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This historic 1931 embryology textbook by Richards was designed as an introduction to the topic. Currently only the text has been made available online, figures will be added at a later date. My thanks to the Internet Archive for making the original scanned book available.

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Outline of Comparative Embryology

By

Aute Richards

Professor 0 7,00/ugy Univrr.r1' 0 0/c/ahomc:

New York John Wiley 8: Sons, Inc. London: Chapman & Hall, Limited 1931

1951

Preface

As the result of a number of years of experience in introducing students to the subject of embryology, the writer has found it desirable to ‘depart from the common practice of studying chiefly the special embryology of the chick, pig, and perhaps of amphioxus and the frog, and to devote much attention to the principles of general embryology. This procedure is based upon the assumption that a broad background of general knowledge forms a desirable starting point for the study of special embryology to be taken up later. As comparative anatomy has proven to be the best basis for the later study of anatomy, so comparative embryology forms a proper basis for a study of special embryology. Unfortunately there has been no book in our language which brings together in a small compass the principles of general comparative embryology.

In this book it is the aim to set up the general principles of development so that the student may see how his special case—chick, starfish, or other form—is merely one of several types and what its relation to the other types may be. The writer holds that the most effective presentation of the subject matter of embryology is the laboratory study of desirable types, supported by text-book and “quiz” work on these forms and accompanied by the consideration from a comparative standpoint of the broad principles of the subject, such as types of egg structure, of cleavage, of gastrulation, and the critical consideration of classical embryological doctrines, etc. To present the material in brief form for this general part of a course in Comparative Embryology this volume is designed. '

In Part One are taken up those phases of development which are passed through in the embryology of most higher forms of animals. A comparative study of these phases no doubt forms the basis of ninetenths of the courses in this subject. Of the vast amount of material from which choice must be made, the decision as to whether any certain topic should be discussed has been based always upon a single criterion. It is sought to show only how the single-celled fertilized egg arrives at the multicellular condition characteristic of the fully formed, but undifferentiated, young organism in which the organ systems are established. In an outline of comparative embryology it is not possible to follow through all the details nor to repeat descriptions whose general features apply to more than one kind of animal. Nor is it possible to follow through for these forms the later stages in their development and differentiation. If completeness were the goal, it would be necessary to include the special embryology of the many forms about which investigators would desire information, an obviously impossible task. It is hoped, however, that a basis is here laid for understanding the detailed embryology of any of the types and for working out later stages in whatever group the student’s interest may lie.

Part Two considers numerous miscellaneous problems that have arisen in connection with the history of embryology. Here the choice of topics has been governed largely by questions asked by students both beginning and more advanced. No attempt is made to cover all the classical topics of embryology, nor to arrange the ones chosen in an order to develop any particular thesis. It is simply thought to satisfy in some measure student interest in them and to make them easily available. Discussions of many of them are not available in brief form in any English text, although they concern matters about which students of zoology should be informed, for they are chiefly problems of normal development.

The embryology of the present day is experimental embryology. Its aim is to secure an insight into the real nature of those steps by which a single-celled egg becomes a completed adult. An understanding of development in its true sense is its goal. Many aids to experiment used only by chemists or physicists in years gone by are now every-day tools of the embryologists, for descriptive embryology alone is inadequate to give a complete understanding of development. Yet experimental embryology runs the danger of considering only processes and those out of relation to structures. It is in structures that functions are inherent, for one cannot understand the workings of a machine with which he is unfamiliar. Morphology in its descriptive phases is therefore basic to a proper attempt to work out functional relations. Our admiration for the attempts to secure an intelligent conception of the mystery which underlies the pageant of development must not allow us to disregard the importance of a foundation of knowledge of descriptive embryology. Perhaps the errors which may be avoided by the study of descriptive embryology in its comparative phases may serve to recompense the student for the time lost from experimental embryology.

Undoubtedly numerous errors are to be found in this volume. Much that is discussed has been the subject of life-long study by many investigators and has concerned material collected from all over the world. To understand all adequately so that each may be mentioned correctly and weighted properly in a brief outline, is a task hardly to be accomplished, and many inconsistencies of treatment must be apparent to the reader. It must be borne in mind, however, that not an extended treatise but an outline of principles is the intent of the book, and the intent places many limitations upon what can be undertaken.

Acknowledgments are due for assistance in almost every section. The material treated in a work of this kind is by no means new, and it would be impossible to present any one phase of it without finding parallel treatment in some source of information available to students of embryology of this day. There is no claim to originality in the book, not even in the treatment of many chapters. The book will have served its purpose, however, if it appears to have sufficient pedagogical background and if it presents in sufficiently compact form these principles, many of which I think are not briefly summarized elsewhere in the English language, and which are needed to give students a basis for work in special embryology,-either medicalor experimental.

Korschelt and Heider’s “Lehrbuch der vergleiehenden Entwicklungs— geschichte” is a great treasure house of embryological learning to which constant reference has been made. In many places (particularly in Chapters VII and VIII, Part One) even their arrangement has been so much the best that only minor departures from it are to be found here. In others an entirely different organization of the same material is here adopted. But the debt to Korschelt and Heider is a very great one. I appreciate very mucl_1-the gracious verbal permission to use their material freely which was given me by both Doctors Korschelt and Heider. .”_

Drawings to illustrate ,the text are from many sources and not many are new. The writer is not impressed with the desirability of replacing pictures made by investigators on specialforms with new ones when the former are well known and of proven value. The drawings in every case are accompanied by a legend giving the name of the investigators to whom indebtedness is due.

Permission has been granted by the following publishers to make use of the figures indicated either as modified, or in the case of numbers 1, 2, 3, 135, 136, 142, 153, 1-81 of the figures themselves. By Henry Holt & Co. permission has been given to use from Loey’s “Biology and Its Makers,” our figures 1, 2, and 3; to use from Kellicott’s “General Embryology” our figure 5; from Kellicott’s “Chordate Development” our figures 135, 136, 142, 144, and 181; and from Lillie’s “Embryology of the Chick” our figures 151, 152. By P. Blakiston’s Son & Co., Inc., permission has been given to use from Folsom’s “Entomology with Special Reference to Its Ecological Aspects,” 3rd edition, our figures 117, 161, and 162; and from Patten’s “Embryology of the Pig,” figure 7. The Oxford University Press has permitted the use from Jenkinson’s “Vertebrate Embryology” of our figures 81 and 173. The Princeton University Press has granted permission to use from Conklin’s “ Heredity and Environment” our figures 7 and 8. The U. S. Bureau of fisheries has given permission to use from Herrick’s “American Lobster” figures 113, and 1973.. And William Wood & Co. have permitted the modification of figures from Bailey and Miller’s “Textbook of Embryology” represented in figures 140 and 141. Macmillan & Co., Ltd., has sold us permission to make use of our figures 103, 109, 110 from McBride’s “Textbook of Embryology, Vol. I, Invertebrate Embryology,” and of our figures 167, 168, and 170 from Graham Kerr’s “Textbook of Embryology, Vol. II, Vertebrate Embryology.” The graciousness of the various publishing companies in thus facilitating our work is deeply appreciated.

Especial acknowledgment for help in many ways including aid with the materials used and the reading of parts of the text is due to 1ny wife, Mrs. Mildred Hoge Richards. My colleagues of the Department of Zoology of the University of Oklahoma, Doctor A. O. Weese and Doctor A. I. Ortenburger have assisted with advice and have read portions of the text. Professor J. F. Paxton of the Department of Greek has aided me with questions of terminology. And finally my assistants Mrs. Celeste Whaley Taft and Miss Kara J. Fullerton have given the most painstaking attention to a multitude of details. To all of these I am deeply grateful.

A. R.

Part Two Embryological Problems

Chapter Vii Polyembryony

By polyembryony is meant among zoologists the production of multiple embryos from a single egg. (This definition does not apply to botanical nomenclature.) The number of individuals produced from a single egg ranges from hundreds as in the parasitic Hymenoptera down to two, although in the latter case the condition is usually known as twinning, and not all types of twins are properly thought of as polyembryonic.- Polyembryony occurs very widely distributed through the animal kingdom in groups which are totally unrelated to each other. Because of this wide distribution the literature to which reference can be made in this connection is really very extensive and the significance of some of the cases extends beyond the realm of embryology. Of the numerous investigators who have studied the question only a few need be mentioned here, but it may be noted that a reference to their work will serve as an introduction to the much broader literature which is not included in this discussion. Patterson has summarized the knowledge of polyembryony in a paper in Volume II of the Quarterly Review of Biology and it is suggested that the student consult this paper in beginning a more extensive study of these problems.

Patterson recognized three types of polyembryony: “ (1) experimental polyembryony, or the production of multiple embryos by artificial means; (2) accidental or sporadic polyembryony, or the occasional production of multiple embryos in the species which is typically monembryonic; (3) specific polyembryony, or the habitual production of multiple embryos in a given species.” The general student of comparative embryology is interested in the last type much more than in the first two, although cases of the first two throw much light upon many fundamental problems of organization of the egg and embryo and offer a very stimulating field for the experimental embryologist. Many types of eggs whose normal development includes no hint of polyembryony can be induced to produce double monsters and even complete embryos by experimental means.

Cases of occasional or sporadic polyembryony are recorded for coelenterates, cestodes, annelids, echinoderms, arthropods, and vertebrates. Perhaps because the latter group is so much more studied, there

353 354 POLYEM BRYONY

have been more cases reported for the vertebrates than for the others and, as Patterson points out, it is here that the occasional identical twins of the human species should be classified.

Specific polyembryony, to which our attention is drawn as the special problem of the comparative embryologist, is found in the following groups of animals: flatworms, bryozoa belonging to the Cyclostomata, earthworms, parasitic Hymenoptera, and the mammals, especially as illustrated by the armadillo. It is of more widespread distribution, however, than is indicated by these groups, but here are included the more important cases.

An inspection of this list is sufficient to show that polyembryony has no phylogenetic or taxonomic significance, for the groups are too widely separated and too diverse in structure to admit of an interpretation of this kind. Moreover, the structural differences both of adults and embryos in these various groups are so great that it is not possible to look for the causes of the phenomena in any simple embryological process common to all. Doubtless in the final analysis all these processes have a common underlying causal factor which is bound up with the innate protoplasmic organization of the egg substance.

As Patterson has pointed out, it is really not much more strange for an egg to give rise to two embryos than to one. The remarkable fact is that a new organism can be produced from an egg at all, and those qualities of the living substance which make possible its reproduction are basic to the one case no more than to the other. We must recognize that specific polyembryony is as much a characteristic of certain animals as is the formation of a coelome. At a certain stage in development there occurs a series of events which lead perhaps to two buds, or more, from each of which individuals grow. Very little thought is necessary to convince oneself that the directive forces behind this type of budding are not much more astonishing than those which cause, in the other case, the archenteron to bud out to form an enterocoele.

A. THE Causns or POLYEMBRYONY

Of the explanations of polyembryony, probably the most prevalent is the theory of blastotomy, according to which the blastomeres of the cleaving egg become separated in the 2-, 4-, 8-cell stage or later, and lead entirely independent existences, each arriving at length at the stage of a completely formed embryo. This theory is the basis of the familiar explanation of the origin of identical twins in human beings, that the blastomeres in the 2-cell stage become separated and each gives rise to an embryo. This is a view with few observational data to support it, but it may be presumed to find considerable support in the THE CAUSES OF POLYEMBRYONY 355

experimentally induced development of isolated blastomeres. Numerous experimental devices are well known by which blastomeres can be separated. To mention only two of these devices as examples, it is possible more or less successfully to cut apart the blastomeres with a knife or delicate thread drawn about the egg, or to keep them apart by subjecting the eggs to calcium—free sea water in which the blastomeres do not cohere to one another. In some of these experiments the isolated blastomeres have lived and developed for a time; in others they have produced partial larvae (ascidians etc.); and in others they have developed into whole larvae (amphioxus, Cerebratulus). But in forms where direct evidence for blastotomy is possible none has been found which forces one to this view for cases of polyembryony. As will be seen presently, the facts in the armadillo make the explanation based on blastotomy untenable there, and render it highly doubtful elsewhere.

A second explanation takes the form of an assumption that polyovular follicles, that is, follicles containing several ova fused together, may provide the mechanism by which polyembryony is accomplished. It is only necessary to say that the evidence for this supposition is entirely insufficient and the theory may now be regarded as abandoned.

In the budding theory we have what is perhaps a closer correspondence with the observed facts, although it is more of a descriptive explanation than an attempt at developmental analysis. After cleavage there arise in the blastulae or gastrulae of lower animals or in the blastocyst of mammals certain areas or “growing points” which are essentially buds. If a single one is produced it becomes the apical or head end of a normal embryo and assumes dominance over the remaining parts. If two are formed they are the beginnings of twin embryos. Similarly secondary buds may be formed and four or more embryos result, depending upon the number of buds. The facts observed in the armadillo will be seen to harmonize well with this explanation.

This ‘theory of budding has been criticized rather severely, particularly by Assheton, on the grounds that budding cannot take place unless there is a stock from which the buds may arise and the presence of a stock Assheton claims was not demonstrated in the cases to which the theory was applied. Rather he interprets the described facts as cases of fission. The blastocyst of several mammals, for he was particularly considering the theory as applied to the armadillo, he described as undergoing direct fission into two embryonic rudiments. As a matter of fact the fission hypothesis does not differ from the idea of budding in relation to the facts as described, but is primarily a redefinition of them, for in both cases the embryonic vesicle is regarded as divided into several primordia, each of which is the beginning of a distinct formative area 356 POLYEM BRYONY

and consequently will give rise to a separate embryo. Whether the emphasis is placed upon the embryonic mass which is thought to bud or upon the separate areas which arise from it as would be the case in fission does not seem to involve any particular difference in the result nor to carry the explanation of the environmental processes much further toward a final analysis.

As an explanation of the process a physiological interpretation must be called on. Stockard has pointed out that if a developmental pause occurs at the critical moment in the formative stages of an embryo, a series of consequences ranging from simple malformation up to the production of double monsters or even two individuals may result. He applies this conception to the explanation of polyembryony, supposing that the reason for the loss of dominance of the first formative region of the embryo and the subsequent ascendency of the primary and secondary buds is connected in some way with the developmental pause. He points out that in the armadillo a period of physiological isolation intervenes at one of the critical moments, making possible the four resultant buds. Certain observed facts bear out this conclusion. In any case within the growing germ the isolation of formative areas occurs and from these formative areas the multiple embryos are produced.

An obvious connection exists between the phenomenon of polyembryony and that of metagenesis which involves at least the alternation of one sexually produced generation with an asexually produced one. It is usually the practice to limit alternation of generations to the lower forms of animal life and to say that it does not occur in higher forms, including the vertebrates. Stockard, however, speaking in terms of the budding hypothesis, has pointed out the fact that the embryonic mass of cells may be looked upon as one generation, namely the one produced from the fertilized egg, and that it is completed when it becomes a stock from which other buds, that is growing points, begin their development. This point of view has its application even to mammals whether only a single organism results or several as in the case of the armadillo. Here the blastocyst would be regarded as the sexually produced individual and the buds which give rise to the four young as those asexually produced from it.

Stockard says, “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. OBSERVED CASES OF POLYEMBRYONY 357

“Alternation of generations is here considered a phenomenon, not limited as is generally taught to lower forms, but occurring throughout the animal kingdom.”

If the blastocyst is to be looked upon as a sexually produced individual from which arises asexually another or several others of a second generation, it is suggested that in this fact lies the explanation

fiG. 219. The development of the ovicell in Crista remosa. (From Patteron, after Harmer.)

A, external view. B, median longitudinal section of young ovicell. fol., follicle formed from polypide-bud; ov., ovary; ovi., oviccll.

of the difliculties that have met every attempt to homologize the early development of the mammals with that of the lower vertebrate classes. These differences have been discussed in Chapter X on the formation of the mammalian embryo; they constitute a problem of major importance to the comparative embryologist.

B. Onsnnvnn CASES or POLYEMBRYONY

We may now devote ourselves to the consideration of the groups of organisms in which specific polyembryony has already been noted to 358 POLYEM BRYON Y

occur, bearing in mind the different types of explanations that have been offered and attempting to discover how the facts conform to the suggested explanations.

flatworms. Among the flatworms are many cases which depart from the usual methods of reproduction. Of these some which do not fully fit into the classification have been described from time to time as polyembryony. There is one flatworm in which polyembryony undoubtedly occurs, however; it is the cestode Taema echmococcus, or Echmococcus coenurus as it is sometimes known. This small tapeworm produces eggs which develop into the usual hooked embryo, the onchosphere, and these in their turn produce a cyst which becomes the bladder worm or cysticercus. As is usual in the further development of a cysticcrcus, an invagination from the outside wall of the bladder into its cavity develops a new scolex and neck region which will later become inverted and form the new tapeworm. However, the matter is complicated in this case in that many scolices may be produced within a single bladder by the budding process, and even secondary bladders which produce multiple scolices in their turn are described. Thus the encysted worm may grow to an enormous size to endanger the life of the host and oftentimes bring about its death.

Bryozoa. Polyembryony occurs in the cyclostomatous bryozoa belonging to the Gymnolaemata of the Endoprocta. These animals live in colonies made up of zooecia

fiG 220- Section through which are in general tubular with densely

a follicle of Cnaza remosa show- - mg primary embryo (1) which calcareous walls. The circular orifices of the

§>ré>dt:)c1I(IiL(zisecorZ<I‘i‘ary erlgnbgnis zooecia give the group their name. At the time son, Xftei’ H$fi;e,_,r°m 3 e ' of the breeding season a curious specialized zooecium develops which is called the oecium

or ovicell. In this is developed the young embryo. Fertilization of a rather unusual sort having been successful, the oecium serves as a brood chamber about the developing embryos and larvae. The primary embryo undergoes a process of budding, and secondary and tertiary buds are EARTHWORM 359

produced all of which may develop into larvae, as many as 150 being on record from a single egg. They develop into ciliated larvae and each is capable of forming a new colony upon escape from the oecium. Earthworm. In the earthworm of the species Lumbricus trapczoides (Helodrilus caliginosus trapezoides) polyembryony results in the production of twins, a process which by Kleinenberg was regarded as universal for the species, but by Vejdovsky was held to be abnormal. It is due to a process of fission of the embryo. The cleavage of the egg

C D

fiG. 221. Twinning in the earthworm, Lumbncus trapezoides. (From Patterson, A, B, C. after Kleinenberg.)

A, Section of young twin embryos. the right one being the more developed. B, Double embryo, in section. C, Double embryo about to break apart. D, Type of double monster formed when embryos fail to separate.

is much modified from the typical spiral form of the annelids and indeed is variable. A blastula is formed, the endoderm and mesoblast cells pass into the cavity and the entire mass begins to elongate. Across the equator of the elongating mass a transverse furrow now appears from one side. As it deepens, the embryo is divided into two hemispheres held together by a few ectodermal cells only. Each half is destined to form one of the twins. Differentiation, gastrulation, and the completion of the internal organization go ahead while the worms remain connected. But at length they separate after a series of rotations which breaks them apart, except in a certain proportion of cases which become double 360 POLYEMBRYONY

monsters of various degrees of union. Thus from the single egg twin earthworms are produced.

In two other genera of oligochaetes double monsters have been described, and one infers that the twinning phenomena, though rare, may occur in a number of families. These cases were Tubifea: tubifex, found by Welch, Tubzfex rivulorum by Penner, and Sparganophilus eiseni by Hague.

Parasitic Hymenoptera. In the parasitic wasps we find some of the most important and complex cases of polyembryony in the entire animal kingdom, and certainly here the results of the process are exemplified in the most striking manner. In a single brood hatching from one parasitized caterpillar hundreds of individuals may be seen to issue all developed from one egg. Our knowledge of polyembryony in insects dates from the work of Marchal in 1898 and of Silvestri in 1906. Although a number of important points still remain to be satisfactorily cleared up, a number of investigators have contributed to this subject since then. Among them are Martin (1914), Patterson (1915, 1917, 1921, 1927), Hill (1922, 1923), and Leiby and Hill (1923, 1924), as well as others. The important genera studied include Litomastix truncatellus, Ageniaspis fuscicollis, several species of Copidosoma, Paracopz'dosomopsis floridanus, and three species of Platygaster. The most recent study is that of Parker (1931) upon the braconid, M acrocentrus. Undoubtedly many other forms of insects show polyembryony, but the ones mentioned include the more important cases studied.

The manner by which a single egg produces multiple embryos in these forms is perhaps best understood by beginning as Patterson has done in the paper earlier referred to with a description of Platygaster hiemalis, a parasite of the Hessian fly, which usually produces two individuals from one egg. From this comparatively simple case we have a gradual increase in complexity which at the end of the series is only incompletely understood, but the simpler cases at least are suggestive of the methods by which the more complicated probably develop.

In this species the parasite lays from four to eight eggs either in the egg or young larva of the host. If fertilized, the egg nucleus divides twice, producing two polar body nuclei; these are not passed to the outside of the egg but remain in the cytoplasm where after a time they come together to form a large polar nucleus called the paranucleus. With the fusion of the male and female pronuclei the egg becomes differentiated into two separate regions. The cleavage nucleus and cytoplasm surrounding it become cut off from the remainder and constitute the embryonic region, for only from it comes the material which will go into the formation of the developing embryos. The rePARASITIC HYMENOPTERA 36 I

mainder of the egg containing the paranucleus and its own cytoplasm entirely surrounding the embryonic region functions to absorb and

fiG 222. Polyembryonie development of Platyoaster hwmalw (From Patterson, after Leiby and Hill )

A. Egg of four hours, showing sperm head and first maturation division B, Egg showing two polocytes. and both pronuclei C, Egg with pronuclei in comunction and polar nucleus D. Embryo, two polar nuclei and a. parasitic body showing four nuclei E, Enclosed within a cyst formed from tissue of the host are two embryonic bodies each surrounded by trophamnion F. A later stage in the development of two embryos G, A thirteen-day-old polygerm in section, showing several embryos.

elaborate the tissues of the host for the nourishment of the young embryos and it is therefore spoken of as the trophamnion. In this species, although it is not characteristic of all, the host tissues form a 362 ‘ POLYEMBRYON Y

cyst wall around this developing body which then is spoken of as the parasitic body.

The paranucleus as development proceeds divides amitotically twice and the daughters distribute themselves in the trophamnion surrounding the embryonic region. The zygotic nucleus of the embryonic region divides first into two, then four, embryonic nuclei. Thereupon, the embryonic region becomes constricted and presently separated into two regions, each provided with two nuclei, and the trophamnion with its paranuclei also divides into two corresponding regions. The two halves of the parasitic body thus differentiated remain held together within a single cyst of host tissue, but develop independently. Four, eight, and sixteen nuclei arise from the division of each of these germs, as they are called, and these arrange themselves into the form of a typical spherical blastula, the cell walls being cut off around each nucleus. The remainder of the development is unimportant, for the process characteristic of these blastulae go forward through regular stages. There is produced from each a new individual which hatches in the next year.

In another species of this genus, P. vernalis, eight embryos are regularly developed. Here the process has a fundamental similarity to that just described, but the details of development differ. The parasitic body develops a number of embryonic nuclei surrounded by an appropriate amount of cytoplasm and a cell membrane. Each of these becomes a germ for the production of the later embryo and the entire mass is spoken of as a polygerm. It happens that the more complex cases were studied before the development of Platygaster was understood and some of the problems that were not clearly worked out would now probably be more easily followed through. It is known now that in Copidosoma gelechiae in the formation of the polygerm many primary germs are produced in a manner similar to the eight of Platygaster vernalis. These separate germs of the last polygerm stage all divide and give rise to two which develop into separate embryos.

Leiby and Hill believe that in Paracopidosomopsis, which is even more complicated, a secondary germ divides to form tertiary ones before the larval differentiation begins.

Armadillo. The final case of polyembryony to be discussed is perhaps the one in which the greatest interest lies. The Texas nine-banded armadillo Dasypus (Tatusia) novemcinctus normally produces four young from a single egg, the quadruplets being identical in respect to sex and to most of their morphological features. The work on the armadillo from the standpoint of polyembryony is of rather recent date, Fernandez concluding in 1909 that it occurs in a South American ARMADILLO 363

species Dasypus hybridus, and Newman and Patterson in the same year publishing the beginning of their important studies on the Texas species. Subsequent publications by these authors working independently have made available an account of the development of this form which is practically complete. The processes have been shown to be identical in respect to all important features in the two species which have been studied. The reader is referred to Chapter X for a discussion of the formation of the blastocyst in mammals.

In the armadillo as in other mammals, a monodermic blastocyst is formed which becomes differentiated into a trophoblastic portion and a formative portion, the inner cell mass. Then from the inner cell mass the endoderm is differentiated off, the remainder becoming ectoderm. Up to this point the blastocyst has remained free in the uterine cavity, but now attaches to the uterine mucosa. Attachment takes place directly over the embryonic ectoderm and the Trdiger forms at this point; then the inward growth of the spherical endodermal mass begins which brings about the so-called inversion of the germ layers. Within the ectodermal mass the formation of the ectodermic vesicle marks the beginning of the amniotic cavity. Above it an extraembryonic cavity is formed in the mesoderm. With progressive development the shift of the ectodermal cells takes place, resulting in the formation of the em~ bryonic shield with its thick ectoderm and the true amnion above it. In this stage the first sign of polyembryony makes its appearance, for from opposite sides of the ectodermal vesicle thus formed blunt projections extend laterally. Patterson called these primary buds. By this time that portion of the trophoblast which has not become involved in the attachment to the uterine wall disappears and the yolk sac endoderm is directly exposed in the uterine cavity. The two primary buds now divide each to form two secondary buds, making in all four buds which are rudiments of the four embryos subsequently to arise. Although they arise in this bilateral fashion and develop in the right and left halves of the uterus respectively, they later come to occupy positions that are about equally spaced from each other.

Meanwhile the vesicle grows, and the endoderm of the yolk sac, including those portions with which the embryonic ectoderm of each bud is in contact and which will form the gut endoderm of the embryo, faces the uterine cavity. The anterior ends of all the embryos point toward the apex of the common ectodermal vesicle, that is, toward the original amniotic cavity, and the entire vesicle may now be spoken of as the common amniotic vesicle. The entire structure now grows very rapidly, especially that portion of it which originated from the trophoblastic knob or the Trdger. This growth finally comes to occupy most 364 POLYEMBRYONY

of the space in the fundus of the uterus. The further history is especially concerned with the embryonic buds. Their posterior ends lengthen out with the growth of the vesicle and finally unite at a point opposite the original attachment and from their point of union the umbilicus later arises. The common amniotic vesicle by this growth procedure is left

fiG. 223. The development of the blastocyst of the armadillo. (After Patterson.) ec., ectoderm; en., endoderm; icm., inner cell mass; mes.. mesoderm; tr., trager; tro., trophoblast; u.. uterus.

as a small structure at the lower apex, in contact with each bud, while most of the remaining portion of the cyst mass with its developing embryos is derived from the growth of the Trdger. The buds meanwhile continue their growth, each forming a primitive streak, which is a substitution for the embryonic shield as described for other mammals. The further development of the embryos and the subsequent changes which lead to the production of the four foetuses need not be traced ARMADILLO 365

since it is simply a problem of organogenesis and of the steps normally following. The relations which are necessary for the understanding of

fiG. 224. A, The uterus and entire blastocyst of the armadillo showing paired origin 01' embryos. B, Half-grown foetuses spread out from uterus wall. (After Patterson.)

the polyembryonic condition all grow out of the budding processes of the embryonic vesicle and are completed with the establishment of the four embryos. There are many interesting problems connected with 366 POLYEMBRYONY

the later stages from the standpoint of later embryology and hereditary correlations of the characters of the four offspring as well as from other points of view, but the special problem of polyembryony is explained by these earlier stages.

C. EXAMPLES or DOUBTFUL POLYEMBRYONY

Two other cases which resemble polyembryony in some respects should be mentioned. As a matter of fact there are some departures in each of them which do not justify their inclusion with this type of development.

Among the trematodes the egg of Fasciola hepatica, the sheep fluke, is fertilized in the body of the adult fluke, makes its way down the bile duct and out of the intestine of the sheep and hatches when it rains as a tiny ciliated larva, a miricidium. Entering the intermediate host, the snail, the miricidium becomes a sporocyst and within it parthenogenetic ova appear which develop into rediae, the next larval form. Within the body of the rediae the process may be repeated but at length cercariae are developed which escape from the snail and bring about the reinfestation of the adult host. This case has been called polyembryony by some, but by others it is regarded as paedogenesis, for here the new larvae are said to be produced not asexually but from parthenogenetic ova, a difierence in method which, if true, would seem to be fundamental. However, the reservation must be made that if the germ masses from which the new larvae arise are not ova, but are produced asexually (as F. G. Brooks believes, having found no maturation phenomena or other egg—like behavior), then we have to do here merely with asexual reproduction, and not even paedogenesis,

A second case which at first thought suggests polyembryony occurs in the tunicates. Among the Thalaceae as represented by the genus Salpa it has long been known that two forms of individuals are to be expected, one a solitary form and the other a colony which is usually found as a chain of individuals. The first of these develops asexually, although produced from a fertilized egg. The second is known to be budded ofi’ from a stolon which grows out from the asexual individual, and among the colonial forms some at least are sexual, producing the eggs and sperm which in their turn start the cycle over again. The genus Dolielum is rather more complicated than Salpa but it seems to illustrate the situation quite well and it is here that the type of development of particular interest to us may be said to occur.

In Doliolum an oozoid is developed from the fertilized egg and it reproduces asexually. By some it is regarded as a larval zooid, and certainly it has not yet reached its final character. If it is correct to EXAMPLES OF DOUBTFUL POLYE-MBRYON Y 367

regard the oozoid as a larval form, this case is very closely akin to polyembryony. From the ventral posterior part of the oozoid a proliferating stolon is formed as a protrusion of the ectoderm into which the mesoderm penetrates. From the stolon buds arise which break loose in a very immature condition and Worm their way upward and dorsally over the surface of the parent. Since the number given off from the ventral stolon is less than the number later found on the dorsal process, it is assumed that the buds must divide en route, and some evidence of this process has been found. Arrived at the dorsal side of the oozoid, the buds attach themselves to a posterior process of the test which arises as a middorsal projection (occasionally spoken of incorrectly as a dorsal stolon). These bodies attach themselves in three longitudinal rows to the outgrowth. The two lateral rows of buds are known as trophozooids or gasterozooids and their only function is to nourish the colony. The dorsal row of buds undergoes metamorphosis into several kinds of zooids. Some become phorozooids or nurse zooids, and according to some investigators they produce from a ventral stalk buds which are protogonozooids or primary sex buds. These latter either by budding or fission produce the sexual animals, the gonozooids or blastozooids, in which with further development sex organs are formed and the cycle is completed. Here an alternation of generations occurs in which there are three asexual generations and one sexual generation in one cycle. Whether it is to be regarded as polyembryony depends upon the interpretation of certain stages. It is very complicated in the extreme, and is at least close to true polyembryony.

Chapter VIII The Determination Problem

A. INTRODUCTION AND STATEMENT or PROBLEM

A problem which has run through the entire history of embryology and is yet a live question at the present time concerns the extent to which the egg represents a fixed and definite system whose course is set when its development has begun. This is the determination problem. It first became a live issue during the controversy between the preformationists and the epigenesists of the seventeenth and eighteenth centuries and was even in that crude form an attempt to solve the question of the organization of the egg. The one school held that development was

simply an unfolding of an organism already present in an infolded con—'

dition; that is, organization was complete and development brought forth nothing new. The other held that the egg was unorganized and that everything in development was new. The history of embryology since these crude beginnings, at least of that part of the science which has concerned itself with the earliest stages in ontogeny, has been an attempt to make clear the manner in which the organization of the egg becomes manifest. Except for historical reasons it is not necessary to trace the course of these studies nor would it be possible except in a much more extensive treatment of the subject than is given here, for the literature is voluminous. It is not even possible to summarize in a short discussion the work which has been done. All that can be attempted is to bring together briefly the lines of work which have been responsible for the present conceptions in regard to determination.

Two sets of facts confront the student who would consider the determination problem. Neither bears directly upon the question at issue, but both serve to limit it. In the first place an egg goes through certain steps with surprising uniformity and produces an individual which is structurally very complex and very much differentiated. It is organized in the highest conceivable degree. In the second place modern genetics has shown that the egg to begin with possesses in the mechanism by which the genes are controlled and distributed a type of organization likewise complex in the extreme; and there is no doubt that this mechanism operates to produce the conditions found in later ontogeny. Yet differentiation is a matter of the cytoplasm of the cells. The repeated

368 INTRODUCTION AND STATEMENT OF PROBLEM 369

mitoses do not affect in a differential fashion the elements of the nucleus and it is clear that the nucleus contains in the chromatin the non differentiating material, the germ plasm, while the cytoplasmic portion of the cell is the basis of specialization and is indeed the somatoplasm which gives rise to all the differentiations later seen in the course of ontogeny. Between these two sets of facts must lie the third set, little understood and often little appreciated at the present time. Almost nothing can be offered in the way of explanation as to the method by which the gene mechanism produces its fundamental effect upon differentiation. The gene mechanism concerns heredity, that is, by it is explained the similarity existing between generations, but as yet its study has offered little which enables us to understand its relation to the differentiating soma. It was earlier said that heredity was the central problem of biology. Differentiation is certainly a central problem of development, and, let it be said, at the present time one of the most elusive. The questions of differentiation and of determination have dominated a surprisingly large part of the embryological work of the last century.

These two questions are really two aspects of the problem of organization, for differentiation is the manifestation of the operative mechanism of the egg as modified by extrinsic factors. To discover the extreme to which this operative mechanism is already present in the early stages and to which it gives a definite “set” to the differentiating embryo is the aim of the work on determination. If differentiations become visible early and are little modified by extrinsic factors with the result that the course of development is orderly in a high degree, we say that the egg is strongly determinative. But if the egg retains its embryonic plasticity and easily adapts itself to environmental disturbances, that is, is easily modified by extrinsic factors, it is said to be indeterminative or of the regulatory type. If differentiations manifest themselves early, little regulatory capacity is retained by the egg. In other words, the capacity of the egg for regulation is inversely proportional to the progress of differentiation.

It must be borne in mind also in considering the problem of differentiation (and hence of determination) that the chemico-physical organization of the cell limits the conclusions which may be reached. Differentiation manifests itself through colloidal materials. The chemistry of the protoplasmic system is so imperfectly known that whatever conclusions are reached regarding these problems must be subject to revision when a better understanding shall have been attained, for example of the phenomena of gelation which plays a very important part in the physical expression of mitotic phenomena. It has been shown by the studies of 370 THE DETERMINATION PROBLEM

Conklin and of others that the position and size of the spindle are matters of great importance in differentiation. Yet the physico-chemical factors which are involved in spindle behavior have not yet been adequately analyzed. Other problems of physico-chemical nature similarly affect our"conclusions. It is not too much to say that in spite of the vast amount of embryological research differentiation and its mechanism is one of the least understood fields of biology.

It has been pointed out that the function of cleavage is to sort out the materials of the egg into cells so that differentiation may progress. The uncleaved egg represents all of the materials present, although not necessarily all that are later to appear. Yet the progressive development of the organism in its usual course at least as indicated by our present knowledge depends upon getting these materials separated from each other. It does not follow, however, that this sorting-out process of cleavage is the cause of the subsequent development. Rather there must be a fundamental underlying mechanism of organization, the manifestations of which are the cleavage phenomena. Primarily determination concerns itself with the manifestations of these underlying methods of organization. It seeks for evidence of pre—1ocalization of areas or substances within the egg or even of an early promorphology which gives visible expression to the underlying mechanism. Some of the questions involved may perhaps be formulated in the following manner: Are there special structures or substances or fundaments which exist in the egg at the very beginning of development and which are independent of each other and of other parts of the egg? If so, how are they formed? Must all appear at the same time, or may some appear later on? Conversely, are the definite structures of the embryo never independent of each other? Are the parts always influenced by the whole of which they are constituents? Or finally, are there some eggs which are determinative in their character and others which are not, or are some parts of the individual embryo determinative and other parts not? Perhaps all these questions may be said to depend most largely for their answers upon this one. At what time and from what sources do differentiations begin in the embryo? It may be noted that the answers to these questions are not the same for all organisms.

B. CLASSICAL THEoR1Es or THE NATURE or DEVELOPMENT

Historically there have been a number of theories developed in response to these questions. They represent the various forms which the answers of different investigators have taken and must be considered from the standpoint of the information available when they were formulated. One of them was the theory of organ-forming regions of CLASSICAL THEORIES OF THE NATURE OF DEVELOPMENT 371

Wilhelm His (1874). This is the doctrine that definitely localized areas perhaps even in the unsegmented egg are the forerunners of the'organs and parts of the embryo, that “the germinal disc [of the chick] contains the preformed germs of the organs spread out over a flat surface, and conversely . . . every point of the germinal disc is found again in a later organ.” Ray Lankester supported His in his views, saying that it was quite possible for the cell to “contain already formed and individualized, various kinds of physiological molecules. The visible process of segregation is only the sequel of a differentiation already established and not visible.” Whitman as a result of his work on Clepsine concluded that the embryo is predetermined, even if not predelineated. Rabl and van Beneden likewise were among the early supporters of the view that even in the unsegmented egg an organization of protoplasmic particles which predetermines the development of the embryo is present. In the bald form in which this doctrine was proposed by His it can scarcely be accepted, however, and as a matter of fact His had no great amount of observational and experimental evidence on which to base it. The study of cell lineage offers what of evidence there is in support of this doctrine but even at best it does not force as rigid an interpretation as His placed upon it. Many authors have denied it in its entirety and there are few who do not criticize it.

Another form of answer to the question of determination is the theory of organ-forming substances, according to which the materials within the egg rather than the areas which they occupy receive the chief attention. Here again it is obvious that not all materials within the egg, even though they be definitely localized, are formative. Yolk, pigment, oil globules, and other inclusions in the cytoplasm are definitely located in many eggs, yet clearly are not formative. In some instances visibly differentiated substances within the unsegmented egg have been shown to have no formative function. At least they have been displaced by pressure or by centrifugal force, and normal embryos have nevertheless resulted. Yet in other forms the materials of the egg have been traced in a precise manner to the organs of the embryo. Surely a principle of determination is here involved which is too important to deny because of a particular formulation of a doctrine. Perhaps the importance of the visible organ-forming substances has been overemphasized and these are but manifestations of a more fundamental mechanism. Yet in very many eggs the mechanism undoubtedly exists, and later researches have certainly demonstrated the essential correctness of the principle involved in the conception that the egg is not without an organization before cleavage begins which conditions the future course of its development. 372 THE DETERMINATION PROBLEM

As a result of the early work on cell lineage by Whitman, Rabl, van Beneden and others the former view that blastomeres were indifferent both in position and significance was gradually replaced by another to the effect that the cleavage pattern was in the nature of a mosaic, and that a blastomere occupied its particular place because of the material contained in it, that is because of its mosaic character. The views were supported not only by the extensive studies on cell lineage, but also by the experiments of Roux, Chabry, Conklin, Wilson, and others. These experiments are discussed later in this chapter.

The mosaic theory of development, if understood in a sense not too rigid to take account of the fundamental plasticity of living protoplasm, certainly expresses one of the underlying truths of embryology. One may, however, raise the question of the manner and the time of origin of the mosaic work and also whether it is so fixed that it is not subject to the regulatory processes of which the organism is capable.

The question of regulation brings up yet another theory of historical significance which We owe to Driesch. His terms prospective significance, prospective potency, his equipotential system, are all well known to those who are familiar with the history of the determination problem. By prospective significance of a cell he meant the actual destiny in the developmental process. By prospective potency he meant the possible fate which the cell might attain. Prospective potency includes the sum of the different developmental possibilities. As the result of numerous experiments, Driesch reached the conclusion which he thought of general application, that the egg is essentially an equipotential system, for the cells of a blastula of a sea-urchin, for example, are of uniform material and might be interchanged like balls in a pile without affecting the result in the development of the embryo; in other words that the fate, the prospective significance, of a particular blastomere is a “function of its position.” Driesch’s theory was in opposition to the mosaic theory of development according to which the fate of the blastomeres did not depend upon their position alone but upon their organization. It was based upon the conception current at the time when work on cell lineage first rose to an important place. Pfliiger had held that the egg and its blastomeres were homogeneous throughout and that cleavage simply multiplied the units out of which diiferentiations were later to arise. This view was also favored by Oscar Hertwig. Driesch, who although chiefly interested in philosophy, set himself to carry out experiments in development upon which certain philosophical decisions might be based, made this conception the starting point of his theory of development.

Some experimental evidence is at hand upon which to a certain MORPHOLOGICAL EVIDENCE 373

extent the theory of Driesch may be based, while there is also evidence upon which the mosaic conception may rest. On the one hand certain eggs possess a high degree of regulatory capacity and are hence indeterminative in their cleavage. On the other hand certain other eggs show the marks of differentiation appearing at a very early stage in the

development of the egg; these eggs possess but slight regulatory ability and are hence determinative in character. Between the mosaic type

and the regulatory type no sharp boundary line can be drawn. Rather there is a gradation from one extreme of the series to the other. If we sum up the results of these researches we may arrange the animal series from the regulatory to the mosaic forms in about this order: amphioxus, teleosts, mammals, nemerteans, urodeles, anura, ctenophores, annelids, molluscs, arthropods, nematodes, and ascidians. But it is to be noted that purely regulatory and purely mosaic types do not occur. In this we are merely emphasizing what has been said before, that the time at which differentiation appears is the real criterion upon which we may recognize the differences between determinative and indeterminative types of eggs. In other words if differentiation sets in early the regulatory capacity of the egg is correspondingly reduced. Conversely, eggs with high regulatory capacity show little evidences of early differentiation.

C. EVIDENCE BEARING ON DETERMINATION

The present status of opinion in regard to the determination problem has been reached as the result of two different lines of evidence. One of these is the morphological study of normal embryological stages. The other line of evidence embodies the results of many experiments which

have sought an insight into the fundamental nature of developmental problems.

1. Morphological Evidence

Of the morphological evidencesthat some type of determination exists to some extent in all eggs reference should perhaps be made first to the contributions of genetics. The breeding experiments of this century have very clearly shown the existence of hereditary units within the germ cells. Although we do not by any means identify these units with visible structures in the egg cell nor do we know how they express their influence upon the developing organism, yet any theory of particulate inheritance (that is inheritance based upon the presence of organized particles within the germ cell) is, in so far as it is justified, evidence in favor of a morphological basis of determinate development. This evidence bears out the conclusion stated above that, although 374 THE DETERMINATION PROBLEM

the regulatory processes may dominate in certain types of eggs, there are none which are purely regulatory.

The first signs of differentiation in the developing egg have to do with the general features of polarity, symmetry, and pattern of the egg. From these’ general features more detailed diiferentiations with respect to polarity, symmetry, location, and pattern of the constituent cells and parts of the embryo arise and later the differentiations of tissues and the beginning of organs. Development is thus progressive, and differentiation, at first relating only to the most general features, gradually passes to specialized details. Since polarity, symmetry, and cleavage pattern are general in character their importance is sometimes overlocked by the student, yet very little thought is required to convince one that they are really basic. Our morphological knowledge with regard to the development of polarity and symmetry has been discussed in the chapters which have to do with the different types of cleavages and reference should be made to those chapters. These observations have been checked by many experiments of the greatest importance and it will perhaps suflice to refer to these experiments under the second line of evidence as listed here.

Morphological studies of the cleavage pattern, as has already been indicated, became the foundation for the mosaic theory of development in the studies at the hands of the students of cell lineage. It early became clear that certain types of cleavage were predominately determinative. Some eggs with bilateral cleavage, certain types of eggs with superficial cleavage, those with spiral cleavage, with disymmetrical, and even an occasional form having radial cleavage, such as Strongylocentrotus, were shown at that time to be determinative in their development. Most forms with radial cleavage, some with bilateral, and even occasional examples of eggs with spiral cleavage show the regulatory type, however. For a discussion of these the student is referred to the chapters on cleavage types.

2. Experimental Evidence

The second line of evidence which bears upon the problem of determination is perhaps no more important than the morphological evidence but is certainly not less so and is much more recent. It is obtained from those special experiments* which have to do with this problem.

‘ As pointed out in the chapter which deals with history of embryology, the present is the period of acperimental embryology. Much of morphological character remains to be learned, but nevertheless the dominant note in embryological research of the present day is the experimental one. It is now sought to discover those underlying principles which account for the form changes by which the structure of the organism is produced rather than simply to give a descriptive account of those characters. EXPERIMENTAL EVIDENCE 375

The first series of experiments of significance for study of determination is concerned with the localization of the median plama. Here as with the other experiments to be summarized the student is referred to Morgan’s critical discussion as well as to the original papers which describe them. It would be impossible in a work of this size to give even a brief account of the experiments themselves. Reference can only be made to those which seem most significant.

The matter of the localization of the median plane is of importance because it indicates the early determination of symmetry and polarity on the part of the egg. According to some the polarity of the egg may be traced back even into an ovarian condition, and some believe that the type of symmetry is determined in the egg cytoplasm before fertilization. Although the evidence of these relationships seems good in some cases it has not been made clear how much the appearance of symmetry_in these early stages is related to the cleavage plane or the symmetry of the embryo. No mechanism for bringing this about has

Experimental embryology has seemed to cover a wide range of topics and the results of the investigations in this field are scattered throughout many journals. There have been but few attempts to bring this material into the scope of a single volume. The first section of Korschelt and Heider’s “Lehrbuch der vergleichenden Entwicklungsgeschichte der wirbcllosen Thiere” deals with the experimental results obtained up to that time (1902). In 1909 Jenkinson published his book entitled “Experimental Embryology” and summarized much of the work available at that time. The contribution to experimental embryology most important now, however, is the volume by Morgan entitled “Experimental Embryology” and published in 1927. Morgan has promised a further volume which will deal with such topics as growth, reflex reactions, tropisms of the larvae, the influence of environment upon the embryos, the source of energy of development, etc. It is to be hoped that this volume will appear at no distant date.

In spite of the diversity of topics which have engaged the attention of experimental embryologists it is possible to classify them into a few divisions. (1) The early students of this field were specially interested in the discovery of the effect that external factors have upon the developing embryo. Among these factors are gravitation, mechanical agitation, electricity, light, heat, atmospheric pressure, osmotic pressure, and the chemical composition of. the medium in which development takes place. (2) In addition to this the growth problem, the problems centering around fertilization, have occupied much attention during the present century. Something of the nature of these problems is suggested in the section of this book which has to do with artificial parthenogenesis, although this is but one of the many topics that have been studied. (3) finally a great deal of attention has been given by experimental embryologists to those problems which are concerned with the forrna— tive changes of the egg. These factors are internal in their nature and seem to take the experimenter further in his attempts at the analysis of the innate, vital character of the organism than do the other problems enumerated. The experiments which bear on the problem of determination come under this head. Those experiments which have sought light on this problem fall chiefly within a few animal groups, namely: the ctenophores, some hydromedusae, the nematodes, annelids and molluscs, echinodcrms, ascidians. It may be remarked that not only are these the forms which seem to have yielded results most capable of analysis but they are also the forms upon which it is easiest to experiment. 376 THE DETERMINATION PROBLEM

been demonstrated. In many cases where a relationship between the first cleavage plane and the planes of symmetry of the later embryo have been made out it is thought that the point of entrance of the spermatozoon and the path traveled by the sperm pronucleus in approaching the egg pronucleus determine the position of the plane. Suggestions have also been made that the pressure of the enveloping membrane of the egg or pressure from the oviduct or other external factors may be related to the position of the plane. Certainly the causal character of such factors has not been demonstrated. It may be said, however, that so long as the possibility remains of a relationship for example at the point of entrance of the spermatozoon it is necessary to assume that the symmetry of the embryo goes back to a predetermined symmetry of the egg.

The question of a relationship of the median plane and the first cleavage plane arises in the history of embryology in relation to the frog egg. As long ago as 1851 Newport had reported that the two coincide, and from that time the question has been often discussed. In the frog egg at the time of cleavage there is already present a bilaterality as shown by the presence of the gray crescent. The real question involved in determining the symmetry relationships is: what in the uncleaved egg causes the material to take the position of the gray crescent, and why does the first cleavage plane cut through the middle of it? Experiments have shown that its position is determined after fertilization and that any meridian of the unfertilized egg may become the median plane. After fertilization the meridians are not equivalent in this respect, however, for usually the crescent forms opposite the point of entrance of the sperm. A long series of experiments can be cited to show that in nearly two-thirds of the cases the first cleavage plane does coincide with the middle of the gray crescent. It is clear, however, that if 30 per cent or more of the eggs fail to show this relationship the mechanism of determination is by no means a fixed one. In other amphibia the experiments of Jordan indicate that the first cleavage plane is at right angles to the axis of the egg of Diemyctylus, and Spemann has found for Triton that the second cleavage plane coincides with the median plane of the embryo. Of course it is immaterial from the standpoint of determination as to whether it is the first or second plane which shows the relationship. In the teleost fishes according to the work of Morgan and of Clapp no definite relation between the second plane and the median plane of the body can be made out. For the sea-urchin a considerable amount of evidence is available. In Toxopneustes, Wilson and Mathews related the appearance of the first cleavage plane to the entrance of the spermatozoon, and Boveri held that it coincided with the median plane of the EXPERIMENTAL EVIDENCE 377

embryo. In the eggs of Echinus, Driesch found the second plane to correspond to the median plane and Runnstrém assumed that the first cleavage coincides with the median plane. Recently Von Ubisch has performed a very ingenious experiment by which certain portions of the egg were stained with intra vitam stains; in three species of seaurchins he obtained results which are in agreement. He was able to demonstrate no fixed relation between the planes of symmetry and planes of cleavage, but found some evidence that the first cleavage plane more nearly coincides with the median plane. On the whole for the sea-urchins perhaps this is the most acceptable conclusion. In his study of ascidian embryology Conklin determined that the first cleavage plane is the median plane of the embryo, and that in Cynthia its position is indicated before the pronuclei meet. In the nematodes, according to Boveri’s work on Ascaris, the third cleavage plane of the dorsal cells is the median plane of the embryo. In Nereis, Just has found that the second cleavage plane usually corresponds to the median plane of the embryo but the first cleavage plane is determined by the position of the entering spermatozoon. Insect eggs are distinctly bilateral so far as their orientation is concerned, the position of the egg as laid corresponding to the symmetry of the mother’s body and definitely indicating the position of the embryo in the egg. For the chick and pigeon Bartelmez has held that bilaterality is present even in the small ovarian eggs.

A second series of experiments related to the problem of determination has sought to discover to what extent localization of germinal areas is present before cleavage, using as a method the development of egg fragments. Eggs of Cerebratulus were out along definite planes by Wilson, Zeleny, and Yatsu. Cuts were made both before and after fertilization and development followed without difficulty. The cleavage of the fragments in general corresponded to the type of cleavage of the whole egg but upon a much reduced scale. It was not found to make a difference from which part of the egg the fragment was taken. The experiments show that either factors which determined cleavage were not yet definitely localized at the time of maturation or the egg is capable of very extensive regulation. According to Yatsu if the operation occurred between the formation of the first and second polar bodies the cleavage was entirely regular but if it was after the second division irregularities developed in some of the cases. Wilson experimented with fragments of the egg of Dentalium. Here the presence of the yolk lobe complicates the result. If the yolk lobe is entirely present and only the apical portion of the egg cut off, the resulting larva is nearly normal; likewise an entirely symmetrical division of the yolk lobe seems not seriously to interfere with the course of development. If the cuts are made in any other manner, however, irregularities appear. The ctenophore Beroé when out into fragments in the unsegmented condition produces in some cases partial embryos and in others whole ones depending on whether the cut is symmetrical or oblique. Driesch, Morgan, Yatsu, and fischel have experimented with this form. With sea-urchin eggs conflicting results have been obtained from experiments with the development of fragments. Taylor and Tennant using accurate methods of cutting with a micro-dissecting machine obtained small pluteae from the developing fragments which were like the normal ones. But Harnley obtained evidence that the materials of the egg were qualitatively different. finally experiments on the development of parts of Triton eggs have been performed by Spemann and Baltzer. Non-nucleated fragments, obtained by separating the egg by means of a hair tied around it, developed but rarely and the nucleated fragments produced dwarf larvae which did not live long enough to undergo metamorphosis although normal in most particulars.

An interesting result of these experiments is the conclusion that the entrance of the sperm into the egg is not of itself sufficient to start development, for, if an egg is cut just after the penetration of the sperm so that the female pronucleus is in one half and the male pronucleus in the other, it is the half containing the sperm pronucleus that develops but not the other. Something else besides the mere initiation of division is accomplished by the entrance of the spermatozoon. The cleavage of egg fragments demonstrates that for many types of eggs the pattern develops along with the mitotic figure.

Another series of experiments has sought to discover to what extent it is possible for a whole embryo to develop from an isolated blastomere. In some few eggs it is possible to cut the blastomeres apart at the time when they are most widely separated from each other as the first cleavage is closing. In others it has been found possible to separate them by shaking them in a tube of water, or by squirting them from a pipette. Again eggs of echinoderms permit the easy separation of the blastomeres if they are kept in calcium-free sea water while the first cleavage is taking place. Isolation experiments have been performed on sea-urchins, the hydroid Clytia flavidula, Cerebratulus, amphioxus, teleost fishes, Triton, and the frog, and in all these cases whole embryos were obtained from the isolated blastomere. On the other hand blastomeres of ctenophores, molluscs, and ascidians when isolated give rise to half embryos only. It would seem that this emphasizes again the distinction between determinative and regulatory eggs.

The question arises as to whether the development of the isolated blastomere is strictly comparable with the results that would be obtained if the material of the missing portion of the egg were present. Experiments have been performed in which the single blastomere has been allowed to develop in contact with the material of its sister cell, which owing to various kinds of injury was prevented from any active participation in the normal result. Experimenting upon the frog, Roux injured one blastomere with a hot needle but did not kill it. He thought that the subsequent development of the uninjured blastomere in contact with the inactive one was in the nature of regulation and that gradually the missing portion of the embryo was restored. This conclusion seems doubtful as there is now good evidence that the resulting embryo is more nearly one-half than a whole. Later studies have shown that, in experiments of this kind, if the plane of the first cleavage goes through the middle of the gray crescent a half embryo results. If the plane is parallel to the gray crescent and the injured blastomere is the one which contains it, nothing recognizable is produced from the opposite one. But if the opposite blastomere is injured the one containing the gray crescent will produce the anterior end of the embryo. Even if the cleavage plane is at other angles the blastomere containing most of the gray crescent will produce an anterior end.

McClendon working on the tree frog, Chorophylus (Pseudacris), sucked out the injured blastomere to determine whether its presence has any effect. In his experiments the remaining blastomere produced a normal whole embryo of one—half size. Injuries to one blastomere of the egg of Ascaris led to the conclusion in the experiments of Stevens, of Boveri, and of Schleip that each individual cell contains the factors which are responsible for its own development; in short, that there is little self-regulation on the part of the blastomeres. The egg of Cyclops after the injury to one blastomere has been studied by Fuchs and by Miss Jacobs. The evidence goes to show that for this egg the contact of the injured blastomere with the uninjured one does not materially affect the result of the development of the latter. This results in a partial embryo. In this same connection the experiments of a number of investigators, of whom Hegner and Reith are typical, on the effect of injury to the eggs of insects should be mentioned. Insect eggs apparently may be among the most determinative with which we have to deal, for these experiments indicate that different cytoplasmic areas on the surface of the egg are fixed in their prospective significance even before the cleaving nuclei with their surrounding cytoplasmic islands migrate to the surface and they have no great powers of readjustment. There is indeed a considerable degree of independence in the development of the respective parts. If the injury is not too severe to a certain localized portion of the egg the remainder will go ahead and develop without much dependence upon the injured portion, but will produce only that part of the embryo which was to be expected. Reith found that, if the posterior end of the egg of the house fly is injured, the parts normally resulting from the anterior end will develop. If the anterior end is the location of the injury, a larva without a head end develops, and in some cases injury to the middle portion of the egg was not so severe but that both anterior and posterior organs developed.

Hegner’s experiments on the eggs of the chrysomelid beetles Calligrapha multipunctata and Leptinotarsa decemlineata are Well known. Here the posterior end of the egg is the location of the pole plasm which Hegner found to pass into the germ cells in development. By injuring this region with a hot needle he was able to secure embryos without germ cells. Evidently the cytoplasmic regions in the eggs of these insects are very early set aside to produce definite parts of the embryo. Yet it must not be inferred that the regions in question retain no powers of adjustment. As cleavage progresses the possibilities of readjustment are lessened but are not entirely lacking.

A very important series of experiments carried out in the laboratory of Spemann on the embryos of Triton have shown that the future of certain ectodermal areas in the gastrula are much modified by their position. The method by which these experiments were carried out was that of transplanting a portion of the ectoderm by means of a micro-pipette from one portion of the body to another and studying its relationship to the development of the neural plate. From this study it appeared that the presence of the endomesoderm beneath the surface of the ectoderm is immediately necessary for the neural plate to be formed. Spemann and his collaborators have shown that the prospective ectoderm is capable of producing entirely different organs upon transplantation. For instance ectoderm from the top of the young blastula or gastrula implanted on the lip of the blastopore and carried to the interior may become notochord, mesoblastic somites, pronephros, or perhaps other organs. Ectoderm taken from a slightly later stage, that is, after the closure of the blastopore, if carried into the mesoderm becomes mesodermal somites; if carried into the endoderm goes to the formation of the archenteron. It should be pointed out that particular organs which differentiate from given substances may through the process of rearrangement be induced in an entirely different direction from that which their normal determination indicated. Throughout the animal kingdom there are many cases which serve to emphasize the difference between the determination of an organ and the actual differentiation which sometimes results from the modification of normal processes. What is shown by such cases, however, is not that the unusual condition is one of indetermination but that the organism has responded to modi— fying factors to produce a result different from that which would have occurred had not the unusual factors been present. Determination of an embryo does not always correspond to its differentiation.

A problem which has given rise to a great deal of experimentation in relation to the question of determination is the influence of pressure upon cleaving eggs. The work began with the experiments of Pflfiger in 1884 in compressing the eggs of frogs between two glass plates. The direction of the first three cleavage planes was found to be at right angles to the plane of compression. When the compression was released, normal embryos developed. Similar experiments have been performed in numerous other eggs with the result that normal development is found to follow the release of pressure on eggs of hydroids, sea-urchins, frogs, and Cerebralulus. In eggs of N ereis, Ciona, and molluscs, abnormal development follows compression. In the first group of eggs it is to be noted that differentiation does not begin until there is relatively a large number of cells present. In the second group signs of differentiation are to be noted very early. This series of experiments was used by 0. Hertwig and by Driesch in support of their opposition to the mosaic theory of development. Perhaps the experiments are less crucial than was formerly believed and merely emphasize the distinction previously insisted upon that there are two types of eggs with respect to their capacities for regulation as distinguished from determination.

finally the literature of experimental embryology contains many records of attempts to bring about the redistribution by centrifuging of egg substances, particularly those which are visibly different. These materials as well as the formed materials of the egg yolk, pigment, fat, and other inclusions are often of different specific gravity and hence respond to centrifuging by redistributing themselves in different zones or strata. This transfer of materials throughout the egg takes place as a rule without injury to its living substance. The use of the centrifuge in experimental embryology began with the work of Lyon in 1906, although it had been utilized for the study of the constitution of the cytoplasm by Gurwitsch in 1904. Lyon’s paper is a classical one in embryology both because of the introduction of a new method and because of his discovery that the redistribution of the egg constituents does not afiect the development of the eggs from the standpoint of determination. Many other investigators have used this method for the study of eggs through a wide range of animal forms. The results of the experiments with the centrifuge have rather uniformly indicated that the visibly stratified substances in the egg are not determinative in the sense that they are organ forming. It would seem that the method, though productive of results which have great value from other standpoints, has failed to give critical evidence as to the determinative character of development in the eggs to which it has been applied.

As a conclusion to this long catalogue of evidences, the position earlier talfen in this chapter may be reiterated. The animal series can be arranged in such a manner as to show a transition from eggs which are highly regulatory to those that are highly determinative in character. N 0 eggs are known which are purely regulatory or purely determinative, but at one end of the series differentiation sets in very late and the regulatory capacity is high. At the other end the marks of difierentiation begin even before cleavage and the powers of regulation are correspondingly lessened.

Chapter IX Ecological Control Of Invertebrate Larval Types

Throughout the animal kingdom many diverse environmental relations surround the eggs and young so that many responses might be catalogued. Environmental influences find expression in the places where the eggs are laid, in the amount and kind of food provided for the young during the early development, in the different devices for their protection, in specialized modifications for accomplishing locomotion and other vital activities, and in many cases in the production of forms of larvae ‘which are totally different from the adult organisms. These special types of larvae present a great range of variations from the modes of direct development with which the student of the embryology of vertebrates is familiar. These larval types involve a metamorphosis to the adult form which may be more or less complete and which in such cases brings the growing organism into an environment quite unlike that of its earlier development; metamorphosis is necessary in those forms in which the food and habits of the adult are unsuitable for developing young. If we assume the sea to have been the original home of most primitive stocks, an assumption which is more or less common and seems in line with the fact that very many types of animals develop in a moist atmosphere or liquid medium, we should expect the majority of unusual larvae to be found in the species living in salt water. The transition from salt water to fresh or to the terrestrial forms of life has presented many difficulties to the developing young and as a result those animals which live in fresh water or are terrestrial are more often characterized by direct development than are the salt-water forms. It may be regarded as an axiom of development that sessile adults have active young. This activity has two obvious results; first, the species secures its dispersal through the migration of the young, and econd, it extends the range. The migration is commonly passive but over a period of time serves to extend the range of the species as successfully as if the adults themselves were able to move. To insure dispersal, vast numbers of gametes must be produced, for the chance method of fertilization that usually accompanies such cases results in a failure of very many eggs and sperm to become further activated, and of those eggs which are fertilized only a few can come to maturity. The larval mortality is very high indeed, for these young are the food of many species as well as the victims of physical forces and of their own inability to find continuously satisfactory conditions for growth. The vast number of eggs produced may be realized by reference to the turbot which produces 9,000,000 in a single season, the cod with 5,000,000, and to the flounder with 1,000,000. In higher animals the reproductive energy is conserved by various means for caring for the young during early life, but in marine forms this is usually not the case. The second consequence of larval activity is to bring the growing forms into an entirely difierent range. This is illustrated in the case of the lobster, which is a bottom feeder, lurking in the crevices between stones and elsewhere to capture whatever prey may come within its reach, or feeding upon such dead forms as may be found nearby. The larvae, up to the time of the fourth molt, however, swim at the surface of the sea water. In addition to this, they are positively phototropic to light of the intensity of ordinary daylight, but the adults are negative. Thus the pelagic larvae are brought into a range of environment where their food, which consists of plankton, especially copepods, is abundant. Special structural modifications adapt these larvae to their surface pelagic life. They are the better enabled to swim on the surface of the water because of the possession of exopods, the outer branches of the walking legs characteristic of lower crustaceans but not present in the adults of the higher forms. These are retained to the fourth molt, when they are cast off, being no longer useful; the larva then goes to the bottom. Herrick makes note of several changes in structure and instincts which take place at the beginning of the fourth stage, which marks the most surprising leap in the whole history of development. Among these are the following: the primitive swimming branches of the thoracic appendages are lost; the cuticle becomes shell— like, containing more lime; the pigments are denser, the colors brilliant, and the color pattern variable; otocysts are present and orientation is perfect; rotation of the great forceps is complete; the animal, during at least a part of this stage, moves toward the light and swims steadily at the surface with the great claws directed forward and held close together; the preying instinct is more marked; the fighting instinct, the instinct of fear, “feigning,” and hiding are all developed by the close of the fourth stage or in the fifth, when the animal goes to the bottom to stay. In many other animals similar gain is accomplished by a specialized form of larval structure. The larva finds itself adapted to-securing food which is suitable for it, and structures which have only temporary employment are present and useful. It follows, however, that the gain to the larva would become a loss if the subsequent stages in the life cycle were not radically changed in such a way as to enable the organism to undertake a new mode of life. In other words, metamorphosis in such cases is a necessity for bringing about those adaptations which fit the organism to live in its permanent environment and

there to undergo those further changes which look toward the production of new germ cells.

It is not usual to find metamorphosis in animals which live in fresh water, although there are some outstanding cases in which larvae totally unlike the adult are developed in species inhabiting fresh water. It may, however, be taken as a general rule that development in freshwater forms with the exception of certain few highly specialized cases is direct. Fresh water presents a great lack of constancy in living conditions as compared with those of the sea. With but few exceptions the bodies of fresh water lack sufficient depth and area to maintain even a low degree of constancy. Fresh water becomes heated much more easily than the sea, it freezes comparatively quickly, its streams are subjected to periods of flood during which the water runs rapidly and scours out the beds or overflows, and when it recedes leaves innumerable forms of life stranded to perish. Every stream has narrows and depths where it runs rapidly and flats where its current is slow, an environment in which the fragile larvae would scarcely be able to maintain themselves, and there are but few animals which go through an independent larval history in fresh water. The lack of suitability of fresh water to larval development without doubt explains why so many groups of marine animals have not been able to gain a foothold in fresh water. A very few sponges, almost no coelenterates, occur in this environment, and many groups of marine fish and ascidians, cephalopods, king crabs, and some of the worm groups have not a single representative in fresh water. Animals that cannot produce eggs which develop into young like the adult are prevented from gaining ‘a. foothold by such a changeable medium as fresh water. In addition to the difficulties of fresh-water life already noted, a zone of brackish water between the fresh water and the sea is itself an almost impassable barrier to the entrance of marine forms.

Terrestrial life presents to developing larvae even more dangers than fresh water. To this situation is due the fact that in land forms development is either direct and fairly simple or else specialized with complicated devices for caring for the young during their immature period. The latter condition is illustrated by many forms of insects in which metamorphosis is complete, often involving very complicated life histories. Some of the disadvantageous features of the terrestrial 386 ECOLOGICAL CONTROL OF INVERTEBRATE LARVAL TYPES

environment are the following: temperature undergoes a wide range of variation, often passing quickly between the extremes. This condition is quite unknown in the ocean and is much less in fresh water. The very changeableness as well as the extremes of heat and cold are hardships for the developing larvae. The weight of the body is no longer buoyed up by water and must be supported at every moment. This involves consumption of much energy as compared with the almost passive drifting of larvae in aquatic habitats, and renders movement much more difficult and restricted. Food no longer streams by as in the contrasting case, but must be actively sought, is often very much more restricted as to kind, and presents an increased toughness since in the composition of land forms the percentage of water is much less, and the food is necessarily more difficult to find. The glare of daylight and the consequent difficulty of avoiding enemies puts the terrestrial larvae to a disadvantage which forms living in the subdued shadows of the water do not share. finally the rapid evaporation to which terrestrial forms are subjected requires special devices for protecting the soft exterior of the organism. A heavy shell, mucous glands, and other similar devices show what a serious drain upon the organism requirements of this kind cause.

These considerations account in a large measure for the lack of uniformity in the occurrence of larval types throughout the different groups of the animal kingdom. The particular forms of larvae within a group are of adaptive rather than taxonomic significance.

The general subject of the care of young animals was briefly but instructively discussed by Gamble in “The Animal World” (Holt and Company). Some of the ideas mentioned on the three preceding pages are suggested by his discussion. Gamble sums up the responses of animals to different environmental conditions in which the young must be produced as follows:* “We find isolated examples of the retention of the young by diminution in the size of the family. Speaking generally, marine animals pour their eggs broadcast, and leave their minute larvae to complete their metamorphosis unaided and unsheltered, but in every group there are malthusian species which not only restrict their families in number, but enclose them by protective envelopes. The varied experience of larval life in this curtailed and direct development supplants metamorphosis. The eggs become larger and the young stronger at birth.

“In fresh water the limitation and protection of the family is more generally the rule. Insects and Amphibia are the only large classes

‘ Quoted by permission of Henry Holt & Co.

which go through their larval life in fresh water. But the protection

is usually of the simplest kind and is confined to the earlier stages of development.

“On land the insects form the only class in which the majority still pursues a free larval history. Others develop directly, either growing up into full stature without guidance or protection, or carried and fed by their parents for some time both before and after hatching.”

Cite this page: Hill, M.A. (2026, September 8) Embryology Book - Outline of Comparative Embryology. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Outline_of_Comparative_Embryology

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