallory's connective tissue stain which tinges the nuclei and surrounding endoplasm, if present, slightly red and the exoplasm of the syncytium intensely blue. We have modified this stain somewhat by omitting the water and int nsifying the blue." The results of the comparative study have convinced me that the intensification of the blue is a mistake in that thereby the differentiation between the cell cytoplasm and the ground substance of the developing connective tissue is largel}^ lost. In material preserved in Zenker and correctly stained with Mallory's original stain there is a clear differentiation between the cells, which are of varying shades of red, and the ground substance which is invariably blue. It is possible therefore in such material to speak with certainty as to the limits of the cell boundaries and the relations the cells bear to the common intercellular ground substance. When the blue is intensified the entire field takes this color and the differentiation becomes obscured or even lost. 5 Mall, 1902, p. 338. CONNECTIVE TISSUE IN AMPHIBIA 453 STUDY OF PREPARED MATERIAL If sections of a frog embryo, taken shortly after the notochord is formed and before the tail bud stage is reached, are studied, it will be found that the notochord of the embryo is surrounded by a mass of transparent gelatinous material (primitive ground substance). Such a stage is shown in figure 1 which was made from a portion of a frontal section of a 3.2 mm. embryo of Rana palustris. The magnification is 285 diameters. Above a portion of the central nervous system is shown. Extending posteriorly, in the median line, is the notochord imbedded in a gelatinous cell-free matrix or primitive ground substance which extends laterally between the muscle plates in either direction. In figure 2 is shown a portion of a transverse section through a 4 mm. embryo of the same species but slightly older than that shown in figure 1. The magnification is 322 diameters. The notochord is shown in the center of the figure. Lying above it is a portion of the ventral wall of the medullary tube. The embryonic muscle cells can be seen lying to the right and left of the notochord. Filling the space between the notochord and the muscle cells is the gelatinous material mentioned above, which constitutes the primitive ground substance. This material also extends dorsally along the sides of the spinal cord and laterally into the muscle tissues. As is shown in the figure, the ground substance in some regions shows a fine fibrillation running through it. In other regions it appears homogeneous. At the stage in embryonic development shown in figures 1 and 2, the mesenchyme cells, which later wander all through the cavities of the embryo, are not present in this region in which the primitive ground substance is first formed. The case is clear, therefore, that the ground substance which is here present surrounding the notochord has not arisen by sj^'ncytial fusion of cell cytoplasm. The primitive ground substance having been formed as shown in figures 1 and 2, it is next found that, in an embryo of a little later stage, cells begin to detach themselves from the cell masses and wander through it. These cells, when they first appear, 454 GEORGE A. BAITSELL are for the most part more or less rounded in shape and contain a considerable amount of yolk. As they move through the ground substance they become amoeboid and also in some cases spindle-shaped; a form which is regarded as being typical of the so-called connective tissue fibroblasts. It is frequently possible to trace the movements of the cells by the altered appearance of the gelatinous ground substance. In figure 3 is shown a portion of an unstained transverse section through a 9 mm. embryo at a magnification of 322 diameters, at the stage when the cells are starting to move into and through the ground substance, as described just above. In this figure a portion of the notochord is sho^Mi with embryonic muscle tissue lying on either side. Both the notochord and the muscle tissue are imbedded in a matrix of the ground substance which is especially heavy in the region just below the notochord. From this region, it spreads to the right and the left surrounding the bundles of muscle fibers and extending to the body wall on either side. At the stage shown numerous cells are to be seen in the portion of the ground substance which lies below the notochord. These cells vary considerably in shape; some are rounded, while others have assumed a spindle-shape. In figure 4 is shown a portion of a transverse section through the tail region of an 11 mm. embryo which was preserved in Zenker and stained with Mallory's stain. The magnification is 760 diameters. The portion of the section shown in this figure lies just below the notochord. At this point there is a considerable area of the primitive ground substance. It is surrounded ventrally and laterally by developing muscle tissue and dorsally by the notochord. In this figure it is desired to lay particular emphasis upon the relations shown between the ground substance and the cells which are present in it. The cells which have wandered into this region have for the most part become amoeboid or spindle-shaped in type and they possess long cytoplasmic processes w^hich stretch out in various directions through the ground substance. The figure shows that, in a preparation of this kind which has been properly stained with the Mallory connective tissue stain, the differential color reaction CONNECTIVE TISSUE IN AMPHIBIA 455 between ground substance and cell bodies is so clear that the cell processes can be differentiated from the ground substance and traced to their definite endings, even though they may be drawn out extremely fine. Attention should also be called to the color reactions of the muscle tissue which bounds the ground substance. It will be noted that the bundles or muscle fibers show the characteristic color reaction of the cytoplasm of the mesenchyme cells lying in the ground substance. These results demonstrate that the Mallory connective tissue stain differentiates definitely the living substance, present in the cells and in the muscle tissue, from the intercellular ground substance. It is worth while to note in this connection that Mall says "In specimens of this kind it is easy to view these cells with their endoplasm as the connective-tissue cells and the exoplasm of the syncytium as the intercellular substance were not the development of the syncytium taken into consideration." And also^ "while my results are now decidedly in favor of Flemming's view, the reader will soon see that if other methods and interpretations are employed (which I now consider false), it will be quite as easy to see the fibers developing between the cells as within them." Inasmuch as in the present work the study of the various stages has shown, in general, that the formation of the ground substance takes place before there is any attempt to form a syncytium by the mesenchyme cells — in fact before they have left the cell groups, wandered into the ground substance and there assumed the irregular shapes characteristic of mesenchyme cells — it is evident that the formation of the ground substance cannot be due to a cytoplasmic syncytium. This fact leads definitely to the conclusion, as Mall suggests above, that the cells with their endoplasm are the connective tissue cells complete and the material in which they lie is not exoplasm but an intercellular substance or ground substance. The cells which move into the common, intercellular ground substance are separate entities; not a part of it but apart from it. «Mall, 1902, p. 336. 'Mall, 1902, p. 330. 456 GEORGE A. BAITSELL The presence of a ground substance formed prior to the appearance of the mesenchyme cells has been demonstrated by various investigators but notably by Szily ('07) who, working on chick and fish material, says^ Vor dem Auftreten der Mesenchymzellen sind die Liicken and Spalten der Embryonalanlage durch ein feines Fasersystem ausgefiillt." He holds, however, that this cell-free, fibrous, supporting tissue, instead of being a secretion from the cells, arises from fine fibrous protoplasmic processes of the epithehal and endothelial cells. The mesenchyme cells appear later and enter into a protoplasmic union with the fibers already present. The embryonic connective tissue is made up of the mesenchyme cells and the fibrillar intercellular material. The mesenchyme cells furthermore serve for the nourishment and growth of the fibers which, in the meantime, have become separated from the cells of which they previously formed a part. Szily therefore believes that, in the formation of the embryonic supporting tissues, all three of the primary germ layers take an active part, and the final product is a mixed tissue in which the product of any one germ layer cannot be identified. The present work agrees with the work of Szily in demonstrating the presence of a primitive ground substance prior to the advent of the mesenchyme cells. No evidence has been found, however, of its formation by means of protoplasmic processes of the surrounding cells. The study of the embryonic cells in the amphibian material, at the early stage at which the primitive ground substance is formed, shows them to have a clear, sharply defined, regular membrane with no processes of any kind. There is no question but that with a differential stain, such as Mallory's which was used, protoplasmic processes could be demonstrated if the}^ were present. It is easy enough to do so a little later with the mesenchjane cells when they wander into the primitive ground substance as shown in figures 4 and 6. The conclusion is, therefore, that the primitive ground substance in the amphibian embryo arises as a cellular secretion and not by the fusion of cytoplasmic processes. 8 Szily, 1907, p. 741. Cf. also Isaacs, 1919. CONNECTIVE TISSUE IN AMPHIBIA 457 In the discussion of the earher stages, it has already been noted that not only is the ground substance formed prior to the advent of the mesenchyme cells but also, in some regions, it shows from the earliest stages a fine fibrillation. In the stage shown in figure 4, this fibrillation of the ground substance has markedly increased and it is found that practically all the ground substance is permeated with the fibers. In some regions the fine fibers have united to form fibrous bundles and in general there appears to be a tendency for this to take place. A careful study of this and similar preparations reveals the fact, as is depicted in the figure, that there is no morphological connection between the cells which are present in the ground substance and the fibers. The latter have formed directly from the ground substance by a modification of the elements of which it is composed. The preparations give evidence that the movements of the cells through the ground substance may supply, in part at least, a mechanical factor which aids in the formation of the fibers. For example, it will be noted in figure 4 that the fiber formation is particularly heavy in the portion of the ground substance lying near the ventral opening in the muscle tissue through which, apparently, a number of the amoeboid cells have wandered into the ground substance. The preparations show many similar instances of regions in which the movements of the cells through the ground substance have supplied a mechanical stimulus and thereby brought about the fiber formation. There is also the possibility that the cells in their movements may have some secretory action which also modifies the ground substance. However, this point is clear from the study of the preparations and should be emphasized that in no case has any morphological connection been found between the cells in the ground substance and the fibers. In this connection attention should be called to the results previously obtained by the author ('17) in certain of the studies on the plasma clot in which it was demonstrated that, under the influence of certain mechanical factors, it was possible to transform a homogeneous fibrin clot into a fibrous tissue which, in its morphology, very closely resembled various types of fibrous 458 GEORGE A. BAITSELL connective tissues. In such cases it was possible to observe all stages in the process from an apparently homogeneous ground substance or matrix to the heavy fibrous tissue. By the use of a microscope equipped for dark field illumination it was possible to analyze the transformation process and to demonstrate that the fibers arose as a result of a fusion and consohdation of the minute elements of the fibrin clot which under ordinary illumination were entirely invisible. In experiments of this type, inasmuch as cells were not present, it was possible to state with, absolute certainty that the fibers had arisen through a modification of the ground substance of the clot. In other types of experiments with plasma clot preparation in which cells were present, it was shown ('15) that a transformation of the clot, and consequently fiber formation, could be brought about by mechanical factors induced by the movements of the cells through the clot. A case of this is shown in figure 5^ which is a drawing of a portion of a plasma clot preparation at a magnification of 950 diameters in which a piece of muscle tissue had been imbedded. A spindle-shaped cell has left the piece of imbedded tissue and is moving through the fibrin clot. In this preparation, the path of the cell can be traced from the imbedded tissue through the clot to its present position, by means of the fibrillar structure' which has arisen in its wake in the ground substance of the clot. In this and similar cases it is clear that the fiber formation in the clot has taken place by the introduction of mechanical factors through the cell movements with a consequent fusion and consolidation of the minute elements of the fibrin clot. In figure 6 is shown a portion of a transverse section through the tail region of a 75 mm. embryo of R. catesbiana at a magnification of 75 diameters. In this embryo the connective tissue is more mature in type with a practically complete transformation of the ground substance into a fibrous tissue. It will be noted in the figure that the densest formation of connective tissue is in the region immediately surrounding the notochord and also 5 Baitsell, 1915. A photomicrograph of this preparation is published in this paper as fig. 19. CONNECTIVE TISSUE IN AMPHIBIA 459 ventrally in the median line. From the median region of the embryo the connective tissue sheet extends laterally in either direction among the bands of muscle fibers — transverse sections of which are to be seen in the figure — until the body wall is reached. Between the body wall and the muscle tissue there is also a compact layer of the connective tissue. The fibrillation of the connective tissue is heaviest around the notochord. In the region of the muscle tissue the fibrillation is comparatively light and the ground substance closely resembles in its structure that found in 3'oung tadpoles as shown in previous figures. Under a higher magnification it will be seen that numerous sp:nd:e-shaped cells are scattered in various regions of the preparation lying in cavities on the connective tissue. In some cases they are stretched along the bundles of fibers. Here again the Mallory stain shows a clear differentiation between the cytoplasm of the cell bodies and the common ground substance in which they are imbedded and in no case has it been possible to demonstrate any morphological connection between the cells and the fibers. DISCUSSION The researches of Studnicka ('03), Hansen ('05), Mall ('02) and others, upon various phases of connective tissue development in different species of animals, demonstrate conclusively that a rigid adherence to the intracellular theory, in which it is held that the fibers develop directlj^ through a transformation of cell cytoplasm, is no longer possible. The result has been that the intracellular theory as originally stated has been modified by the Hansen-Mall theory which holds, as stated earlier in this paper, that the fibers develop, in general, in a common, living intercellular material, termed the exoplasm, which has been formed as a result of a syncytium of the mesenchyme cells. At the present time, the inadequacy of the unmodified intracellular theory has been recognized even by Flemming, whose work on the development of connective tissue fibers in the Salamander larvae ('91) is generally regarded as the corner stone of the intracellular theory. Flemming admits that the results 460 GEORGE A. BAITSELL of his researches, in which he showed that the fibers arose through a direct transformation of the cell cytoplasm, do not explain authenticated cases in which it is known that, in the later stages of development of certain connective tissues, an independent growth of the bundles of connective tissue fibers takes place. In such cases Flemming holds ('06) to the Hansen view of a living exoplasm which can nourish itself, grow by intussusception and differentiate new connective tissue fibers. ^° A review and summary of the question is given by Heidenhain in his Plasma und Zelle ('07). He says^^ that the general opinion of most of the recent investigators is that the first fibrillar differentiation in connective tissue formation arises by a transformation of the cell protoplasm. It is also generally held that a secondary fiber differentiation arises from an organized living matrix independently of cells. Cases are known however (e.g., V. Ebner's work) where the cells apparently first produce a structureless intercellular substance which secondarily produces fibers. With regard to the activities of the organized living intercellular substance (exoplasm), Heidenhain says:^^ >" Flemming, 19C6, p. 7. To quote: ". . . . ; wie sind aber die spateren Zustande zu denken, wo die schon gebildeten Fibrillen aus der Zelle herausgeriickt und Intercellularsubstanz geworden sind? Ich antworte darauf mit Hansen . . . . : dann liegen eben die Fibrillen in dem Territorium von Intercellularsubstanz, das von der betreffenden Zelle geschaffen worden ist, das aus ihrem 'Ektoplasma' (Hansen) hervorgegangen ist; diese Territorien sind mitlebendig, wie ich mir die ganze Intercellularsubstanz so denke . . . . , es konnen in ihnen Vorgange fortspielen, die zu einem Langenwachstum der Fibrillen durch Intussusception fiihren. Ein solches, intussusceptionelles Langenwachstum der Fibrillenbimdel waren wir ja tibrigens auch genotigt anzunehmen, wenn wir eine freie intercellulare Fibrillenentstehung voraussetzen woUten. Denn wenn ich mir eine embryonale Sehne in ihren friihesten Zustanden denke und dann spatere dagegenhalte, wo die Biindel 10- und mehrmal so lang geworden sind als zu der Zeit wo sie eben entstanden waren, wie soil das ohne eigene Wachstumverlangerung der Biindel abgegangen sein?" And again (p. 9): "Es bestande danach die gesamte Intercellularsubstanz des Bindegewebes aus solchen vereinigten Ektoplasmen von Zellen, die fibrillar umgewandelt wurden und die, wie ich mit Hansen glaube, mitlebend fortbestehen unter dem vitalen Einflusz der produzierenden Zellen und zur Entwickelung neuer intercellularer Formteile im stande bleiben." 11 Heidenhain, 1907, p. 37. •12 Heidenhain, 1907, p. 37. CONNECTIVE TISSUE IN AMPHIBIA 461 Innerhalb der einmal angelegten Intercellularsubstanz wachsen die Bindegewebsbimdel spaterhin selbstandig weiter fort, in die Lange sowohl in die Dicke. Dasz das Langenwachstiim ohne eine besondere Tatigkeit der Zellen vor sich geht, ist wohl immer angenommen worden ; was das Dickenwachstum anlangt, so war friiher die Anschaimg vielfach verbreitet, dasz die Zellen neue Fibrillen auf die Oberfliiche der erstmals angelegten Bimdel abscheiden. Indessen ist dies niir eine Formel, welche zwar aiis dem cellularen Prinzip sich ergibt, aber immoglich iiberall zutreffen kann. Tatsache ist, dasz man im lockeren Bindegewebe die Zellen oft nur sehr sparsam eingestreut findet, und dasz die Bindegewebsbimdel auf weite Strecken bin mit ihnen in keiner Berlihrimg stehen, also nur durch eigene Assimilation wachsen konnen. Heidenhain further holds^^ that the living intercellular material is of a more passive nature, in fact a sort of a living framework which lacks many of characteristics of true cell cytoplasm, but still retains at least for a time the power of growth through intussusception, and the ability to differentiate new fibrillar structures. To quote: "Die Metaplasmen sind in Verhaltnis zu den eingeschlossenen Zellen eine lebende Substanz besonderer Art, welche in eine andere Bahn der Entwickelung iibergegangen ist, ohne dasz eine Moglichkeit der Riickverwandlung in Protoplasma besteht." The adoption of this view of a living exoplasm wdth the power to assimilate food material and the ability to differentiate new fibrillar structures independently of the typical cells at once raises a number of questions. In the first place this living exoplasm must be regarded, as stated by Heidenhain, as being of a lower grade of living matter than that found in the regular cell bodies. It possesses only certain of the vital activities and entirely lacks others. In other words, it is necessary to conclude that, in the connective tissues of an animal, there are different degrees of living matter, beginning with the nucleus of the cell as the highest type, then grading into the endoplasm of the cell bodies and finally into the common exoplasm. In the second place, the postulation of a living exoplasm modifies the cell theory to a great extent, and delegates many of the powders supposedly possessed only by cells to masses of intercellular material, in considerable areas of which even nuclei " Hpidenhain, 1907, p. 48. THE AMERICAN JOURNAL OF ANATOMY, VOL. 28, NO. 3 462 GEORGE A. BAITSELL may be entirely lacking. This raises at once the question as to how such a material is governed. Heidenhain believes^* that "den Intercellularsubstanzen jene eigenartige Automatie des Lebens zukommt, welche alle lebenden Telle besitzen." He also holds that there is an external regulation of some sort but "Woher diese Regulation stammt, ist uns einstweilen verborgen." A consideration of the intracellular theory as modified by the studies of Hansen and Mall and the intercellular theory reveals a quite close agreement. This rests upon the recognition in both theories that a common intercellular ground substance is present in which fibers form independently of cells. The difference between the two theories lies in the question of the nature of the intercellular ground substance; the intercellular theory holding that it is a secretion product of the embryonic cells and the Hansen-Mall theory holding that it is a special type of living matter formed by a syncytium of the mesenchyme cells. In this connection, the present researches give evidence that the ground substance is nothing more than a type of secretion given off by the embryonic cells. This conclusion is based, first, upon the fact that ground substance is formed originally before any syncytium of mesenchyme cells could have been formed and, second, that material which has been properly stained with JMallory's connective tissue stain shows, at all stages, a clear and consistent differentiation in the color reaction between cell cytoplasm and the ground substance. The ground substance having been formed, the next step we have to consider is the formation of the connective tissue fibers. In this connection from the intracellular standpoint, Flemming and others have clearly demonstrated that the cells in developing connective tissue contain intracellular fibers which they regard as being connective tissue fibers. The presence at times of these intracellular fibrillar structures cannot be questioned but conclusive proof is lacking that they become the connective tissue fibers; there is the possibility that they are transient fibrillations due to various factors, examples of which can be found in many types of cells. •* Heidenhain, 1907, p. 48. CONNECTIVE TISSUE IN AMPHIBIA 463 In connection with this point, MerkeP*^ rightly calls attention to the fact that it is only the formation of the fibers in the first stages of connective tissue development concerning which there is a difference of opinion at present. For a long time there has been no question but that in the later stages of connective tissue development there is a complete separation of fibers and cells. Since this is the case, the logical conclusion appears to be that the fibrillar structures which are present in the connective tissue cells at certain times are not connective tissue fibers, for there is no apparent reason why the fibers in the early stages of connective tissue development should be formed in the cells and later the process change and the fibers be formed in the ground substance independently of cells. Considerable stress has been laid at times upon the spindle shape of the connective tissue cells or fibroblasts. I regard the shape of the connective tissue cells as almost entirely dependent upon the external factors which the cells encounter. The study of the material shows that when the mesenchyme cells first enter the ground substance they are, in general, rounded, typically-shaped cells. Later, in the ground substance, they assume various irregular shapes, as shown in figure 4, among which will be found a number of spindle-shaped fibroblasts. The idea has been more or less prevalent that cells of the spindle type are in some way 'spinning fibers' and therefore the shape has been regarded as a constant factor for the fibroblasts. The studies of various types of cells in tissue cultures^ show that the shape of the cell is not a constant factor. Certain cells w^hen grouped together in the cell masses show a typical cell body. The same cells when separated assume various irregular shapes, such as stellate, amoeboid and spindle, depending upon the nature of the medium and whether the cell is exhibiting movement. If the cell begins to move in a certain direction along a fiber or other support it will elongate in a direction parallel to the movement and assume thereby a spindle shape. ^^ It is believed, i^Merkel, 19C8, p. 381. « Harrison, 1914, pp. 535-8. Cf. Figs. 8-12. Matsumoto, 1918, pp. 555 et seq. 17 Harrison, 1914. Cf. figs. 4-7. 464 GEORGE A. BAITSELL therefore, that the spindle shape of a cell indicates a response to the environment and not a process of fiber formation. With regard to fiber formation the present work demonstrates that the fibers may develop in regions of the ground substance which are free from cells. At later stages when cells are present the study of the material gives no evidence of any morphological connection between them and the fibers in the ground substance. The complete [^process of fiber formation from the ground substance can be observed and the transformation into a fibrous tissue appears as a gradual development from a fine fibrillation through various stages until the well developed fibrous condition is reached. The previous studies of the author, with the plasma clot, show clearly how a morphological transformation of this character may take place, by means of a fusion and consolidation of the minute elements of which the clot is composed, in response to mechanical factors such as tension or pressure. It was suggested at that time^^ that "a reaction of this kind plays an important part in the ontogeny of the individual in the formation of the various connective and supporting structures. The well known fact that they, in general, are laid down in exact correspondence to the definite stresses of the organism leads to the conclusion that in their formation some arrangement must have been present which would respond to the various mechanical factors introduced during development, such as has been shown to be the case with the plasma clot. The generally accepted view of the intracellular origin of the connective tissues does not give any adequate explanation of this fact." From the morphological standpoint, the results of the present study indicate that the formation of connective tissue in the amphibian embryo is similar to the process which takes place in transformation of the plasma clot. The intercellular ground substance of developing connective tissue may therefore be compared in its morphology to the plasma clot. This ground substance when first formed appears homogeneous or with a '8 Baitsell, 1917, p. 130. CONNECTIVE TISSUE IN AMPHIBIA 465 fine fibrillation. The process of transformation into a fibrous tissue is a progressive one. The fibrillation increases, bundles of fibers are formed and in time the entire ground substance, which at first showed such a high degree of homogeneity, becomes transformed into a fibrous tissue. It is indicated that this transformation occurs as the results of the introduction of mechanical factors in the embryo. These factors may be due to certain lines of tension in the embryo corresponding to the inherent polarity of the organism or, just as in the plasma clot, the movements of the cells through the ground substance may introduce mechanical factors which aid in the transformation of the ground substance into a fibrous tissue. The cells, however, are to be regarded primarily as assimilative and secretive agents, chiefly concerned in the formation of the undifferentiated ground substance. SUMMARY 1. The primitive forerunner of connective tissue in amphibian embryos is a gelatinous material (primitive ground substance) which can be demonstrated around the notochord soon after it is formed and shortly before the embryo has reached the tail bud stage. A little later this material, which is to form in general the ground substance of the connective tissues, surrounds the medullary cord and a layer of it, following the body wall, extends ventrally on either side and in time completely encircles the body cavity. The formation of this matrix around the notochord takes place before there is any syncytium of the mesenchyme cells in this region. It is evident, therefore, that this primitive ground substance of connective tissue has arisen as an intercellular secretion of the embryonic cells and not by a cytoplasmic syncytium. 2. The ground substance having been formed, cells begin to move into it and wander through it. These are spherical at first, but as they move through the ground substance, they soon change into various shapes, becoming stellate, spindle-shaped, etc. The study of the sections shows that, in general, individual cells do not separate from the cell masses and move out into the 466 GEORGE A. BAITSELL various cavities and open spaces of the embryo until after the formation of the secreted ground substance which is the forerunner of the connective tissues. It appears evident therefore that cells, whether in tissue cultures as shown by Harrison^^ or in the developing embryo, have need of a supporting framework of some kind in order for movement from the main masses to take place. 3. The connective tissue fibers begin to arise in the ground substance soon after it has formed. In some cases, they form in the ground substance in regions which are free from cells so that it is certain that they have not arisen by an intracellular action. The fibers are evidently formed by a gradual transformation of the ground substance, first, into a delicate net-like structure and then into the long fibers which are typical of connective tissue. In its morphological features, at least, this process gives evidence of being identical with the one previously observed in the transformation of the plasma clot. 4. In those regions of the ground substance into which the cells have wandered, the formation of the fibers can also be shown to be due to changes in the ground substance and not to a sloughing off of the cell protoplasm. A differential stain, such as ]\Iallory's connective tissue stain, shows definitely the boundaries of the cell cytoplasm and of the ground substance. It is suggested that the movements of the cells through the ground substance introduce mechanical factors which aid in fiber formation just as has been shown to be the case in plasma clots. In any case, the fact that the fibers can form in a cell-free ground substance shows that no part of the cell cytoplasm is necessary for their differentiation from the ground substance; nor is it possible to demonstrate any morphological connection between the cells and the fibers in the ground substance, 19 Harrison, 1914, pp. 540 and 543. CONNECTIVE TISSUE IN AMPHIBIA 467 LITERATURE CITED Baitsell, G. a. 1915 The origin and structure of a fibrous tissue which appears in living cultures of adult frog tissues. Jour. Exp. Med, 21, 455. 1916 The origin and structure of a fibrous tissue formed in wound healing. Jour. Exp. Med., 23, 739. 1917 A study of the clotting of the plasma of frog's blood anc^ the transformation of the clot into a fibrous tissue. Am. Jour. Physiol., 44, 109. 1920 The development of connective tissue in the amphibian embryo. Proc. Nat. Acad. Sci., 6, 77. Ebner, V. V. 1897 Die Chorda dorsalis der niederen Fische und die Entwick elung des fibrillaren Bindegewebes. Zeitschr. f. wiss. Zool., 42, 469. Flemming, W. 1897 tjber die Entwickelung der collagenen Bindegewebs fibrillen bei Amphibien und Saugetieren. Arch. f. Anat. u. Phys. Anat. Abt. 21, 171. 19C6 Der Histogenese der Stiitzsubstanzen der Bindesubstanzgruppe. Handbuch der vergl. und experim. Entwickelungsges. der Wirbeltiere. Herausg. von O. Hertwig, Jena. Bd. 3, Teil 2. Hansen, Fr. C. C. 1899 tJber die Genese einiger Bindegewebsgrundsubstan zen. Anat. Anz., 16, 417. 1905 Untersuchungen liber die Gruppe der Bindesubstanzen. I. Der Hyalinknorpel. Anat. Hefte., 27, 538. Harrison, R. G. 1914 The reaction of embryonic cells to solid structures. Jour. Exp. Zool., 17, 521. Heidenhain, M. 1907 Plasma und Zelle. Jena. I. Abt. 1. Hertzler, a. E. 1915 The development of fibrous tissues in peritoneal adhesions. Anat. Rec. 9, 83. Isaacs, R. 1919 The structure and mechanics of developing connective tissue. Anat. Rec, 17, 243. Mall, F. P. 1902 On the development of the connective tissues from the connective-tissue syncytium. Am. Jour. Anat., 1, 329. Matsumoto, S. 1918 Contribution to the study of epithelium movement. The corneal epithelium of the frog in tissue culture. Jour. Exp. Zool., 26, 545. Merkel, Fr. 1908 Betrachtungen iiber die Entwickelung des Bindegewebes. Anat. Hefte, 38, 323. Nageotte, J. 1916 Les substances conjonctives sont des coagulums albumin oides, etc. Comp. Rend, de la See. de Biol. 79, 833. 1920 Croissance, modelage et metamorphisme de la trame fibrineuse dans les caillots cruoriques. Comp. Rend, de I'Acad. des Sci. 170, 1075. Studnicka, F. K. 1907 tJber einige Grundsubstanzgewebe. Anat. Anz., 31, 497. SziLY, Al. v. 1908 tJber das Entstehen eines fibrillaren Stiitzgewebes im Embryo und dessen Verhaltnis zur Glaskorperfrage. Anat. Hefte, 35, 107. For complete bibliographies of this subject see references to Heidenhain, Flemming or Merkel above. PLATE 1 EXPLANATION OF FIGURES 1 Portion of sagittal section of 3.2 mm. embryo of R. palustris. X 285. The notochord (N.C. ) is shown imbedded in the cell-free connective tissue ground substance (G.S.). The latter extends laterally on either side into the muscle plates. 2 Portion of transverse section of 4 mm. embryo of R. palustris. X 322, A section of the notochord (N.C.) is shown imbedded in the connective tissue ground substance (G.S.). 468 CONNECriVE TISSUE IN AMPHIBIA GEORGE A. BAITSEIX PLATE 1 9J^^W^^i&t ^W^i ■ ml It-,. ^V^- «*^V».-^s^v- :^r:^^^^y: **^^-^^ '^-:,M S. itp '■ f-^"^ ^ ::^^t^-:'^.:Ji^l^^Mbi.^ G.S. N.C. 4tj9 PLATE 2 EXPLANATION OF FIGURES 3 Portion of 9 mm. embryo of R. sylvatica. X 322. The ground substance (G.S.) is shown lying below the notochord (N.C.) and extending laterally on either side between the muscle plates to the body wall. At this stage some of the mesenchyme cells are beginning to move into the ground substance. 6 Portion of a transverse section through the tail region of a 75 mm. embryo of R. eatesbiana. X 75. The figure shows a portion of the notochord surrounded by the connective tissue ground substance (G.S.). In this late stage the ground substance, particularly around the notochord (N.C), is largely composed of bundles of typical connective tissue fibers. 470 CONNECTIVE TISSUE IN AMPHIBIA GEORGE A. BAITSELL PLATE 2 '\.-^ -••-TBI, ?■ -v m ^5«.*'^-^ N.C. G.S. ^G.S. W^%^ N.C. G.S. PLATE 3 EXPLANATION OF FIGURES 4 Portion of transverse section of 11 mm. embryo of R. sylvatica. X 760. Mallory connective tissue stain. The region shown lies just below the notochord as in figure 3. The connective tissue ground substance (G.S.) contains a number of wandering mesenchyme cells which take the same stain as do the surrounding muscle fibers (M.F.) — both types of cells being clearly differentiated by the stain from the secreted connective tissue ground substance which is formed before any cells are present in it. 472 CONNECTIVE TISSUE IN AMPHIBIA OEOBGE A. BAITSBLL PLATE 3 wis (wimlt^M 4 473 PRESS WORK BT FRED K OOF.B PLATE 4 EXPLANATION OF FIGURE 5 Section of plasma clot from a tissure culture. X 950. The figure shows the transformation of the fibrin net (F. N.) of a plasma clot into a fibrous tissue (F. T.) as a result of the mechanical factors induced by the movements of the cell through it. Cf. Baitsell, 1915, fig. 19. 474 CONNECTIVE TISSUE IN AMPHIBIA GEORGE A BAITSELL PLATE 4 \ X ■^' ^ \ \~ -F.N. /■■ / X--^F.T. «.*ssr^ ■^, ^.£.^.v<*tr 475 PKESe WOKK HY I'HKD K DOKH SUBJECT AND AUTHOR INDEX ABDUCENS nerve in human embryos. Recurrent branches of the 373 Acinus tissue. Cytological studies of Langerhans's islets, with special reference to the problem of their relation to the pancreatic 1 Activity. The development of the mammalian spleen, with special reference to its hematopoietic 281 Albino rats. Studies on the effects of thirst. I. Effects of thirst on the weights of the various organs and systems of adult 401 Amphibia. A study of the development of connective tissue in the 447 Abai, Hay.vto. On the cause of the hypertrophy of the surviving ovary after semispaying (albino rat) and on the number of ova in it 59 BAITSELL, Geoege A. A study of the development of connective tissue in the Amphibia 447 Birds. Studies on the gonads of the fowl. I. Hematopoietic processes in the gonads of embryos and mature 81 Bremer, John Lewis. Recurrent branches of the abducens nerve in human embryos 373 CELLS of tissue cultures. The effect of potassium permanganate on the mesenchyme 431 Connective tissue in the Amphibia. A study of the development of 447 Cultures. The effect of potassium permanganate on the mesenchyme cells of tissue, 431 Cytological studies of Langerhans's islets, with special reference to the problem of their relation to the pancreatic acinus tissue 1 DEFORMITIES, and the interaction among embryonic organs during their origin and development. Developmental rate and structural expression: An experimental study of twins, ' double monsters' and single 115 Development of connective tissue in the Amphibia. A study of the. . . .' 447 Developmental rate and structural expression: An experimental study of twins, 'double monsters' and single deformities, and the interaction among embryonic organs during their origin and development 115 Digestive system just before and soon after birth. The early establishment of the intestinal nutrition in the opossum — The 343 ' Double monsters' and single deformities, and the interaction among embryonic organs during their origin and development. Developmental rate and structural expression: An experimental study of twins, 115 Downey, Hal: Thiel, Geo. A., and. The development of the mammalian spleen, with special reference to its hematopoietic activity 281 EFFECTS of thirst. I. Effects of thirst on the weights of the various organs and systems of adult albino rats. Studies on the 401 Embryos and mature birds. Studies on the gonads of the fowl. I. Hematopoietic processes in the gonads of 81 Embryos. Recurrent branches of the abdudens nerve in human 373 FOWL. I. Hematopoietic processes in the gonads of embryos and mature birds. Studies on the gonads of the 81 GONADS of the fowl. I. Hematopoietic processes in the gonads of embryos and mature birds. Studies on the 81 HEMATOPOIETIC activity. The development of the mammalian spleen, with special reference to its 281 HetjSER, Chester A. The early establishment of the intestinal nutrition in the opossum — The digestive system just be^ fore and soon after birth 343 Human embryos. Recurrent branches of the the abducens nerve in 373 Hypertrophy of the surviving ovary after semispaying (albino rat) and on the number of ova in it. On the cause of the. ... 59 INTERACTION among embryonic organs during their origin and development. Developmental rate and structural expression: An experimental study of twins, ' double monsters ' and single deformities, and the 115 Intestinal nutrition in the opossum — The digestive system just before and soon after birth. The early establishment of the. . . 343 Islets, with special reference to the problem of their relation to the pancreatic acinus tissue. Cytological studies of Langerhans's 1 UDO, TOKUYASU. Studies on the effects of thirst. I. Effects of thirst on the weights of the various organs and systems of adult albino rats 401 K LANGERHANS'S islets, with special reference to the problem of their relation to the pancreatic acinus tissue. Cytological studies of 1 Lewis, Warren H. The effect of potassium permanganate on the mesenchyme cells of tissue cultures 431 MESENCHYME cells of tissue cultures. The effect of potassium permanganate on the : 431 NERVE in human embryos. Recurrent branches of the abducens 373 477 478 INDEX NoNiDEZ, Jos6 F. Studies on the gonads of the fowl. I. Hematopoietic processes in the gonads of embryos and mature birds. . 81 Nutrition in the opossum — The digestive system just before and soon after birth. The early establishment of the intestinal 343 OPOSSUM— The digestive system just before and soon after birth. The early establishment of the intestinal nutrition in the 343 Ovary after semispaying (albino rat) and on the number of ova in it. On the cause of the hypertrophy of the surviving 59 PANCREATIC acinus tissue. Cytological studies of Langerhans's islets, with special reference to the problem of their relation to the 1 Permanganate on the mesenchyme cells of tissue cultures. The effect of potassium. 431 Potassium permanganate on the mesenchyme cells of tissue cultures. The effect of . . . . 431 R ATS. Studies on the effects of thirst. I. Effects of thirst on the weights of the various organs and systems of adult albino 401 SAGUCHI, S. Cytological studies of Langerhans's islets, with special reference to the problem of their relation to the pancreatic acinus tissue 1 Semispaying (albino rat) and on the number of ova in it. On the cause of the hypertrophy of the surviving ovary after 59 Spleen, with special reference to its hematopoeitic activity. The development of the mammalian 281 Stockard, Charles R. Developmental rate and structural expression: An experimental study of twins, 'double monsters' and single deformities, and the interaction among embryonic organs during their origin and development 115 Structural expression: An experimental study of twins, ' double monsters' and single deformities, and the interaction among embryonic organs during their origin and development. Developmental rate and. . 115 System just before and soon after birth. The early establishment of the intestinal nutrition in the opossum — The digestive 343 THIEL, Geo. A.,. and Hownet, Hal. The development of the mammalian spleen, with special reference to its hematopoietic activity 281 Thirst. I. Effects of thirst on the weights of the various organs and systems of adult albino rats. Studies on the effects of . . . . 401 Tissue cultures. The effect of postasium permanganate on the mesenchyme cells of. . 431 Tissue. Cytological studies of Langerhans's islets, with special reference to the problem of their relation to the pancreatic acinus 1 Tissue in the Amphibia. A study of the development of connective 447 Twins, ' double monsters' and single deformities, and the interaction among embryonic organs during their origin and development. Developmental rate and structural expression: An experimental study of 115