Book - Vertebrate Embryology (1949) 1

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McEwen RS. Vertebrate Embryology. (1949) IBH Publishing Co., New Delhi. 1949 McEwen: 1 Germ Cells and Amphioxus | 2 Frog | 3 Teleosts and Gymnophiona | 4 Chick | 5 Mammal | 1949 Vertebrate Embryology

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Part I The Germ Cells and Early Development of Amphioxus

Introduction

IT has long been an axiom with biologists that all organisms consist either of single cells or of cell aggregations. often with the addition of various cellular products. It is also well known that even in the case of multicellular animals or plants, each individual starts from a single cell. This cell may be one that has recently fused with another in the process called fertilization, or it may develop without such fusion by a process called parthenogenesis. The latter is a natural prmi-mlure in some instances and may be artificially induced in othgfi,


With the foregoing facts in mind it may then be stated that the development of any multicellular animal or plant involves three fundamental processes which go on more or less coincidentally. These processes are: The increase in cell numbers b cell r d division (usually mi


totic‘ ; the i erentiation of the cells and sometimes their products ‘;

e arran ement o t ese ce 5 an tissues to constitute parts and organs. It is therefore the study 0 t ese processes which com» prises embryology. Stated thus baldly and reduced, so to speak, to its lowest terms, the subject may appear rather d _v and prosaic. Such, however, is furthest from the truth for anyone with any real interest in living things, and in the problems of existence in general. F or there is no more astounding and fascinating drama which one may view than to watch the development of certain eggs. This is particularly true of relatively srnall transparent ova which it is literally possible to see through in the living state, such as those of many of the Invertebrates, like Sea Urchins or Molluscs, and even some Vertebrates, like many Fish. Here one may observe under the microscope the active division of the cells and their gradual differentiation and rearrangement. Thus in certain rapidly developing forms, there may be seen in a.few hours the transformation of an apparently structureless blob of jelly into a clearly recognizable and relatively complicated organism. Careful and accurate descriptions of these and many other cases more dilficult to observe have been recorded for a long time, and this constitutes descriptive embryology. It was inevitable, however, that after observing this veritably magical performance man should begin to inquire how it was done, and this inquiry has led to the growing and very active field of experimental embryology. Hence at first by relatively crude acts of interference with normal development, and later by more cleverly planned procedures it was and is being sought to analyze the fundamental processes involved. As in the analysis of all life phenomena the goal has constantly been to reduce them to physico-chemical terms; and though this end is by no means attained, workers everywhere are constantly pressing toward it. Hence, though the primary aim of this book is to present a description of normal embryological phenomena, opportunity will frequently be taken to indicate how experiment has helped to throw light on many of the basic mechanisms concerned.

.lLhaLb;.en_§t§t§é-£!29!..,:1ev91Qprssnt starts from 3_9§l1.an.<lt_11at cells c_o_nstitute the ‘units or building ':b_locks:of living structures ar_e mafie. Vileiimighlfthereforelspend some time in a discussion of cell structure and physiology. For the purposes of this book, however, it is assumed that the student is already familiar with this subject, and with the phenomenon of normal cell division or mitosis. We shall therefore omit further reference to this matter. It does, however, seem desirable to make some comment as to the origin and history of the germ cells. Let us then begin with this topic.

The Gonads and the Germ Cells

The germ cells or- gametes are certain cells in both cytoplasm

and in fileusgare speciéiliied:iv5_r:thelpill-pose‘ of reproduction. They are

thus distinguished ££oz'fi"'Is6d§% or somatic cells which are specialized for other functions in the life of the organism. Before considering the detailed development of the germ cells it will first be necessary to give a

brief history and description of the organs in which they are finally located.

THE GONADS T_l_1_e__germ cells of__tl,_1eMadult occur in _orga<ns”_lcnqw_n_has_ _g9riq§s, the

_tisIn most true Vertebrates these are paired structures, same pair are aanmycai the §9m¢_..§2x- In their earliest condition both ovarieswanidiitesties ap;’$飣aiiikej as a pair of ridges (the genital ridges) consisting largely of thickened coelomic epithelium (the germinal epithelium). Beneath this epithelium thereoccur a mass of loose mesodermal cells known as mesenchyme.


Presently these cells give rise to real connective tissue which soon increases and constitutes the supporting element of the organ, termed the medallary tissue or stroma. Each genital ridge lies along the back on either side of the dorsal mesentery of the gut between it and the embryonic excretory organ. Within the germinal epithelium there presently appear


Fig. 1. Cross section of the ovary of a fledgling of Numenius arcurzrus 3-4 days old. From Lillie after Hoilmarm. The region of the germinal epithelium is toward the bottom of the figure. f. Follicle. o. A very young ovum around which the epithelial cells have formed a definite follicle. str. Stroma. pr.o. Primitive ovum within a portion of the germinal epithelium.

certain cells which are often distinguishable from their fellows by their larger size and also by their relatively larger nuclei. These are the primitive or primordial germ cells in which sex diiie1'entiation, at least as regards the cytoplasm, is not yet apparent. The origin-and later development of these cells will he discussed after completing our descrip tion of the gonads. ‘ The Ovary. --lt'l_l:~lt§_£§§§_M9f‘ltl'i§_pY§.}f}:, as the germinal epithelium graduzilly _ing_r_§eses in thickness it is in someliiristances dividedlby the 4 ' iooigeurdus cords. In any orinests of the epithelial


fllgecorriew scetteretl about


event, during cells, each con aining a priini

it e ourse of growth


thr u‘gh_gvutm_t_he connective tissue. Each germ cell then proceeds to de

as the epithelial/celllslwhicli‘surround‘ it,’ known asfitsd 1), servhelto convey’ it iiiru‘i£n‘Erit.t ' ' l H K


Fig. 2.—-Cross section through the periphery of the testis of a just hatched Chick. From Lillie (Development of the Chick). After Semon. The sexual cords have acquired a lumen, and the walls of the canals thus formed are lined within by the spermatogonia. Next to the latter come a layer of supporting or Sertoli cells, and outside of these a thin layer of connective tissue, the theca (not labeled). The remaining connective tissue (stroma) lying between the sexual cords (now seminiferous tubules) connects at the periphery of the testis with the special layer of connective tissue (albuginea) which covers the entire organ beneath the thin outermost layer of coelomic epithelium.

Alb. Albuginea. c.T. Connective tissue of the stroma, or septulae testis. Ep. Remains of the germinal epithelium now forming the outermost or serous covering of the testis. l. Lumen of the sexual cords. pr.0. Spermatogonia. s.C. Sexual cord, lined by supporting cells and spermatogonia.

The Testis. — __ ithin the_young gonad which is to become a testis there develqp. .th£9P..s.h9yLth§ ,s,tr.€>iIrié; ‘sfiands o£..fiésfié it. this . case té??7§3'ls§aey.zl eerie Th9y.sh._..thsi: 9_1ti.si.nin s°rr!9..i.n§t§p9es is 'd0’l“l1"btfl1l’


ih5Y..%ERaF9.¥!lX. %‘.5§¢= Tik.f9.F1}E=.v .9Y}s¢r.9t1.,s .9,91.1<1..s, trorx1.th¢_.g9rmi:1a1,ePi thelium._Whatever their iorigin,Ahowever;theyapresently become filled

cords then with the germ cells whi§ltL§eern,to migratejnt __t 6 I INTRODUCTION

become tubular, and the tubes are lined by the germ cellsteither arranged in layers oreinclosed in cysts (some Amphibia). Certain of the cells constituting the walls of the cysts, or tubes, as the case may be, are homologous in function to the follicle cells of the ovary, i.e., they bring: nutriment to the growing germ cells. These nourishing cells in this case are often termed supporting or Sertoli cells. Externally each tube is covered with a thin layer of connective tissue termed the theca, and the whole is known as ‘a seminiferous tubule ig. 2).

more detailed’ description of the development and structure of a typical vertebrate ovary and testis will be found in our treatment of this subject in connection with the Chick. Likewise short discussions of these organs are included in the accounts of the other animals to he studied. With this as an introduction the student is now prepared for a description of the history of the actual germ cells.


The Origin of the Primordial Germ Cells

There have been two theories regarding the origin of the germ cells. lt was originally believed that they arose through the modification of certain cells of the germinal epithelium. In the earlier part of the century. however, it was discovered that in some animals, at least, the primordial cells were not first seen in the germinal epithelium at all, but were discernible as far away as various parts of the gut wall. From thence they were seen by some to migrate to the gonad through the mesentery of the gut. as in the Turtle and Gar-pike (B. Allen, ’O6, Fig. 3), or to be moved thither by shifting of parts due to growth as in the Amphibia (Humphrey, ’25l . or to be carried by the blood stream as in the Chick (Goldsmith, ’28) .

While evidence for this sort of thing has continued to accumulate. other observers have questioned the ultimate fate of these migrating cells. In many cases it is claimed that such cells are not the ones which form the actual or definitive germ cells. lt is asserted on the contrary that the so-called primordial cells degenerate, and that the definitive germ cells arise later by the transformation of indifferent epithelial cells as was originally supposed. This is said to be the case for the Rat by Hargitt (’25, ’30) , for the Cat by Sneider (’40) , for the Opossum by Everett ("42), for the Guinea Pig by Boolchout (’45), and in various other cases. Also in some instances, there are opposing views concerning the same animal as in the case of the Cat in which Kingsbury (’38) claims, contrary to_ Sneider, that all definitive germ cells come from the primordial ones.


t appears too that the situation may vary in different animals since Everett (’43) thinks that in the Mouse, contrary to his View regarding the Opossum, the primordial cells furnish all the definitive germ cells. Thus it is evident that this question is still an open one, and hence subject to continued research. The reason for reference to it here is that much of this interest in the origin of germ cells in Vertebrates stems from certain well-known cases of apparently very early origin of these cells in some of the Invertebrates, e.g., the Coelenterates (Weismann, ’83) and Ascaris (Boveri, ’l0). These cases in turn were long used to bolster the famous Weismannian theory of the fundamental separateness of the germ plasm and somatoplasm, and also the correlated theory by that author concerning the mechanism of development. Modern genetical and experimental embryological research has pretty much outmoded Weismann’s notions as to the nature of the germ cells and the mechanism of development in their original form. The actual source of the germ cells, however, is still obviously a subject of considerable interest to biologists. Let us now turn to a consideration of the structure and development or maturation of a typical female and typical male germ cell.


Fig. 3.——-From Morgan (Heredity and Sex. Published and copyrighted by the Columbia University Press). After Allen. Origin of germ-cells in certain Vertebrates, viz., Turtle (Chrysemysl, Frog (Rana), Car-pike (Lepidosteus), and Bow-fin (Amid). The germ-cells are seen migrating from the digestive tract (endoclerm). End. Endoderm in various localities. Int. Intestine. S.C. Sex (germ) cells. S.gl. Region of the gonads.



The Ovum

The fully developed female germ cell is termed the ovum. The ova of different Vertebrates vary widely in size, in the amount and arrangement of their deutoplasm, and in their coverings. They are uniform, however, in their relatively large size and inertncss as compared with the male reproductive cell (Fig. --L). They also rosemhle both the latter and each other in one particular, i.e.. the behavior of their chromatin. This latter point involves a rather compli~ cated aspect of maturation termed meiosis which conipriscs two special cell divisions, the meiotic divisions, sometimes known simply as the maturation divisions.


Fig. 4. —GVeneralized diagram of a slightly telolecithal egg ready for fertilization. The only membrane represented here is the vitelline. :1. Animal pole. 2'2. The nucleus containing a nucleolus and a linin network along the fibers of which chromatin appears. 0. An oil vacuole. v. Vegetal pole. vt. Vitelline membrane. yg. A yolk granule.

Inasmuch as these divisions are not only complicated, but also of great significance, they will be considered later under a separate heading. The other features of maturation in an ovum and then in a spermatozoon will now be discussed.

It has already been noted that the primordial germ cells which migrate into the germinal epithelium are not readily distinguishable as to sex, at least as regards their cytoplasmic morphology. Their male or female character becomes apparent, however, as the gonad develops THE GERM CELLS 9

and they become distributed through the stroma of the ovary, or take their places in seminiferous tubules as the case may be.

In the former instance which is now under consideration the young female germ cells in and near the epithelium proceed for a time to multiply quite rapidly. They do this by means of typical mitotic divisions, and during the process are known as oiigonia. This stage of

Fig, 5.———Egg of the Teleost, Fundulus heteroclitus. From Kellicott (General Embryology l. Total view, about an hour after fertilization.

c. Chorion. d. Protoplasmic germ disc or blastodisc. 0. Oil vacuoles. p. Perivitelline space. 2;. Vitelline membrane. y. Yolk.

simple multiplication usually continues at least until the time of birth or hatching of the animal in which they are contained. According to most accounts the multiplication of cells then ceases, so that at this time the animal in question contains as many——-though only partially grown -— ova as it will ever have.

The next period is one of growth during which the cell becomes surrounded by its follicle, and is termed an oficyte.

The Nucleus. ———The nucleus during this second period enlarges greatly, and is known as the germinal vesicle. It is relatively clear, though it usually contains a line reticulum, and may possess one or more conspicuous nucleoli. The latter may he of either the plasmosome or the karyosome type or both, and their significance is not well understood. It probably varies in dilierent cases. At the end, and also sometimes at the beginning of the growth period, certain changes occur in the nucleus which are connected with meiosis. These will he described below.

The Cyzopla.sm.——Meantime the cytoplasm is increasing considerably in bulk, chieilv as a result in many cases of the accumulation of deutoplasm or yolk. This substance usually first appears in the shape of granules and droplets. Later it assumes various forms and contains a variety of chemical substances, consisting in general of proteids. nucleoalbumins, fats, carbohydrates, and certain salts. Not only does the composition of the yolk vary, but also its amount and distribution. Thus where the amount of deutoplasm is large the oocyte becomes relatively enormous as in the eggs of Birds and some Fish. In such forms the yolk comes to be situated on one side of the ovum — the vegetal pole, whereas the remaining cytoplasm containing the nucleus occupies a greater or less part of the opposite side, or animal pole. Ova of this type are said to be telolecithal, and in those instances where this arrangement is most marked the relatively yolkless cytoplasmic cap at the animal pole is called the blastodisc (Fig. 5). In other ova, such as those of the Mammal, there is relatively little yolk and this is scattered throughout the cytoplasm. An egg of this type is termed homolecithal.

The manner in which the yolk originates and grows is of some interest. The actual new material for its formation is of course supplied from without, probably throughithe medium of the follicle cells. The organization of this material into yolk, however, often seems to take place in connection with a certain body known as a yolk-nucleus-Corr» plex. The nature and even the exact origin of this body is rather uncertain, and indeed seems to vary iri different cases. Frequently, however, it is seen near the true nucleus as a clear spheroidal mass, similar to if not identical with an idiozome} containing a granule or granules (centrioles), and surrounded by a layer (pallial layer) consisting partly of Golgi bodies and mitochondria. Whatever its nature when present it seems to exercise some influence over the building up of the nutritive material. T

The Central Body. — Concerning this body in the oiicyte there is considerable question. In some eggs, as just indicated, the oégonial divi 1 This is a special term applied to the centrosome during certain stages in the development of the germ cells. THE GERM CELLS 11

sion-center appears to persist for a time as a part of the yolk-nucleus complex. Before the yolk has finished forming, however, this complex generally disappears, and with it the division-center also usually vanishes. At the time of meiosis a new center forms, apparently in connection with a new (?) centriole, the origin of the latter in these cases being uncertain.

The Egg Membranes

Following growth the oiicyte, or ovum, as it may now be called, is often surrounded by as many as three different types of coverings, whose character and development are as follows. The first of these is a thin envelope immediately surrounding the egg, termed the vizelline membrane. It is doubtful in the eggs of many Vertebrates whether or not this covering is really present. When it is present, however, it is characterized by the fact that it is a secretion from the ovum itself. The second covering is the chorion, which is secreted by the follicle cells. It varies much in structure and again may be entirely lacking, as is probably the case in the Chick. Finally there are frequently one or more tertiary coverings. These may be jelly-like as in the Frog, or one soft and the other calcareous as in the Bird. When present they are always secreted by some portion of the oviduct through which the egg must pass on its way to the exterior.

The Spermatozc-5n.-—The mature male germ cell is called the sperm.atozoiJ'n. In general it is characterized by its extremely minute size, its lack of any nutrient material within itself, and its equipment for active locomotion through a semi-fluid medium. More particularly such a typical sperm consists of the following main parts (Fig. 6) :

I. The Head. —— This is chiefly composed of concentrated chromatin enclosed in a thin envelope of cytoplasm. It varies greatly in shape in different animals, but is often a more or less ovoid disc. To its anterior end is attached a tip, usually rather pointed, but also subject to much variation in form. It is the acrosome or perforatorium, apparently derived from a part of the centrosome or idiozome. Thus the head may be said to consist essentially of the nucleus and a very little cytoplasm.

II. The Middle Piece. — This has long been a convenient descriptive term rather than an accurate designation of a part which is truly homologous in different forms, and is in general the region immediately posterior to the head. According to Bowen (’24), however, this part may be more accurately described as that portion of the spermatozoiin which is composed of the following materials:_cytoplasm, mitochondria, the axial filament, and a centriole or centrioles, to one of which the filament is attached. Of these items, moreover, the mitochondria and ep. ep.

Fig. 6.——A diagrani of a generalized flag:-llate spermatozoiin based on the Mammalian type, showing the flat side of the head and also its edge.

H. Head. M. Middle piece. T. Tail. a. Acrosome. af. Axial filament. c. Centrosome. cy. Cytoplasm forming an envelope for the head and middle piece. ep. End piece. mi. Mitochondria arranged in the form of a spiral thread. s. Sheath of unknown origin and

’ constitution covering the axial filament of the main piece of the tail, and extending up inside the cytoplasm of the middle piece. :1. Neck.

centrioles are supposed to be confined to the middle piece, thus defining it. Sometimes at its anterior end is 3 short clear region of the middle piece attaching it to the head. It is termed the neck, and when it exists one or niore of the centrioles lies in it.


III. The Tail or Flagelhtm.-—Continuing with the definitions of Bowen. this part of the sperm extends posteriorly from the point win.-re the cytoplasm and mitochondria of the middle piece and. It thus consists of that mgiszni of the axial filament which though lat.-1-ting these.» mv<,-.rings is nevertheless enveloped by a sheath, plus a short final portion of naked iilaxrwiit. The sheathed region is termed the main piece. and the naked filament the end piece. The former, along with the middle piece, may also possess a fin-like membrane which is supposed to arise from the axial filament. lt should be noted that according to this description some sperm. e.g., those of the Urodeles, have no main piece, the middle piece extending all the way to the end piece.

It must now he added that though the chiei features thus described may be regarded as typical of spermatozoa in general, there are numerous, and sometimes quite bizarre, variations. Indeed in certain cases even the characteristic flagellum is lacking, and the cell depends upon amoeboid movements for its locomotion. A suggestion of the varieties of forms which occur is indicated in Figure 7.

With this idea of the general structure of a sperm in mind, it is now possible to consider the stages through which such a cell passes in its development or maturation. The primordial germ cells have already been described in the

study of the ovum, and it was noted that during this early period their appearance is practically alike in both sexes. Thus no further account of this stage is necessary in describing the history of the male cell. 64

Fig. 7.—-—Variou5 types of spermatozoa. From Kellicott ( General Embryology). A, B._ The Teleost, Leuciscus (Ballowitz). C. D. The Birds, Phyllopncuste and Tadorna (Ballowitz). E, F. Two forms of the sperm of the Snail, Paludina (Von Bmnn). C. The Nematode Ascaris (Van Beneden). H. The Annulate, Myzostoma (Wheeler). 1. The Bat, Vesperugo (Ballowitz). J. The Opossum, Didelphys (Wilson). K. The Rat (Wilson). L. The Urodele, Amphiuma (McGregor). M. The Crustacean, Ethusa (Grobben). N. The Crustacean, Inuchus (G1-obben). O. The Crustacear;, Sicla (Weismann). P. The Crustacean, Bythotrephes (Weisrnann .

1:. End knob. m. Middle piece. n. Nucleus. p. Perforatorium. u. Undulatory membrane. Not drawn to same scale. A—F, I-K, from Wilson.

By the time the male germ cells have become located in the seminiferous tubules, they have become clearly distinguishable as such. They then enter upon a period of multiplication in which they are known as spermatogonia. This stage corresponds in all essentials to the similar period of multiplication of the young ova (oogonia) .

Following this stage is a time of growth which also corresponds to a period of like change among the ova (oiicytes) . The cells at this time are therefore called spermatocytes. In this case, however, the growth, though noticeable, is naturally much less marked than was observed in the oéicytes, and there is, of course, no accumulation of yolk. The nucleus, nevertheless, goes through processes very similar to those which characterize the ovum at this period, at the close of which it undergoes meiotic divisions. Although these divisions are fundamentally the same as those of the oiicyte, they differ in certain important details which will be considered more fully when that topic is discussed.

Other Difierences between the Development of the Sperm and the Ovum. — It will be recalled that in the case of the ovum the end of the growth period found it practically completed. This, how ever, is one of the points in which the spermatocyte cliiiers strikingly from the female cell. After meiosis the products of the second division are called spermatids, and instead of being complete they are just ready to enter upon their remarkable metamorphosis into the highly specialized spermatozoa. This process varies considerably in different animals as regards its details, particularly with respect to the exact method of formation of the middle piece and tail. Indeed there is still so much difference of opinion on the matter, that it seems inadvisable in a text of this type to attempt a description beyond an indication of the general constitution of each of the main parts as already stated. The student interested in the details of metamorphosis as it has been described in a particular form is referred to the account of the process in the seal by .l. R. Oliver (’13).

Two further dilierences between the history of the egg and sperm may finally be noted as follows: One of these is the fact that the multiplication of spermatogonia does not cease during the sexual life of the animal. This of course is correlated with the almost continuous production of vast numbers of spermatozoa in comparison with the relatively much smaller production of eggs. As a result of this condition, all the various stages of developing sperm are always to be found in the seminiferous tubules. Where there are no cysts, theyoungest cells occur next THE GERM CELLS 15

to the epithelium, and the older ones successively nearer the central lumen. Where there are cysts, on the other hand, any one, at a given time, usually contains only cells of one stage. In view of the very great number of spermatozoa thus produced, there is perhaps even more question in their case than in the case of the ova, whether all are.derived from the original primordial germ cells. Instead it seems probable that some at least arise directly from the division of apparently indifferent epithelial cells.

The second dilierence is the arrangement of the developing sperm relative to their source of nutriment. it has already been indicated that the cells iSertoli cells) which furnish this do not, except sometimes in the earliest stages, surround each spermatozoiin. Instead they form the lining to either a tubule or’ cyst containing many such germ cells. Then as the development of these

Fig. 8.—-Diagrammatic outline. of the spermatogenesis of the Rat in thirty-two stages. From Kellicott (General Embryology). Aiter v. Ebner. Theca of tubule toward the left. Lumen of the seminiferous tubule toward the right.

I-8. Period of multiplication (the number of cell generations is actually very large). 9-18. Period of growth. I9-24. Period of meiosis. 25-32. Period of metamorphosis. b. Basal cells or Sertoli cells. I -I 6. Spermatogonia. 17, I 8. Primary spermatocytes preparing for division. 19. First spermatocyte division. 20. Secondary spermatocytes. 21. Secondary spermatocyte division. 22-25. Spermatids. 26-31. Transformation of spermatids. 32. Fully formed spermatozoa. 15 INTRODUCTION

cells proceeds, they become arranged in bundles, all the heads of one bundle becoming imbedded in a single nutrient cell. When the sperm are mature the cyst wall, if there be one, breaks so that their tails project freely into the lumen of the tubule. At the same time the spermatozoa become loosened from the _Sertoli cells and are tlius ready to he released into the above mentioned ‘lumen (Fin. 8).

MEIOSIS

It is now necessary to return to the consideration of a process whit,-h is common to both ovum and sperm, i.e., meiosis. As has alrvmly i.tt"l*'.‘Il indicated, the phenomenon is a rather complicated one. Furthcrrnme, it varies somewhat in diilerent animals, and the exact i'ne.axiiii;:s of statue of its stages are still in considerable doubt. For the sake of nec:e:ssary brevity and clearness, therefore, it will be nece:-sary to limit rather sharply the varieties described, and the possible interpretations of which their stages are susceptible.” Also, inasmuch as there are differ‘ences in the behavior of the ovum and sperm, it will be necessary to describe them separately. The male germ cell will he considered first.

Meiosis in the Spermatocyte.

I. The Leptotene Stage. —— Shortly after the last spermatogonial division, the chromatin of the enlarging nucleus arranges itself in spireme or lepzotene threads (Fig. 9). These threads are relatively very fine, and appear as a tangled maze in which it is difiicult or impossible to determine where any particular thread begins or ends. This often leads to the impression that the threads consist of a continuous network, but this is probably not so. Rather, the most favorable cases indicate that this network really is composed of the thread-like components of the chromosomes known as chromonemaza (singular chromonema) (Figs. 9 [2], 11, I). It is, of course, difficult to determine their exact number, but at this stage there is probably one representing each chromosome, and the number would be the same as that of the chromosomes in the somatic nuclei of the organism concerned.

11. The Synaptene Stage.—At this point it should be recalled that the somatic chromosomes of most organisms, with the exception of one chromosome to be noted later, occur in pairs. The members of a given pair appear alike, and were derived, respectively, one from each parent of the organism in question. Such a pair of chromosomes are called

'~’ For a full discussion of this subject with references to the complete literature the student is referred to The Cell in Development and Heredity by E. B. Wilson. MEIOSIS 17

homologous chromosomes as contrasted with a’ pair produced by mitotic division of a single chromosome, and known as sister chromosomes. It then happens that during this stage the chromonemata come to lie side by side in pairs ‘which are thought to represent pairs of homologous chromosomes. Usually these chromonemata converge to the nuclear membrane on the side nearest the centrosome, and extend thence toward the other side of the nucleus (Fig. 9 [4]; Fig. 11, II). Presently the members of the pairs begin to fuse or synapse. If this is the correct interpretation the number of pairs should be just half the somatic number of chromosomes. Unfortunately, however, the threads or chromonemata in this stage are still so fine and tangled that they give only the general impression described above, and it is impossible to determine their number exactly.

Even so, in instances where the pairs of threads are well lined up with their ends toward one pole, a fairly close count can be made; in such cases the results confirm the interpretation indicated. Another type of synaptene occurs in some animals and many plants which is termed syrzizesis or contraction. Here the leptotene threads or chromonemata become drawn into a tangled mass, usually somewhat to one side of the nucleus. In this type of synaptene the side by side pairing of the threads is much less clear; yet even here there is some evidence that it is occurring as the contraction into the mass begins, and this is generally assumed to be the case. Sometimes, also, the contraction is not so complete as to obscure the fundamental nature of the process. Whichever appearance this stage may have, there is plenty of indirect proof that a close union of the homologous members of chromosomal pairs is occurring here, and hence the name synopsis or fusion (Fig. 9 [4—5] ; Fig. 11, 1 I, Ila).

III. The Pachytene Stage.——— In this stage the threads appear much thicker and often somewhat fuzzy (Fig. 9 [6-7] ; Fig. 11, III). They are also obviously fewer in number than in the leptotene, and though an accurate count is again difficult, the number at this time appears to be about half that of the chromosomes in somatic cells. Indeed according to the interpretation generally accepted and here given, this number is exactly half, except for the possible presence of the one odd chromosome to be mentioned later; this has been brought about by the more or less complete fusion of the, paired threads of the synaptene. This half number of chromonemata, or of chromosomes, of which they are the equivalents, is known as the haploid number, as compared to the number formed in the somatic cells and termed the diploid number. It should 18 INTRODUCTION

he noted, however, that the reduction here indicated is not really a genuine reduction since all the threads are still present in a fused condition. The true reduction comes later. This is emphasized by the fact that in some cases, as in the Orthoptera, for instance, there is always, in


Fig. 9.—Prophases of the heterotype division in the male Axolotl. From Jenkinson (Vertebrate Embryology).

’ 1. Nucleus of spermatogonium or young spermatocyte. 2. Early leptotene. 3. Transition to synaptene. 4. Synaptene with the double filaments converging toward the centrosome. 5. Partial synizesis or contraction figure. 6, 7. Pachytene. 8. Early. 9. Later diplotene. I0. Heterotypic chromosomes with disappearing nuclear membrane and with one figure showing its quadripartite character.

properly stained preparations, a slight indication of the duality of the fused threads.

TV. The Diplotene Stage. -——Following the pachytene stage the chromatin threads no longer converge toward one pole, and again appear definitely double. Indeed, especially toward the latter part of this stage, each pair of chromonemata may appear fairly clearly quadripartite, at MEIOSIS V 19

which point each one of the four threads is called a chromatid, and the group of four is called a tetrad (Fig. 9, [9—10] ; Fig. 11, IV, IVa). This quadripartite condition is due to the fact that sometime during the pachytene or early diplotene each chromonema of an homologous pair has duplicated itself to form a sister thread. At the same time the four chromatids in each tetrad have become twisted about one another in a


Fig. 10.—First meiotic division in the male. 2. Salamander, the remainder Axolotl. From Jenkinson (Vertebrate Embryology). 1, 2. The heterotypic chromo somes on the spindle (metaphase). 3. Anaphase. 4, 5. Telophase. 6. Resting nuclei. 4-6. Cell-division into two secondary spermatocytes.

peculiar way to be explained later, this twisted condition being called strepsinema (Figs. 9 [9]; 11, IV). On the basis of the four-part situation just described one might ask why this stage is termed diplotene, meaning double thread. It is because, though the groups may be quadripartite, one of the lines of separation is usually much more evident than the other, and it is along this line that the first meiotic_ division occurs. '

It used to be thought of considerable interest, whether this line represents a separation of the formerly synapsed homologues, or whether it represents a new _line of separation between duplicated sister chromonemata, now chromatids. If it is the former, the first meiotic division is said to be reduczional because it appears to separate the original homologous members of chromosomal pairs. The second division, then, must 20 INTRODUCTION


Fig. 11.—Diagrams of possible prophases of meiosis, involving three pairs of chromosomes; 1, barred, light, dark; 2, dots, rings; .3, szippled, white. I. Lepmrene. Chromosomes in form of thread-like chromonernata. II. Synaprenc. limnnlngous chromonemata fusing. Ila. Synizesis, another form of synapsis. III. Pzu-l:,we~ne. Chromonemata fused, each one starting to duplicate itself, and also starting to ex~ change parts in 2 and 3. Only “ pre-reduction” situation shown in this stage (see below). I V, I Va. Diplotene, shown enlarged in 1, la, etc. Members of pairs starting to separate, each chromonema now definitely duplicated to form a chromatid of a tetrad. In the barred pair pre-reduction is shown in IV and 1, post~rc-duction in I Va and 1a. In the other two pairs exchanges have occurred between the members of the pairs as indicated. Hence though the arrangement of parts varies, as shown in 2, 2a and 3, 3a, each separation in these cases is partly reductional and partly equational. In 4 and 411 two exchanges between a single pair of chromonemata is

shown, a case not represented above or in Fig. 12. There are other possibilities. V, Va. Diakinesis, show possibilities of this stage following IV and I Va, respectively.

presumably separate the sister chromatids produced by duplication, and hence like any ordinary mitosis is equational. This order of events is called pre-reduction. If the sequence is reversed, it is post-reduction. (Fig. 13). Actually, since all four chromatids of a group usually look alike, and since the number of remaining chromatids is the same in

‘ either case, there is generally no way of telling which type of division

has occurred except in a few peculiar situations such as illustrated in Figs. 20, 21, and 22. Here post-reduction, though probably the more unusual‘ type, can clearly be seen to have taken place. Obviously, however, the final result following the second division will be the same in either case. Also, because of certain further events, the terms “ pre- ”

and “ post-reduction ” often lose their significance. These events are as follows:


Fig. 12.-— Continuation of diagram in Fig. 11, showing the I and II meiotic divisions. In I and II the barred tetrad, as in Fig. 11, is undergoing pre-reduction. In la and [Ia the same tetrad is undergoing post-reduction, i.e., the II division is reductional (see text and Fig. 13). As indicated under Fig. 11, for the other two tetrads each division is partly reductional and partly equational. The groups of chromosomes bracketed under a given letter (A, A etc.) are those to be found in each cell following the division immediately above. Each tetrad behaves independently of the others, e.g., in 1, cell A happens to receive the lightly barred pair of chromatids (chromosomes), but this is a matter of chance, and is unrelated to which pairs from the other sets of tetrads go to this cell. This is called independent assortment, and applies similarly to the single chromosomes of the II division. Hence many more combinations are possible than are shown above.


\ oauouo D

At some point after the quadripartite condition has developed, apparently in the pachytene or early diplotene, it is believed that exchanges of parts (genetic cross-overs, see below) frequently occur between the homologous chromonemata (chromatids) of a tetrad. While such exchanges may occur between one pair of homologues at one or more places simultaneously, and possibly between one pair at one place and the other pair elsewhere simultaneously, exchanges between members of both pairs seem never to occur simultaneously at the same place (Fig. 11, [2, 3, 4]). It should now also be noted that following such exchanges the initiation.of repulsion between corresponding parts

4‘lP..W,WK'Tt.’>s A. Pre - Reduction First Division Second Division Reductiona! EQU3t|°“3l


First Division P°st' Reducthn Second Division Equational Reductional

.0 .0


Fig. 13.——-A stereoscopic diagram representing the two possible types of behavior of one of the three pairs of chromosomes indicated in Fig. 12 during the first and second meiotic divisions. The letter a designates one member of the pair and b the other member. For the sake of clearness, the plane of the second division is indicated in both types before the first division has actually started, in this manner producing a tctrad consisting of four chromatids. These chromatids are often definitely separate at this stage, or even as early as the diplotene stage (see text and Figs. 11, 12, 14).

In the upper set of four figures the first division (that on the left side) is reductional, i.e., a and b are separated from one another, while the second division (that on the right side) is equational, i.e., a and b are each split in half (Prereduction). In the lower set, on the other hand, the first division (that on the left side) is equationnl, i.e., a and b are each split in half, but in each instance the half of it remains attached to the half of b. The second division (that on the right side) then follows and in each half which resulted from the first division the a portion is separated from the b portion (Post-reduction).

of chromatids leads to a crossing of the chromatids as in Fig. 11. In the case of “ pre-reduction,” the repulsion will be between the corresponding parts of the hornologues which attracted one another during synapsis, while in “ post-reduction ” it will be between corresponding parts of


Fig. 14.—Tetrad formation in the spermatogenesis of Ascaris megalov cephala bit/alerts. From Kellicott (General Embryology). After Brauer. x 795. A—'G. Stages in the division of the primary spermatocyte. A, B. Splitting, and C, condensation of chromatin thread, seen in side view. D. shows, in end view, that the splitting is double. Centrosome divided. E. Migration of centrosomes and formation of spindle. F, G. Division of the cell body and of the two tetrads. H.,Secondary spermatocyte containing two dyads. I. Division of secondary spermatocyte. J. Two of

the spermatids, each with two “ monacls ” or single. univalent, chromosomes.

n

sister chromatids. In either case, crossing results, and the point of crossing is called a chiasma (pleural chiasmata) , the general situation being termed chiasmazypy.

In some forms the diplotene is followed by a so-called confused or diffuse condition in which the threads become less distinct, and approach the state seen in a “ resting ” nucleus. Either with or without the interpolation of this diff use condition, there may _also ensue a second contraction stage in which the threads are again drawn into .a clump quite similar in appearance to that of the original synizesis in those cases where the ‘latter occurs. 24 ‘ INTRODUCTION

V. The Diakinesis Stage. -In this stage the chromatid threads, as in the case of any chromonemata approaching the metaphase stage of a cell division, undergo great shortening and condensation of chromatin. In the case of meiosis, however, the forming chromosomes difier from those of a similar mitotic stage in that they assume peculiar shapes, e.g., crosses, rings, etc. (Fig. 9 [10]; 11, V; 15: D, E), and are hence Said to be heterotypic. This is due partly to the quadripartite nature of the chromatid groups, and partly to the twisting of the chromonema indicated above. The number of tetrad groups is of course haploid.

V I . The First Meiotic Division. —- The above chromatids are presently arranged at the equator of an ordinary amphiaster, but, because of the quadripartite character of the groups and the chiasmata involved, the metaphase figures, like those of diakinesis, have a peculiar appearance and are also termed heterotypic (Figs. 12, I, la; 15, A, B) . As has been stated this division occurs along the more prominent of the diplotene separations, and in the case of a tetrad where no exchanges of chromonemal sections have occurred, the division will be exclusively reductional or equational, depending upon whether the separation is between homologous or sister chromatids. Even so, since all four chromatids of a tetrad look alike, there is usually nothing to show which type of division has occurred. Also where exchanges have taken place between homologues, each division is inevitably partly reductional and partly equational. In any event the resultant number of double chromatids, like the number of tetrads, will be haploid.

VII. The Second Meiotic Division.--Until the completion of the first division, the spermatocyte is known as primary. After that it is called secondary. The secondary spermatocyte generally enters upon a brief period of rest preceding the next division (Fig. 12, II, Ila). During this time the nucleus is often reconstituted, and the chromatin assumes to varying degrees the typical resting condition. Presently. however, the haploid number of double chromatids emerges from this stage in the usual manner, and becomes arranged on the spindle preparatory to the second division. Upon this occasion they generally present a normal appearance, aside from the important fact that their number remains haploid, and hence this division is termed homotypical.

From preceding discussion and reference to Figs. 11 and 12 it should now be clear why the question of pre- and post-reduction, as stated, often loses its meaning. Thus it may even be that the situation is different for dilferent tetrads in the same nucleus. The only cases where pre- or postreduction applies to the entire nucleus would be in organisms like the male of Drosophila where, for some unknown reason, there are no exMEIOSIS . 25

changes between any of the chromonemata. In instances where there are exchanges, however, reference to Figs. 11 and 12 makes it evident that in these cases two meiotic divisions are needed to effect complete separation of all homologous parts. Thus, considering parts 2, 2a and 3, 3a in the above figures, it is evident that each division as diagramed is, as noted, partly reductional and partly equational, and this is probably the


Fig. 15.-—Meiotic divisions in certain Insects, showing forms of chromosomes and their relation to tetrads. From Kellicott (General Embryology). After de Sinety. x; 1125. A, B.'Two stages in anaphase of primary spermatocyte division in Stenobothrus parallelus. Rings opening into Vs which diverge. C. Anaphase of spermatogonial division in Orphania denticauda, showing differentiated chromosome, x. D, E. Preparation for first spermatocyte division in Orphania, showing “tetrads”

in various stages of formation from rings and crosses, i.e., diakinesis figures.

situation in the majority of cases, not only with respect to particular pairs of chromosomes, but with respect to all the pairs in a nucleus. The above situation might be cited as a reason why two meiotic divisions are necessary, but this is not so. It is rather the duplication of chromonemata, probably in the pachytene previous to the exchanges of parts, which requires a subsequent second division in order to secure distribution of all homologous sections to separate nuclei. It is, therefore, the original duplication which needs explaining, and it appears that this phenomenon is simply inherent in all prophases. Hence a second division is inevitable whether needed to effect complete reduction or not. In any event, regardless of when reduction occurs, it is now evident that the 25 INTRODUCTION

final result is the same; that is, there are produced four spermatids,

’ each containing one haploid set of chromosomes with unique parts.

This last statement, it should be added, is frequently not precisely true. The exception is exceedingly important, but it has been omitted for the time being for the sake of clearness. It can be better appreciated, furthermore, when described in connection with the condition

in the ovum. We shall reserve this point, therefore, until after the description of meiosis in the female.


Fig. 16.-—-From Kellicott (General Embryology). A. Chromatin extrusion from the nucleus into the cytoplasm in the oiicyte of the Medusa, Pelagia noctiluca. After Schaxel. B. Extrusion of chromatin into the cytoplasm during the maturation of the oiicyte of Proteus anguineus. After Jiirgensen. x 1080.

Meiosis in the Ovum.——-Meiosis in the ovum is fundamentally similar to that in the sperm, with certain variations in detail. lt will be possible, therefore, to make clear the process in the oiicyte by simply "indicating the points in which it differs from that just described. These points"may be stated as follows:

1. Length of Early Sta,ges.———In some instances at least, the early meiotic stages up to and including synizesis occur immediately after the lastoiigonial division. As previously noted, however, these divisions are said in some cases to cease at the time of the hatching or birth of the female containing the cells in question. As indicated this is now denied with respect to Mammals, and is in doubt as regards all Vertebrates. In so far as it may occur, however, there follows the fact that certain of the meiotic stages must, in the cases of the last ova to mature, MEIOSIS 27


Fig. 17.—Meiosis and fertilization in the Nemertean, Cerebratulus. From Kellicott (General Embryology). After Coe. C, D, x 375, others 3: 250. A. Primary oficyte. Part of the chromatin has been condensed into chromosomes, only five of which are shown (the number present is sixteen) . The remainder of the chromatin is thrown out into the cytoplasm. The centrosomes, each with a small aster, are diverging, and the nuclear membrane is commencing to disappear. B. First polar spindle fully formed and rotated into radial position. Chromosomes in equatorial plate. The extra chromatin (vc) is seen scattering through the cytoplasm. C. First oiicyte division; anaphase. D. First polar body nearly separated. E. First polar body completely cut 0E; second polar spindle formed and rotating into radial position. Spermatomiin within the egg. F. Second polar body completely separated. Egg pronucleus forming, surrounded by large aster. Sperm pronucleus, also with a large aster, enlarged and approaching the egg pronucleus. These steps connected with the be havior of the egg and sperm nuclei (pronuclei) will be fully explained later on in the text.

c. Chromosomes. o. Nucleolus, vacuolated and commencing to disappear. 5. Spermatozotin just within the egg. v. Germinal vesicle. vc. Extra chromosomal chromatin being scattered through the cytoplasm. I, II,

First and second polar bodies. 0” Sperm nucleus (pronucleus). 9? Egg nucleus (pronucleus).

occupy very considerable periods of time. This is apparently not true of these stages in any of the sperm.

II. Loss of Chromatin.—— In the oiicyte, a loss of chromatin into the cytoplasm has been alleged in a few special cases during the growth period, particularly in the diplotene stage (Fig. 16, B). That this phenomenon actually involves a loss of parts of the diplotene threads, however, seems unlikely for these threads or chromonemata presumably carry the genes, and any indiscriminate discarding of genes at any time INTRODUCTION

Primordial germ cell ("Primitive Ovum")

Period of multiplication. s chromosomes. The number of cell generations is much

greater than indicated here _ Danni, Period of growth, ending in tetrad formation or its equivalent Primary Oocyte Secondary Oocyte,

Period of maturation divisions. the first in this case being reductlooal

and first polar body

Mature ovum and 2 three polar bodies, ‘ each with ‘g’ chromosomes

Fig. 18. —~Diagram of the chief events of oogenesis. Modified from Kellicott after Boveri. The chromosomes are assumed to consist of two pairs represented by letters. AA represents one pair and BB the other. It is to be noted that the members of a chromosomal pair are not always dissimilar as the light and dark letters in

i this case suggest. They are so represented here in order to make apparent the ¢lis~

tinction between the equational and reductional divisions during meiosis. Also as indicated in connection with Fig. 12, the dissimilarity of the members of one pair has no necessary relation to the dissimilarity of those of the other. Finally, it should be remembered that when dissimilarity between members of a pair of chromosomes

does exist, it can rarely be detected by observation of the bodies themselves, only by the efiects they produce.

is highly improbable. It, therefore, seems more reasonable that whatever loss there is in these cases concerns only the matrix material surrounding the coiled chromonemata, storage karyosomes, or the like (Figs. 16, A; 17) . Such losses as these are not observed in spermatocytes.

III. Size of the Division Pratlucts.- Perhaps the most striking of all the differences between meiosis in the ovum and that in the sperm is the difference in the size and fate of the products of the two divisions. In the sperm, as, has been noted, the two meiotic divisions are equal MEIOSIS 29

Period of multiplication. s chromosomes. The number of cell generations is much greater than indicated here

Period of growth, ending in tetrae formation or its


equivalent Primary spermamma Period of maturation divisions, the first in this S°°°"d“" "’°'""‘°°’" case being reductional Spennatids \ 1, ‘\ \ I \ I Period of metamorphosis. ‘t I ‘. II ‘\ I ‘..' spermatozoa

-§- chromosomes present ‘E’ E’ ‘E’ 3'

Fig. 19.—Diag_ram of the chief events of spermatogenesis. Modified from Kellicott after Boven. The chromosomes are represented in the same manner as in the

case of the ovum in Fig. 18. It will be noted that the light and dark members of the pairs are differently arranged relative to one another in the primordial and subsequent cells. This was done to indicate that this phase of the arrangement is purely a matter of chance. It might be the same in the case of the ovum or as suggested in that case the A and B might both be light or both dark in all the cells. Likewise starting with the combination shown in the primary oiicyte or the primary spermatocyte the four final cells in either instance might have had AB in two and AB in the other two instead of the combinations indicated. All that is required is that there be one member of each pair in each mature cell or polar body.

and the resulting four cells are all alike and functional. In the ovum, on the other hand, the cytoplasmic divisions in both cases are extremely unequal and only one of the four final products is a functional egg cell. The others are relatively minute and are known as polar bodies, the one resulting from the first division being termed the first polar body and that resulting from the second division the second polar body. This condition of inequality is brought about by the fact that at each divi30 INTRODUCTION

sion the nucleus and division mechanism take up a position at the periphery of the cell instead of at its center. Thus one set of chromosomes remains in the main cell, while the other set is pinched off in a very small bit of cytoplasm (Fig. 17).

Although there is this great discrepancy in the distribution of the cytoplasm, there is good reason to believe that the nuclear content is thg same in every case, just as it is in the sperm. In other words, the performance is in every way homologous with the two spermatocyte divisions except for the inequality in the distribution of the cytoplasm. This idea is borne out by the fact that in many cases, as might be expected, the first polar body divides again as does its larger sister cell, thus

Fig 20 -—A diplotene nucleus in Lygaeus bicrucis. Producing one Olvum After E. B. Wilson. Note the condensed condition of and three P013; bodies.

~ - X d Y. Th ' ' h - . . . ‘.lI}i(()3s:fr1xe‘5:,h‘:l1lnt‘llse‘):(l-Jltelltlir hzziiid, are stiallrfgigalilllrilgef oliile T]-115 behavlor 1“ the

of them, a, as well as the sex-chromosome X, show- case of the ovum is ing the characteristic diplotene split. This split in h ah t b d the case of the X is obviously equational. The plas- t ‘mo '3 ° 3 an 3 3P‘

mosome, 121., is only partly visible. tation to secure the greatest amount of cytoplasm and nutriment in a single cell.

IV. The Time of the Meiotic Divisions. —— In the sperm, as has been seen, meiosis is entirely completed within the testis and before the spermatid even enters upon its final period of development. In the ovum, on the contrary, meiosis is the last thing to occur. Sometimes division takes


' place while the ovum is in the ovary. More frequently, however, espe cially among the Vertebrates, at least one of the two divisions occurs after the ovum has left the gonad. Indeed in many cases the second division does not take place until after the egg has been entered by a spermatozoiin (Fig. 17). A comparison of the chief processes involved in the development of the sperm and ovum is presented diagrammatically in Figures 18 and 19.

The _Sex-Chromosome-s.—-We are now prepared to return to a consideration of the exception in chromosomal behavior which was MEIOSIS ' 31

Fig. 21.—Meiosis during the spermatogenesis of the squash-bug, Anasa tristis, showing the behavior of the X-chromosome or idiochro~ mosome. From Kellicott (General Embryology). A, After Wilson, others after Paulmier. A. Spermatogonium. Polar view of equatorial plate showing twenty-one chromosomes (ten pairs, plus one). The X-chromosome is not distinguishable at this time. B. Primary spermatocyte. Tetrads formed. C. Equatorial plate of first spermatocyte division. X-chromosome divided. D. Anaphase of same division. The daughter X-chromosomes have also diverged. E. Equatorial plate of second spermatocyte division. F. Anaphase of same division. The Xchromosome lies, undivided, between the two groups of daughter chromosomes. G. Late anaphase of same division. The undivided X-chromosome has passed to the upper pole, lagging behind the others. H. Telophase of same division. X~chromosome still distinct.

noted but not described at the end of the account of meiosis in the sperm.

In the somatic and germ cells of many animals, both male and fe- ‘ male, there are found one or more chromosomes which in many cases behave quite differently from their fellows. They often stain more ' deeply, and are especially peculiar in that they frequently remain in the condensed condition during the entire growth period of the germ cells. 32 INTRODUCTION

On this account they sometimes appear at this time like nucleoli with various distinctive shapes (Fig. 20). A150, during the 3“aPha5e stage of cell division, they are noted for a tendency to lag behind on the spindle (Fig. 21). One of the most striking things about these chromo 6'


Fig. 22.—A diagram of the behavior of the chromosomes during the meiotic divisions in the male of Protenor belfragei. From Morgan (Heredity and Sex, published and copyrighted by the Columbia University Press). The sex-chromosome throughout is represented in outline, the others in solid black. A. The chromosomes in the somatic cell of a male. B. The chromosomes united in synapsis prior to the first meiotic division of a germ cell. The single sex-chromosome is without a mate. C. The first meiotic division, which for the sex—chromosome is certainly equational. D. The second meiotic division, “ reductiona1” for the sex—chromosome, i.e., the latter goes to one pole or the other. It is impossible to say, certainly in this case, which division is really reductional for the ordinary chromosomes (autosomes). E,

E’. The distribution of the chromosomes in the four spermatids resulting from the two meiotic divisions.

somes, however, is the fact that in some animals in the male, each somatic cell, as well as each unmaturated germ cell, possesses only one of them, while each cell of a similar type in the female has two. Under such conditions the one or two eccentrically behaving chromosomes are termed X-chromosomes. In such cases it follows of course that in the male the total number of chromosomes in each cell of the types indi cated is odd, whereas in the female the number in each cell of a similar type is even. ’ MEIOSIS ' 33

Thus in the male of the insect Protenor the somatic cells and the unrnaturated germ cells each possess 13 chromosomes, while similar cells in the female have 14 (Figs. 22 and 23). Under such circumstances it is obvious that when the male germ cell undergoes meiosis, its X-chro Q Protenor



Fig. 23. —A diagram of the behavior of the chromosomes during the meiotic divisions in the female of Protenbr belfragei. From Morgan (Heredity and Sex, published and copyrighted by the Columbia University Press). The sex-chromosomes throughout are represented in outline, the others in solid black. A. The chromosomes in a somatic cell of the female. B. The chromosomes united in synapsis prior to the < first meiotic division of a germ cell. Note that in this case the sexchromosome has a mate. C. The first meiotic division, probably equational, at least for the sex-chromosomes. D. The second meiotic division, which, if the first division was equational, is presumably reductional. E. The distribution of the chromosomes in the two polar bodies and the egg. The first polar body is represented as just under going the second division.

mosome will be without a mate. Apparently as a result of this fact the odd chromosome in the male only divides at one of the meiotic divisions, e.g., in the instance in question the first; and since this chromosome has not had a mate, its division must presumably be equational (Fig. 22, C). Following the second division, the final result, as usual, is four male germ cells, but their content is obviouslynot quite equal. Two of them possess six ordinary chromosomes (autosomes), while each of the other two possesses a similar six autosomes, and in addition an X chromosome, i.e., a total of seven (Fig. 22, D, E, D’. E’). INTRODUCTION


Fig. 24.——A diagram of the behavior of the chromosomes during the meiotic divisions in the male of Lygaeus bicrucis. From Mor an (Heredity and Sex, published and copyrighted by the 3:"§:l!lm1la University Press). A. The chromosomes in the somatic cell of at male. Note the large X and the small Y sex-chromosomes. B. The chromosomes united in synapsis prior to the first meiotic division of a germ cell. The X and Y do not usually unite at this time so that it is not indicated in the diagram (see Fig. 261. C. The first meiotic division in this case, so fat as the sex-chromosomes go, is evidently equational. D. The second meiotic division, which for the Ee!-Cl’ll‘0mosomes is evidently reductional. E, E’. The distribution of the chromosomes in the four spermatids resulting from the two meiotic divisions, two receiving an X-chromosome and two a Y.

In the female since there aretwo X~cht-omosomes in the germ cells previous to meiosis each egg after meiosis ‘will contain an X. This will also be true of course of the three polar bodies, but these being nonfunctional may be disregarded. Obviously, then, whether a fertilized egg is to contain one X or two will depend upon whether it is united with an X~bearing sperm or with one without an X.

There are numerous variations of this basic situation, the most com~ man one being the type illustrated by the insect Lygaeus. Here the X-chromosome in the male does have a mate called the Y-chromosome, but it is different from the X, in this instance smaller, and can thus be distinguished from it (Figs. 24., 2Q, $6). A similar situation as regards MEIOSIS 35

an X and Y pair of chromosomes occurs in Man. A slight variant of this arrangement is seen in Drosophila, the fruit fly, where the mate of the X in the male differs from it in shape rather than size (Fig. 27). There are still other situations where the X and Y are quite similar in appearance to each other, and even to the autosomes, but can be distin



Fig. 25. —A diagram of the behavior of the sex-chromosomes in the female of Lygaeus bicrucis. From Morgan (Heredity and Sex, published and copyrighted by the Columbia University Press). A. The chromosomes in the somatic cell of a female. Note the two X-chro-i mosomes. B. The chromosomes united in synopsis prior to the first meiotic division of a germ cell. C. The first meiotic division, probably equational. D. The second meiotic division, probably reductional. E. The distribution of the chromosomes in the two polar bodies and the egg. The first polar body is just undergoing the second division.

guished from the latter by their behavior, as already noted. ln this last case, where the X and Y are not visibly distinguishable from one another, there is of course no obvious difference between the chromosomal condition in the male cell with its X and Y and in the female cell with a double X. There is good evidence from other sources, however, that even here fundamental qualitative differences do exist between the presumed X and Y chromosomes.

A more fundamental and striking variation in the relationships of these chromosomes occurs in Moths, Birds, and some Fishes. Here it is

the female which has the odd chromosome, while the male has two of a kind. Since this arrangement was first observed in the moth Abraxas it

is known as the Abraxas type. Also to avoid confusion the peculiarly behaving chromosomes are here termed Z and W instead of X and Y. INTRODUCTION


Fig. 26.——Division figures from the meiosis of the germ cells in the male of Lygaeus bicrucis. After E. B. Wilson. A. A polar View of the first meiotic division. In this insect the synapsis of the Xand Y-chromosomes not only does not occur while they are in a threadlike condition, but is postponed until almost the end of the first meiotic division. Even then it is evidently very slight, indicated lay the figure. B. A side View of the second meiotic division in the same animal. The chromosomes in this case do not lose their identity during interkinesis (i.e., the interval lwtweeii the two divisions), and it therefore is possible to determine that the X and Y which united in synapsis 81 the end of the first division. as shown in A, are now being separated from one another. Thus for these chromosomes in this instance the second division is clearly reductional.


Fig. 27. -— From the Mechanism of Mendelian Heredity, after Brildges. The female and male groups of chromosomes in Drosophila Re ¢z)SJ{gasi.:er,_ showing the four pairs of aiitosome chromosomes plus

e pair in the female and the XY pair in the male. In this ani afighe members 0i 93°11 Pair are usually found together as indiTHE SIGNIFICANCE OF MEIOSIS 37

It is then the ZZ combination which is found in the males and ZW in the females. Because of the evident relationship to sex which these XY or ZW chromosomes have, they are also termed sex-chromosomes. Something more concerning this important relationship will be said in a subsequent paragraph, but first a word is required regarding the entire ‘meiotic phenomenon as so far described.

THE SIGNIFICANCE OF MEIOSIS

It is assumed that the student is aware of the evidences from his study of heredity and cytology that chromosomes are qualitatively different from one another with respect to chemical entities called genes or determiners. These genes, as is well known, are distributed from one end of a chromosome to the other, and with the exception of the sex chromosome in the male they normally occur in pairs. One member of a pair of genes is in one member of a pair of chromosomes, and the mate or allelomorph of that gene is in a corresponding position on the other chromosome of that pair. Thus‘ it happens that at the reductional meiotic division one complete haploid set of chromosomes, and hence of genes, goes into one cell and another set into the other. The only normal exception to this is the case of the sex chromosome in the XY male, and the genes it carries.‘They go to one cell only. The non-reductional meiotic division is then similar to ordinary mitosis, and merely doubles the number of cells containing haploid sets. Fertilization of course involves the fusion of two germ cells, an egg and a sperm, and obviously the reduction of the chromosomes and genes at meiosis prevents them from being progressively multiplied at successive fertilizations. How this ingenious state of affairs came about is not known, and the speculations concerning it would take us too far afield in this text.

A further very significant parallel between the behavior of the genes and that of the chromosomes is as follows: It will be recalled that in the discussion of the heterotypic chromosomal figures of late diplotene and diakinesis it was suggested that part of the explanation for such figures was the fact that an exchange of sections had occurred between the homologus chromonernata (chromatids) of tetrads during the pachytene or early diplotene. It now remains to add that genetic evidence indicates that exchanges of blocks of genes, technically termed cross-overs, take place somewhere during the interval when the pachytene and diplotene stages are visible. The exact time is uncertain, but that these gene exchanges are in some way definitely related to the exchanges between homologous chromoneniata is generally admitted as beyond doubt. 3;; ' INTRODUCTION

’ ssx DETERMENATION

It is known that the XY chromosomes, more particularly the X, also carry genes and some of them apparently concern sex. Genetic evidence 7

V N 81

Fig. 28.—~Diagrsm to illustrate crossing over. From Morgan (Mechanism of Mendelian Heredity). The white and the black rods (a) twist and cross at two points (1)). Where they cross they are represented -as uniting (shown in c). That an interchange of pieces has taken place in the region between genes W and Br is demonstrated from the standpoint of inheritance by breeding experiments. The results of these are most readily explainable on the assumption that the gene M

has gone over to the other chromosome.

from normal and abnormal cases itidicatcs that the sex genes in the X-chromosome tend to produce female characteristics, or at least to produce the initial impulse in that direction, while those in the autosomes tend to produce male Cll3I‘i1(‘I£3i'S. This of course applies only to the nonAbraxis type of sex inheritance wlicre the male contains the single X. Tin: sitnaticm is evidently reversed in the other types. Thus the determination of sex is a matter of balance between two types of gene influence. Normally a diploid set of autosomes balanced against a single X pru« duces a male, while the addition of another X is enough to produce a female. All genes, however, produce their eficcts by interacting not only with each other, but with their surroundings. Thus we know that various inherited tendencies can be modified by the proper environment.

' So it should not be surprising that sex also

is subject to environmental influences. In some animals, like Birds and Mammals, it is rather hard to alter the surroundirigs of the developing organism very rnuclt. In other animals like Amphibians, however. this is easily possible, and in such crea tures various environmental changes have been tried. It has thus been found that proper temperatures at critical periods (Witschi, ’29, see Chap. VI) , and other appropriate procedures are able to reverse the sex which a particular chromosome complex would have produced in a more normal situation. Also changes in the internal environment, such as

3 endocrine secretions, might be expected to alter the development of sex

characters, and experimental evidence shows that this is true, not only in Amphibians, but In both Birds and Mammals. ERTILIZATION AND EARLY STAGES IN "DEVELOPMENT

FERTILIZATION

B EFORE proceeding to an account of development in any par-'

ticular animal, it may be well to discuss certain processes which are always involved, and to note the chief methods of their occurrence. rtilization in all hitvher forms consists of the union of

n; This union may occur within some cavity of the female into which the sperm have been introduced, or it may occur outside. The latter is the more common method among animals which live in the water. In either case, thousands of the relatively minute sperm are required to insure the fertilization of each single egg by one spermatozoon. We shall now turn to a generalized account of the process.

PENETRATION

The Action of the Sperm. —— Both eggs and sperm contain certain substances similar to hormones, hence called gamones. Those in the sperm are androgamones, one which prevents premature excessive sperm activity thus conserving their energy, and another which dissolves the gelatinous membrane surrounding many eggs. Those in the egg, on the other hand, are gynogamones, one of which, at an appropriate time, counteracts the first androgamone, thus increasing sperm activity, and a second which makes the sperm heads sticky, causing them to adhere to the egg surface. In addition, some eggs may secrete something which attracts sperm. Penetration of the egg may take place at any point of the surface, or the sperm may enter through a special orifice, the micropyle. Usually only one sperm enters (monospermy), and in case more do so development is generally abnormal. Sometimes, however, in relatively large yolked eggs, several sperm normally enter, a phenomenon called polyspermy. Even in such cases only one of the spermatozoa takes active part in the further events of fertilization. The remainder eventually degenerate and disappear; previous to this they may divide several times, and perhaps aid in breaking up the yolk to make it more assimilahle. 4o, FERTILIZATION, EARLY DEVELOPMENT

In such cases they are referred to as merocytes. The method by which the extra sperm are excluded in the event of monospermy will be discussed presently.

As soon as the head of the sperm has punctured the surface of the egg the swimming movements of its tail cease. In some cases the latter , e is regularly drawn into the egg along with the head and middle piece, while in others it is left outside. In either event it soon degenerates and

takes no more part in the fertilization process.

The Reaction of the Egg. The Perivitelline Space and the Fertilization Membrane. —— Probably

the first and most characteristic reaction of almost all eggs to puncture by a sperm is the formation of a space between the egg surface and its innermost covering (i.e., in most instances the vitelline membrane) . lt is called the perivitelline space and seems in some cases to be due to the pushing away of the membrane by a secretion from the egg. In other instances it may be due to shrinkage of the egg or to absorption of water by some substance between the membrane and the egg surface. In any event such a separation of the egg from its covering of course makes the latter more conspicuous, and even in such eggs as have seemed previously to lack a membrane, one now becomes visible. Because of this increased visibility following fertilization, the membrane about the perivitelline space, whether it be the original vitelline membrane, one apparently newly formed, or a fusion of both of these, is frequently called henceforth the fertilization membrane (Fig. 46, D). The significance of the phenomenon just noted is not well understood. It was thought at one time to aid in preventing polyspermy. Since eggs from which the membranes have all been entirely removed continue to be impervious to further fertilization, however, it is evident that this condition is not the result of the existence or the location of any membrane. It has also been maintained that the obvious alteration in position of the membrane is accompanied by increase in its permeability to gases and other substances. That‘ there is considerable basis for this belief is indicated by the fact that in some instances there is a decided increase in oxidative processes and other phenomena requiring such a change.

The Changes in the Egg Cytoplasm. ——Aside from these phenomena connected with the inner egg membrane, fertilization also initiates certain other changes in the egg proper. Almost simultaneous with the appearance of the perivitelline space there is frequently evident an out. of the sperm which it contains are apparently drawn down into the

PENETRATION 41

Fig. 29.——Enu-ance of the spermatozoiin in the fertilization of the Annulate, Nereis limbata. From Kellicott (General Embryology). After Lillie. A. Spermatozoon. B. Perforatorium has penetrated egg membrane; entrance cone well developed. Fifteen minutes after insemination. C. Thirty-seven minutes after insemination. D. Entrance cone sinking in and drawing the head of the spermatozoiin after it. Forty-eight and onehalf minutes after insemination. E. Head drawn in still further. Fortyeight and one-half minutes after insemination. F. Entrance completed. First meiotic division in anaphase. Fifty-four minutes after insemination. The middle piece, as well as the tailnremains outside.

c. Head cap. e. Entrance cone. h. Head of spermatozoiin (nucleus). m.

Middle piece. p. Perforatorium. v. Vitelline membrane. 1. First polar division figure.

pushing of the cytoplasm at the point where a spermatozoéin has penetrated the fertilization membrane. This protuberance is then entered by the sperm, and because of this fact it is often termed the entrance cone (Fig. 29, B). Following these events both the cone and the parts

deeper egg substance ( Fig. 29, C, D, E). Besides this somewhat localized activity on the part of the cytoplasm, however, there are.alse—evidances of other efiects which seem to he more widespread. Thus, since Fig. 30. -—Total views of the egg of Tunicate Cynthia partita, showing (‘l111!1§:f'fi in arrangement of materials of egg subsequent to fertilization. From Kr-lli<-on Werneral Embryology). After Conklin.‘ x 200. A. Unfertilized egg, before fading out of germinal vesicle. Centrally is gray yolk; peripherally is protoplasmic layer with yellow pigment, and surrounding egg, the test cells and clmrirm. B. Almut lire minutes after fertilization, showing streaming of superficial layer of prntuplo-em toward lower pole where spermatozoon enters, and consequent exposure of gray yolk of upper hemisphere. The test cells are also carried toward lower pnlt-. C. Side view of eggs showing yellow protoplasm at lower pole: at upper pole at small clear region where polar bodies are forming. The location of sperm prnnucleus (nucleus) is also indicated. D. Side view of egg shortly before first cleavage, showing posterior collection of pigmented protoplasm (yellow crescent) and clearer area above it. E. Posterior view of egg during first cleavage, showing its relation to the symmetry of egg. ‘

a. Anterior. c. Clear protoplasm. layer, with yellow pigment. g._u.&3 ' Jge Polar bodies. t. Test cells. y. Ydk 0“ Sperm nucleus. "'4?



w crescent. e. Exoplasm or cortical e. 1:. Chorion. p. Posterior. p.b.

0 terial). y.h. Yellow hentisplrerc. ‘D FERTILIZATION: LATER STAGES 43

polyspermy is not prevented by the fertilization membrane, it is held that such prevention may be due to a general alteration in the egg cytoplasm.

More specifically, according to one theory the entrance of the sperm is made possible by the interaction of a substance in or on its head with another substance on or near the surface of the egg. This latter substance is called fertilizin, and such part of it as is not used up in the interaction with the sperm is supposed to be immediately eliminated by interaction with another substance called antifertilizin. This latter material is thought to be located more deeply within the egg cytoplasm, and is brought into contact with the fertilizin by a rearrangement of the egg materials produced by the entrance of the sperm. All the fertilizin having thus been eliminated, no f_urther fertilization is possible (Lillie, ’19). Though this explanation of events is still theoretical there is considerable experimental evidence for it in certain organisms. Also, whether or not this be true, evidence is not wanting that in some cases at least, all of the egg cytoplasm is profoundly disturbed by the sperm entrance. It seems likely indeed that this is more or less true of all egg but the disturbance is particularly obvious in certain instances because in these instances different regions of the egg cytoplasm are diflerently colored and thus distinguishable. In such eggs it has therefore been possible to observe that, following fertilization, a sudden and marked rearrangement of these parts of the cytoplasm takes place. Such, for example, is the case with the egg of the Tunicate, Cynthia (Styela) partita (Fig. 30), and also with that of Amphioxus (see below).

THE LATER STAGES

The later steps in the fertilization process which are now to be described are all more or less directly connected with the fusion of the nuclei of the sperm and egg.

The Egg Nuc1eus.———The meiotic divisions of the egg are some-l

times entirely completed previous to’ fertilization. More usually. however. as in the case of most Vertebrates, only one of these divisions occurs before the sperm entrance, and in some instances (e.g., Nereis) both are delayed until after this event (Fig. 32, B, C ). In these cases where meiosis has not begun, or is unfinished prior to the penetration of the sperm, the latter event seems to act as a stimulus which causes the meiosis to proceed. As soon as it is completed the egg nucleus is defi nitely formed, and the centrosome which took part in .the second division disappears. ‘ 44 FERTILIZATION, EARLY DEVELOPMENT

Fig. 31.-—A generalized diagram of the penetration of the sperm and the fusion of the egg and sperm nuclei, the haploid number of chromosomes being assumed

in this case to be two. The trail of pigment marking the path of the sperm actually 7

occurs only in the case of the Frog’s egg. The egg membranes are not represented. Compare the stages with those in Fig. 32 showing corresponding processes in the egg of Nereis.

.-A. The first polar -body has been given off, and the second meiotic division is in progress. The sperm head and middle piece have entered the egg, leaving the tail outside. B. The first polar body has divided and the second has been given off, while the completed egg nucleus has started to move toward the center of the ovum. The sperm nucleus consisting of the sperm head has enlarged somewhat, has partially rotated, and is also moving toward the center of the egg. The new divisioncenter has appeared in the region of the middle piece. C. The two nuclei are enlarging and approaching one another. The sperm nucleus, having completed its

‘ rotation, has altered the direction of its movement somewhat (not always neces sary), to hasten their meeting, and the division-center is dividing into two parts. D. The nuclei, each containing the haploid number of chromosomes, have started to fuse. The division-centers, each consisting of a centriole and centrosome and stfirrfitinded by its aster, have taken up their places preparatory to the first division o t e egg. ,

cp. Copulation path. ec: Entrance cone. en. Egg nucleus. ep. Entrance path. h. Head of sperm. m. Middle piece of sperm. ms. Meiotic spindle of the second

meiotic division. pbi, pbz. First and second polar bodies. sn. Sperm nucleus. 1:. Tail of sperm. FERTILIZATION ‘ 45

Fig. 32.——Photomicrographs of sections of Nereis eggs, showing stages in fertilization meiosis and cleavage. The photographs were made in the Anatomical Department of Western Reserve University Medical School from specimens presented to that department by Professor 0. Van der Stricht, and are reproduced by the courtesy of Professor Van der Stricht and Dr. E. W. Todd.

A. At the top of the figure the spermatozoon is shown just entering the egg; The ,

egg membrane is broken, and separated from the egg‘ at various points. B. The first meiotic division spindle. C. The first meiotic division has been completed, and the first polar body lies outside the egg beneath the egg membrane. it appears at the top of the figure and slightly to the right. Just within the egg in the same vicinity is the second meiotic spindle, while at about the center of the egg is the sperm head with its aster in front of it. D. The egg and sperm nuclei in the upper left hand part of the egg are fusing, while just beneath the egg membrane is one of the polar bodies. E. The division spindle for the first cleavage. F. The first cleavage is completed and parts of the asters for the second cleavage are indistinctly visible in the two daughter cells. ~ 46 FERTILIZATION, EARLY DEVELOPMENT

The Sperm Nucleus and the Division-Center. —— While this completion of the meiotic divisions is taking place, the head and the middle piece of the sperm advance into the egg.‘ Also, as this u(:cux's these parts rotate through an angle of 180° so that the middle piece is in the lead (Fig. 31, A, B). The advance then continues along a course whose first portion is called the entrance or penetration. path, and which, in the case of the Frog, is marked by granules of pigment. Meanwhile the acrosome which etiected the entrance of the sperm has disappeared, while marked changes are also taking place in the nuclear pnl‘~ tion of the head and the middle piece. The former is cnlmgirig. and within it the chromatin is forming a typical nuclear reticulum. in the region of the middle piece, on the other hand, a rrentriole and u.-utmsome appear and are presently surrounded by a small astcr. it has lwvn, claimed that this centriole is identical in whole or in part with the contriole (or one of the centrioles) which entered the middle piece during the transformation of the spermatid. This is very doubtful, and in many cases is certainly not true. It does seem, however, that in most instances the new division—center at least arises under the influence of the middle piece.

The Fusion of the Egg and Sperm Nuclei. — Previous to or during the above processes, the second meiotic division of the Pgg has been concluded, and the egg nucleus has moved from the periphei _v of the cell into approximately the midst of the active cytoplasm (Fig. 31, D; Fig. 32, D). Of course in telolecithal eggs with a large yolk, this point will be just below the surface of the animal pole. rather than at the actual center of the egg. The new sperm division-center and nucleus, which have meanwhile been advancing along the penetration path. now move directly toward the egg nucleus. This in many instances may involve a slight change in the course of the sperm, and when such is the case the latter portion of its course is termed the copulation pat}: as distinguished from the first portion or entrance path (Fig. 31, C ) .

As the nuclei meet each other their membranes disappear. Also there has appeared in each the haploid number of chromosomes "' I'l’i,r_r,. 31, C, D). Meanwhile the sperm division-center and aster divide, if indeed they have not already done so, and form a typical division spindle. Upon this spindle the restored number of chromosomes arrange the-.m~

1 In some instances; e.g., Nereis, the middle piece, as well as the tail, remains outside. 3 In many cases the chromosomes are not actually visible as such until after the

fusion of the pronuclei. In these instances the number appearing in the single fusion nucleus is then diploid as would be expected. CONSEQUENCES . OF F ERTILIZATION 47

selves, and each is then divided in the usual manner preparatory to the first cleavage of the egg (Fig. 32, E). It should be noted that in this process there is no fusion of the chromosomes. On the contrary, this event, presumably the actual climax of the entire phenomenon, does not

occur until the period of synapsis in the germ cells in the new individual, as described above.

THE CONSEQUENCES OF FERTILIZATION AND THEIR IMPORTANCE

We may now consider briefly some of the apparent results of this process and their possible importance. There have been three main consequences of fertilization which have been held to be of vital signifi cance, though as will appear, none of them has proved to be necessarily dependent on this phenomenon. They are as follows:

I. Reproduction. —— It has been said that the chief result of fertilization is to bring about reproduction, (a) by restoring the diploid number of chromosomes, and (b) by furnishing or causing to develop a new kinetic division-center. This argument is unsatisfactory for the following reasons:

1. Granting that these events take place in connection reproduction, the answer is, nevertheless, superficial. For the question immediately arises, why should the egg lose half its chromosomes and its division-center, thus making fertilization necessary before reproduction can occur?

2. There are numerous cases of both artificial and natural parthenogenesis, showing that neither the extra chromosomes nor the new divi'sion-center is absolutely necessary.

3. Finally the fact that the union of two cells so frequently precedes reproduction may be explained thus. Let us assume that there is some reason, such as those indicated below, why a mixture of different strains of protoplasm is beneficial. It then follows that in a Metazoan, the only time such a mixture _can possibly occur is when the protoplasm of the animals concerned is in the form of single cells, i.e., the germ cells. Then since the animals are in fact Metazoa, the union of the germ cells must eventually be followed by cell division in order that the Metazoan condition may again be reached. Under such’ circumstances, the multiplication obviously is not proved the result of the fertilization.

II. Rejuvenescence.-—It has been widely held that the fusion of different strains of protoplasm which occurs during fertilization is necessary to bring about a revivifying of any given race of animal or plant. 48 FERTILIZATION, EARLY DEVELOPMENT

Without this, it is held, cell division will gradually become less frequent, and will finally cease. The chief argument for this view has been furnished by certain experiments on Protozoa. Thus, Calkins C19) seemed to prove this by work with Paramecium, although earlier studies by Woodruff (’14) had appeared to show that some strains could. be kept going indefinitely by an internal reorganization called erzulonzrxzs. Later work by Jennings (’4-44), Sonneborn ,('39), and others has shown the situation to be even more complicated than had first appeared. Thus, con— jugation sometimes prolongs the life of certain lines. and sometimes not. At all events it is evident that the mixing of different strains of protoplasm is at least not universally necessary for revitalization.

III. Variation.——Fundamentally, of course, variation depends upon changes in the genes. As modern genetics has shown, however, the actual appearance of these variations in an animal or plant may sometimes depend upon the shuffling and recombinations of the genes which meiosis and fertilization bring about. Also in some instances significant variations may result from the abnormal behavior of whole chromosomes or sets of chromosomes, which in a few instances is definitely known to have produced new species. Weismann was entirely ignorant of the details of all these processes as now understood, but he did have some rather elaborate theories concerning normal meiosis and fertilization. He termed the recombinations of genetic determiners. which he correctly believed came about through these latter events, amphimixis, and he considered that variations so caused were an important source of material upon which natural selection might act. Others, e.g., Hertwig, believed that the shuflling and recombining processes tended to cancel out the effects of gene mutants and thus helped to keep the race constant. As a matter of fact it is now clear that both points of view are correct in different‘ cases. It also appears that evolution could occur without the fertilization process, though probably not

so rapidly.

Conclusion. —— In view of the above facts, the general conclusion as to the function of fertilization may perhaps be stated thus: While it seems reasonable that the process is an important one in view of its wide occurrence, we do not as yet understand its full significance. lt does appear likely, however, that recombinations of genes favorable to renewed vigor, and also to production of -variations, are involved. Advantages of this nature, while not essential for life, may well have been

great enough to have favored the evolution of sex and the correlated phenomenonof fertilization. GENETICS AND EMBRYOLOGY 49

EARLY STAGES IN DEVELOPMENT

RELATIVE INFLUENCE OF EGG AND SPERM ON. THESE STAGES

In the above discussion of the germ cells it has been stated that despite the great disparity in the cytoplasmic content of the ovum and sperm, their influence upon development is approximately equal. The abundant egg cytoplasm is simply for the purpose of supplying food and material for the nuclear factors to work upon, and varies according to requirements in these respects. The sperm cytoplasm, on the other hand, is only for the purpose of bringing its nucleus to that of the inert egg, and possibly of initiating division. Indeed the very features which characterize the cytoplasm of a particular egg or sperm are presumably determined by genes within the chromosomes, just as are the features which characterize the adult animal.

Nevertheless, it must now be n_oted that the character of the egg cytoplasm does determine in a rather obviously mechanical way, and apparently sometimes in more subtile ways, the nature of the early stages in development which we are about to consider. The cytoplasm of the sperm, however, though often strikingly variable in form, is apparently without any such influence. Because of this fact, in the case of most of the animals whose embryology is to be studied, it will be necessary to give a rather full account of the ovum and its development. The various kinds of spermatozoa, on the contrary, will need little further attention.

RELATION OF GENETICS AND EMBRYOLOGY

Before proceeding with a general description of the first steps in development, it is perhaps pertinent to say a few words at this point concerning the relationship between the field of genetics on the one hand and that of embryology on the other. This text deals primarily with the latter, yet the term gene or determiner has been frequently employed, and quite evidently these entities are supposed to be significant controlling elements in development. As a matter of fact the subject matter of these two disciplines, i.e., genetics and embryology, like that of physics and chemistry, is becoming constantly more interrelated. In

the earlier days of these subjects the geneticists were more concerned

with showing how genes were distributed during the reproductive proc- - ess. They also sought to prove that their occurrence in certain combinations always resulted in the appearance of certain; the . V .,

I r""' ““‘ V?‘

A./, ‘--l /" -\ I‘ 50

FERTILIZATION, EARLY DEVELOPMENT

Fig. 33.—Cleavage in the Sea-urchin, Strongylocentrozus lividus. From Jenkinson, after Boveri. Animal pole uppermost in all cases.

a. Primary oiicyte surrounded by jelly, and containing large germinal vesicle with nucleolus. Pigment uniformly distributed over surface. 1). Ovum after formation of polar bodies. Pigment forms a band below the equator. c, 41. First cleavage. e. Eight-cells. Pigment almost wholly in

lower quartet (vegetative blastomeres). f. Sixteen-cells. The lower quartet has divided latitudinally and unequally, forming four micromeres at the vegetal pole; the upper quartet has divided meridionally forming a plate of eight cells. g. Section through blastula. h. Later blastula, showing formation of mesenchyme at lower pole. i, j, 1:. Three etages in gastrulation, showing the infolding of the pigmented cells to form the hypoblast (archenteron). In j the primary mesenchyme is separated into two masses, in each of which a spicule is formed (k).

In k_ the secondary, or pigmented, mesenchyme is being hudded ofl from the inner end of the archenteron. GENETICS AND EMBRYOLOGY 51

Fig. 34.-—Meroblastic cleavage in the Squid, Laligo pealii. A, B. Egg viewed obliquely, showing animal pole. x 45. From Kellicott (General Embryology).Jifter Watasé. C, D. Surface views of animal pole, more highly magnified, to show bilateral arrangement of blastomeres. From Wilson, “ Cell,” after Watasé. A. Four-cell stage. B. About sixty cells. Cells at the animal pole very small, lowermost cells incomplete, cell walls extending down toward the uncleaved lower pole. C. Eight-cell stage. D. The fifth cleavage (sixteen to thirty-two cells).

a—p. Marks the plane of the first cleavage and the median plane of the organism. l—-r. Marks the second cleavage, and the transverse plane of the

organism. adult animal or plant.'The embryologists, on the other hand, were occupied mainly with describing the steps in development. Presently, however, both groups came to ask the question: How do the genes act to produce their end results? This has led to a rapid rapprochement between the students of the two fields. The geneticists have tried to find out how genes interact with each other and with their cytoplasmic environment to cause the development of the adult characters. Also, as already suggested, the embryologists on their side have ceased to be interested in merely describing what happens, and are now actively engaged in experiments to find out how it happens. Thus both groups are, 52 FERTILIZATION, EARLY DEVELOPMENT

so to speak, approaching the same goal from opposite sides. When they meet, and we know how all the genes act to produce all the end results,

the problems of embryology will be solved. Meantime, enough remains

to be done from both directions to keep us all busy for a long time.


C

Fig. 35.--Cleavage in the Sea-bass, Serranus amzrius. From H. V. Wilson. A. Surface view of blastoderm in two-cell stage. B. Vertical section through four-cell stage. C. Surface view of blastoderm of sixteen cells. D. Vertical section through sixteen-cell stage. E. Vertical section through late cleavage stage.

c.p. Central periblast. m.p. Marginal periblast. s.c. Segmentation cavity (blastecoel).

SEGMENTATION

Subsequent to the first division of the egg which has been indicated, further divisions follow each other, often in relatively rapid succession. The period of these early divisions is termed that of segmentation or cleavage.

Types of Cleavage.—-As has been suggested above, the type of cleavage is largely determined by the nature of the egg cytoplasm, particularly as regards the amount and distribution of the yolk which the latter contains. In a homolecithal egg with relatively little yolk, the cleavage is total or holoblastic, and approximately equal (Fig. 33) GASTRULATION 53

The equality in the size of the cells decreases, however, as the amount of yolk increases. This follows from the fact that where there is much yolk present, it is never equally distributed. Instead it gathers on one side, i.e., the vegetal side, so that the ovum becomes telolecithal. Then since yolk-filled cytoplasm divides with more difliculty than cytoplasm that is free from ‘yolk, inequality of division necessarily results. It is termed simply unequal cleavage (Fig. 61). Finally in cases where the amount and density of the yolk is very great, as in many Fishes and Birds, that part of the egg which contains it does not cleave at all, or only very slightly. In such eggs, as already noted, the yolk-free cytoplasm exists only as a small accumulation at the animal pole of the em called the blastodisc. It is then chiefly this disc which divides; after

DO’ division it is called the blastoderm. Cleavage of this type is known as

meroblastic, or discoidal (Figs. 34, 35).

The Blastula.-— After cleavage has continued for a time in an egg of the homolecithal type a hollow sphere of cells results, with a cavity___ at or near its center (Figs. 33, g; 36, A). Such a sphere is called a blas- 7 tula, and the cavity at its center is termed the segmentation cavity, or blastocoel. In eggs of the markedly telolecithal type there also exists at

' the completion of cleavage a sphere, but in this case, as has been noted,

the greater part of it consists of undivided yolk. It is nevertheless termed a blastula, and the segmentation cavity will lie at the animal pole between the largely unsegmented yolk mass and the blastoderm (Figs. 35, D, E; 37, A). Although cell division continues the cleavage stage may be said to end when the blastula condition has been reached.

GASTRULATI ON

Gastrulation, as the name implies, has to do with the formation of the primordial gastric or gut cavity called the archenteron. In many cases this cavity is entirely separate from the blastocoel from the beginning of its formation, but in others complete separation comes later. In any event in addition to the formation of the gastrular cavity the process also usually involves the setting‘ apart of two of the three primordial germ layers with which all higher animals start their differentiation. These first two layers are sometimes referred to as the ectoderm and enzloderm, the former being on the outside and the latter lining the archenteron. This, however, is not quite correct because the thirdlayer, called mesoderm, to be referred to presently, is necessarily derived from one or the other or both of the two already formed. Hence at least one of these is really ‘more than ectoderm or endoderm for it contains the 54 FERTILIZATION, EARLY DEVELOPMENT

elements of the mesoderm. Therefore the one from which the third is derived in cases where this origin is clear, is often temporarily termed mesectoderm or mesentoderm as the case may be. Another pair of terms frequently applied to these two layers are eptblast for the outer layer and hypoblast for the inner one. These terms are noncommittal so far as indicating which is to give rise to mesoderm, and it is therefore convenient to use them, up until the time that this last-named layer appears. After that each of these layers can be referred to by its definitive name, ectoderm, mesoderm, and endoderm. This is the procedure which will be followed in this text. It should be further added that in some Invertebrates, like the earthworm, the mesoderm actually arises before gastrulation by the budding off of cells into the blastocoel. After this budding off of the mesoderm, the remaining wall of the hlastula might then be called ectoendoderm, since it is this wall which later becomes differentiated into definitive ectoderm and endoderm during gastrulation. Among Vertebrates, however, events appear to be always in the order indicated. _

Gastrulation having been thus defined, it now becomes necessary to indicate briefly and in a general way the processes through which it may occur. For the sake of clearness and convenience these processes will be described separately, though it should be noted that in the majority of actual cases two and often more of them take place together.

Invagination. — Probably the simplest method of gastrulation is by invagination, a method which is sometimes spoken of as being typical. As_a matter of fact, however, the accomplishment of gastrulation by this means alone is rather exceptional even among the Invertebrates, and among the Vertebrates it never occurs to the exclusion of other methods. Indeed within the latter phylum it is found in a relatively unmodified form only among a few of the very lowest members of the group. In all the higher animals it is very largely altered and aug ’mented by other means, and in many instances appears not to be present at all. In its simplest and most unmodified condition, however, it may be described _thus:

Let the blastula be thought of as a hollow sphere, "one hemisphere of which is to be regarded as the animal half and the other hemisphere as the vegetal half, while the cavity within the sphere is the blastocoel ( Fig. 33, g; 36, A). Now, imagino the vegetal half.to be pushed in or invaginated until it almost touches the animal half opposite to it. The sphere has thus become a gastrula. The original blastocoel has been virtually obliterated and a new cavity has been formed by the imaginaGASTRULATION M 55

tion. This is the archenteron, and it is lined by the original vegetal cells which may now be termed hypoblast (Figs. 33, k; 36 B). The cells which constitute the animal hemisphere, on the other hand, are now called epiblast. The opening of the archenteric cavity to the exterior is then in this case the blastopore, and the rim of this opening the lip of the blastopore. It must be immediately stated, however, that only in, eggs of a relatively yolkless character, is the blastopore thus a wide-open orifice. As the amount of yolk increases it tends to fill both the archen


Fig. 36.—Diagrammatic representation of gastrulation by invagination. A. Ideal meridional section of a blastula. B. Ideal meridional section of a gastrula. (1. Animal pole. arrh. Archenteron. blast. Blastocoel. bl. Blastepore. ep. Epiblast. hyp. Hypoblast. lp.b. The lip of the blastopore or germ ring. 21. Vegetal pole. The cells at the vegetal pole are usually larger because they contain more yolk.

teron and its opening more and more, until in eggs of the extremely tel- t

olecithal type there is very little left of the archenteron as a cavity or of the hlastopore as an openinu. Thus in eggs of this sort the boundary of the blastopore, i.e., the blastoporal lip, is really the edge of the blastoderm. To cover all cases, therefore, it is perhaps better to describe the lip of the blastopore as the line of undifferentiated tissue where epiblast and hypoblast merge with oneanother. This description it will be found applies to the edge of the hlastoderm asxwell as to the rim of a blastopore which possesses a wide opening, It» may now be added that the lip of the blastopore is also often called by another name, i.e., the germ ring. The reason for this is the fact that it was once thought that a very large portion of each side of the embryo always originated from this ring in a manner to be described below (see concrescence). A further word will be said on this topic when the latter process is discussed. 56 FERTILIZATION, EARLY DEVELOPMENTT

Invc1ution.——A second process of gastrulation may be described as involution or inflection. It is very common among the Vertebrates, and, within this group at least, it probably always accompanies any invagination which may occur. In many cases also it appears to be the chiefifactor involved, particularly among forms arising from a telolecithal egg. Therefore we shall study involution in a telolecithal egg.


Fig. 37.— Diagrammatic representation of gastrulation by involution in the case of an egg with a large yolk mass which does not segment. A. Ideal meridional section of a blastula. B. Ideal meridional section of a partially completed gastrula, bisecting the dorsal blastoporal lip. arch. Archenteron. blast. Blastocoel. bld. Blastoderm. ep. Epiblast. hyp. Hypoblast. lp.b. The

lip of the blastopore. The arrow points to the blastopore, and indicates the movement of involution.

In such eggs it has been noted that the yolk usually does not segment at all, and that in correlation with this the blastocoel will be greatly reduced (Fig. 37, A). Under such conditions it is evident that gastrulation cannot occur by simple invagination because the mass of yolk filling the center of the blastula will not permit it. What does happen, therefore, is this: At some point on the edge of the blastoderm (see above), the dividing cells, instead of extending out over the unsegmented yolk, begin to be turned over the blastodermal rim, i.e., involzited into the segmentation cavity. These inturned cells then constitute the hypoblast, while those which remain without are epiblast ( Fig. 37, 3). According to definition, therefore, the edge of the rim, in this case the edge of the blastoderm, is the blastoporal lip or germ ring, while the movement over this lip is designated as involution. As suggested above, however, this process is not confined to animals with a large yolk mass, and it is to be clearly understood, therefore, that the GASTRULATION 57

only essential feature concerned is the passage of cells over the lip. It is this movement, which, as stated, comprises involution, and this remains true whether the active cells be arranged in the form of a blasto

derm or otherwise. In some instances where the yolk mass is very great, as in many Fishes, the movement is accompanied by no invagination. In others (Amphioxus and Amphibians), the latter process also takes place to a greater or less extent. In any event the inflecuon or involu4

eplblast archenzeron infiltrating

blasroderm hypobhfl

,,- m.+-rat-s:a._,,“ V .


A B

Fig. 38.———Diagrammatic representation of gastrulation by infiltration in the case of an egg with a large yolk mass which does not segment. A. Ideal meridional section of a blastula as in Fig. 37. B. Ideal meridional section of a partially completed gastrula, showing some of the cells of the blaste derm creeping inside the former blastocoel, and spreading out there to form the hypoblast.

tion is most active in that portion of the blastoporal lip which eventually proves to be dorsal. The degree and character of its occurrence in other parts of the lip vary considerably in different animals, and can best be indicated later in specific cases. . Mechanisms Concerned in Invagination and Involution. —— Before proceeding to a description of the next methods of gastrulation, it seems well to pause here to consider the possible mechanisms involved in the processes already described. As has been indicated the essential feature in either invagination or involution is the movement of cells over or around an edge or lip. This type of movement, moreover, is an important aspect of various other cell rearrangements in embryology, as for example in the enterocoelic formation of mesoderm and the de velopment of neural folds to be described later. Hence an effort to discover the mechanism involved here has been one of the important points of attack by the experimentalists. What makes a ball of cells invaginate? What makes cells roll over a margin? The answer seems to be that it is due to a change in the shape of the cells as suggested long ago by Rhum. bler, Butschli, T. H. Morgan and others. This can be easily understood if one imagines a hollow ball of cells such as depicted in Figure 36. If one notes especially the bigger cells in this figure it is clear that they are larger atutheir outer ends. It is also clear that so long as they retain this shape :'t will be very difiicult or impossible for them to roll inward. If. however, the cells at 0116 P013 °f the 883a 0’ in the case Of tel‘



Fig. 39.—Diagrammatic representation of gastrulation by delamination. A. Ideal meridional section of a blastula, or as it is called in Mammals, the blastocyst. B. Ideal meridional section of a gastrula. arch. Arehenteron, as yet only partially lined by endoderm and lacking a blastopore. blast. Blastocoel. ep. Epiblast. hyp. Hypoblast. icm. lnner cell mass, virtually homologous with the blastodenn of blastulas with much yolk. rroph. Trophoblast, an embryonic layer peculiar to Mammals. t, See chapter XIV.)

olecithal eggs, at the blastoporal lip, should become smaller at their outer ends, the case would be difierent. Then their tendency would be to behave just as they actually do behave in invagination or involution, l.e., to 1‘0'll inward around the margin of a lip. That this is what occurs, seems now to be quite evident. The question remains, however, as to what makes cells in such situations change their shape. Here experiment is still seeking a complete answer. However, according to some investigators it is most probably due mainly to a higher alkalinity of the blastocoelic fluid. This in turn causes a change in surface tension in different regions of the cell membranes of the cells concerned (Holtfreter, ’44, Lewis, ’47). Thus if the tension at the inner ends of these cells became relatively less than at their outer ends, the inner ends would tend to become larger. One can and must of course then go still further back and ask why the tension changes in these cells and not in GASTRULATION I 59

others. This and related questions have not all been satisfactorily an swered, but their asking points the way in which investigation must proceed.

Infiltration. ——Heretofore this process has not been recognized as

.a method of gastrulation. Recent investigations on the Chick, however,

seem to indicate that possibly such a term is appropriate to describe what takes place there and'perhaps also in the Mammal. In any case it involves simply the inwandering or infiltration of cells from the blastederm, or its homologue, into the space beneath (the blastocoel). This space may or may not be largely filled with yolk. In the Chick of course it is so filled, while in the Mammal it is not. In either event the cells thus originating soon spread out to form a continuous layer of» hypoblast, and the former blastocoel becomes the archenteron. The infiltration process, if and where it occurs, is, like invagination and involution, probably -due to the change in shape of some of the cells of the original layer. Each cell concerned, becoming larger at its inner end, tends to form, as it were, a sort of pseudopodium, and crawl into the blastocoel (Fig. 38).

Delamination. —= A fourth process by which gastrulation may occur is that of delamination, and so far as Vertebrates are concerned it has

been supposed to take place most typically in Mammals. However, just _

as. infiltration may be involved to some extent in this group, so may delamination occur to a certain degree in the Birds. According to Brachet, delamination of a sort also plays a small part in a rather special manner in the gastrulation of the Amphibian. This "will be considered more fully when we come to the Frog. At any rate the process, wherever it may occur, consists simply in the separation or splitting ofi' of cells from a pre-existing layer or mass. These cells then become confluent, as in the case of those derived by infiltration, to form the hypoblast (Fig. 39).

It should be noted that where gastrulation occurs wholly, or almost wholly, by either infiltration or delamination, or both, no real blastepore exists, at least at first, and hence apparently there can be no blasteporal lips. It will be recalled, however, that the blastoporal lips have

been defined in general as the region where the epiblast meets and

merges with theyhypoblast. Furthermore it may be stated that even in

the cases of gastrulation almost or wholly by infiltration or delamina- .

tion the epiblast and hypoblast do ultimately come_into cgntact around the rim of the blastoderm, and also in another region to be noted later.

Hence the essential part of the definition of a blastoporal lip stillholds _ 60 FERTILIZATION, EARIY DEVELOPMENT

for both the places referred to. This problem will be discussed at greater length when the cases of the Chick and the Mammal are reached.

Accessory Processes. —-Two other processes are probably always to some extent involved in gastrulation, and in most instances are of considerable prominence. As will presently appear, however, these movements, at least among Chordates, are not strictly a part of gastrulation

1 1


Fig. 40.—Diagrams illustrating four stages in the formation of the Teleost embryo (having an extremely teiolecithal egg), and the growth of the germ ring or lip of the blastopore around the yolk mass (epiboly). From Kellicott (General Embryology). After 0. Hertwig. e. Embryo. gr. Posterior margin of the germ ring. y. Yolk mass. 1, 2, 3, 4, successive positions occupied by the germ ring as it advances over the yolk. proper;'i.e., they do not actually differentiate hypoblast from epihlast, though they aid in the extension and disposition of both these layers. Hence they may be more correctly regarded as accompanying or accessory _ activities. I. E piboly.——This is the first of these accessory movements, and occurs most typically in the development of eggs possessing abundant

yolk, e.g., those of Fishes and Amphibians. It merely involves the grad ? ual growth of the blastoporal lip over the yolk, or the yolk-filled vege tal cells. It pray be roughly pictured (Fig. 40) by imagining a solid sphere, the yolk, over which a rubber cap, the blastoderm, is being stretched, the rim of the cap representing of course the lip of the blastopore. The movement,

however, is not due ap parently to any actual '

process of stretching, but rather to active cell division in the overgrowing layers, and this activity is thought to be most intense in the region of the lip itself, i.e., the germ ring. It may be also that in this case, too, the movement is augmented by surface-tension changes which produce a creeping of the cellular rim over the yolk. At all events the result of such a process will obv1ously be eventually to enclose the yolk as in a sac (the yolk sac); the completion of this process necessarily involves also the closure of the blastopore (Fig. 40). II. Concrescence and Convergence. The process of concrescence as contrasted with that of convergence is one whose

occurrence, - as previously suggested, is now seriously ques tioned. At least this is


Fig. 41.——Diagram of the formation of an embryo

by confluence (concrescence). From Kellicon (General Embryology). A. Germ ring before formation of the embryo is indicated. The letters a-e represent symmetrical portions of the germ ring. B. Beginning of confluence. C. Embryo forming. AA, BB represent regions of the embryo formed out of the materials of the germ ring at aa, bb. D, E. Later stages in the formation of the embryo. The germ ring regions cc and dd, have been differentiated into the embryonic regions, CC, DD.

true with the conception of it originally held. Nevertheless in order to understand what is now believed it seems best to indicate the essentials of the original theory. It may be described thus. As the process of epiboly goes forward there always results, as noted, a gradual drawing together of the blastoporal lips, so that the size of the blastopore itself is dimin62 FERTILIZATION, EARLY DEVELOPMENT

ished. Furthermore, in the course of this procedure there is not, contrary to what might be expected, any noticeable puckering or thickening of the lips as their circumference decreases. This fact may be readily accounted for by assuming that much of the material which they contain is required to furnish the layers which they are leaving behind them. Aside from this, however, there was held to be another source for the consumption of at least part of the surplus substance of the germ


Fig. 42.———-Diagrammatic representation of the process of convergence as contrasted with that of confluence or concrescence illustrated in Fig. 4-1. .4. Surface view of the blastoderm at the beginning of the process. B. Asimilar view near the completion of gastrulation. Note that here most of the originally marginal material indicated by the letters, has simply moved medially and slightly posteriorly, i.e., has converged toward the median line of the future embryo and toward the dorsal hlastoporal lip. Some of it represented by letters a and b has been involnted over the dorsal blastoporal lip, and hence is no longer visible. “Invisible” letters shown in dots.

ring. Thus as gastrulation proceeds it was thought that the two sides of the germ ring were flowing together at a certain point upon the margin of the blastoderm, this movement being aptly designated as confluence or concrescence (Fig. 41). In this manner, as previously noted, it was held that each side of the original ring actually came to form a lateral half of the axial structures of the embryo. Thus the halves of the ring or blastoporal lips could be thought of as the “ germ ” of the future embryo, and hence the name germ ring. The theory was originally applied more especially to telolecithal eggs with a very large yolk as the description and figures suggest. It was not, however, confined to these types. ‘

The present View is that actual concrescence in the manner just described is very limited. Indeed in no case can the complete side of the axial structures of an embryo be said to arise in this manner from a half of the blastoporal rim. Actually what seems to happen in most MESODERM AND CO‘-ELOM 63

cases‘ is more in the nature of a flow of material from each entire posterior half of the blastoderm toward the median line and to some extent over the dorsal blastoporal lip (involution). In this manner, much of the substance forming the axial structures of the embryo is brought into its definitive position (Fig. 4-2) . This process of movement toward the median line may perhaps he aptly described in part as convergence, but hardly as concrescence in the original sense, and the latter term is now seldom used. Also in correlation with this point of view it seems scarcely appropriate any longer to speak of the blastoporal lips as the germ ring. This is because, though materials destined for certain parts do, as we have said, pass over the lips, these materials are not, to any great extent, actually furnished by them. Nevertheless the term is still

employed by many embryologists especially in’ connection with telolecithal eggs.

FORMATION OF MESODERM AND COELOM

All animals whose tissues are formed from three fundamental cell layers are said to be triblastic. The Chordates belong to this group and therefore, as already indicated, possess a third embryonic layer, the mesoderm, which eventually lies between the other two. The source of this layer has also been mentioned, and it was stated that among Vertebrates it always arises from one or both of those previously differentiated by gastrulation. After its emergence as a separate layer the three primary layers may then, as noted, he definitely referred to as ecto derm, mesoderm and endoderm. It is now necessary to describe the ways '

in which mesoderm may arise. There are four chief methods, and the first is rather intimatelyconnected with the origin of the coelom. The

’ remaining three, as we shall see, are not quite so closely correlated.

I. The Enterocoelic Method.——This method, though common among certain Invertebrates, occurs in connection with only a few of the lowest members of the Chordate phylum. In its general aspects it may be described thus: Along each side of the archenteron in its dorsal region there arises from the hypoblast a longitudinal outpushing or fold lying between the epiblast, now ectoderm, and hypoblast, now endoderm. This is indicated diagrammatically in Figure 43, A. Later each fold develops a space between its two layers as shown in the diagram. Then, as a result of the downgrowth of the folds on either side, the two spaces presently meet ventrally and fuse (Fig. 43, B"). The common cavity thus formed is the coelom, and its lining is mesoderm. The lining next to the ectoderm is called somatic’ mesoderm, and this somatic 64 FERTILIZATION, EARLY DEVELOPMENT

Fig. 43.—A diagram of the origin and early differentiation of the mesoderm, and of the notochord and nerve cord in a generalized Vertebrate.

A. The mesoderm is arising by means of enterocoelic pouches which are pushing out from the archenteron and are not yet separated from it. B. The enterocoelic pouches have separated from the archenteron, their walls forming the splanchnopleure and somatopleure, and their cavities the coelom. The notochord is beginning to develop and the meclullary folds are approaching each other. C. The regions of the vertebral plates, which are divided transversely into somites, the nephrotomes and the lateral plates are marked out, and the various parts of the somites are distinguishable. D. The closing of the neural tube or nerve cord is completed. The somites are further developed and the myocoel is nearly obliterated. The notochord is separated from the archenteron, and the mesentery has formed. The pronephros or embryonic kidney is developing from the neph~ rotome.

coel. Coelom. dt. Dermatome. ect. Ectoderm. e.ca. Enterocoelic cavity. end. Endoderm. lp. Region of the lateral plate. me. Myocoel. mf. Medullary folds. mg. Medullary groove. mp. Medullary plate. mes. Mesoderm originating in th1s case by the enterocoelic method. mest. Mesentery. mt. Myotome. nc. Neural canal. neph. Region of the nephrotome. neph.c. Region of the nephrocoel. not. Notochord. nt. Neural tube or nerve cord. prn. Rudiment of the pronephros or embryonic kidney. 5. Region of segmental or vertebral plate (somites). scl. Sclerotome. s.mes. Somatic mesoderm. spl._mes. Splanclmic mesoderm. MESODERM AND COELOM 65

mesoderm with the adjacent ectoderm are sometimes referred to together as the somatopleure. The lining of the coelom next to the endoderm on the other hand is called splanchnic mesoderm, and it together with the adjacent endoderm may be designated as splanchnopleure. In this case it will be noted that it was the hypoblast which gave rise to the mesoderm. Hence in this instance the hypoblast would be mesentoderm if one were using that terminology. Meanwhile dorsally the splanchnic mesoderm from either side has pressed in above the endoderm and has fused to form a double sheet of tissue called the mesentery. Thus the enteric canal or enteron, formally the archenteron, is, so to speak, slung from the dorsal wall of the coelomic cavity by this sheet.

It remains to be observed that, despite the rarity of this method of mesoderm formation among the Chordates, it is regarded nevertheless as of considerable zoological interest. The reason for this is the fact. already suggested, that it is found abundantly in some of the large Invertebrate groups (e.g., the Echinoderms and Prosopygia), and is then repeated among the lowest Chordata. This is significant because such repetition in these members of the Chordate phylum is suggestive in helping to determine from which class of Invertebrates the Vertebrate group arose. a

II. The Method of Delamination.—The production of a cell layer by a method whose essential feature was a splitting off or delamination of cells has already been noted in connection with the diiTerentiation of the first two layers. It now remains to be stated that a similar process is quite frequent among Vertebrates with respect to the generation of mesoderm. Here again the layer from which the mesoderm arises is the hypoblast, only in this case the origin is by splitting of? instead of evagination (Fig. 44, A). Later the coelom forms by still another split within the mesoderm itself, giving rise as before to a somatic and splanchnic layer. The relations of these somatic and splanchnic layers to the body wall and to the enteron and the subsequent development of other parts are the same as in Method I.

III. The Method of Pro1iferation.—This method involves simply the budding OH of cells from the sides of a linear thickening in the outer of the two primary layers (epiblast), along what will be the longitudinal axis of the future embryo. This thickening in these cases is termed the primitive streak, of which more will be said in connection with specific forms, and the cells budded from its sides soon spread out between the two primary layers, and constitute the mesoderm (Fig. 44-. B). Presently as in the previous cases this mesoderm splits into two 66 FERTILIZAT ION, EARLY DEVELOPMENT

mesoderm

endoderm


ectoderm

I I ‘endodcrm

Fig. 44.--Diagrams illustrating three other methods of mesoderm origin. A. Method II, delamination, shows mesoderm split ofi from the underlying hypoblast. It is characteristic of the Frog and other Amphibians. 13. Method III. proliferation, shows mesoderm budding off from a median longitudinal thickening of epiblast, the primitive streak. This was formerly supposed to be characteristic of the higher Vertebrates. C. Method IV, “invagination” or involution, shows mcsodernt being involuled through the primitive streak from the overlying epiblast. This is now thought to be the method in Birds, and probably in the other higher Vertebrates. In all cases the single layer of mesoderm later splits into two with the coeiom between them.

sheets. As usual the one next to the outer layer, now ectoderm, is somatic mesoderm, and that next to the inner or endodermal layer is splanchnic mesoderm with the coelom between them. It is to be noted that it is only in this last instance that the epiblast rather than the hypoblast gives rise to mesgderm. Hence on the basis of the older terminology the mesoderm in this case is mesectodermal in origin.

The method just described is one which has been supposed to prevail generally among the highest Vertebrates, i.e., the Birds and Mammals. According to the most recent evidence, however, it now seems probable that it plays little if any part in the Birds, and quite possibly this is SOURCES OF TISSUES 67

also true of the Mammals. Instead, considerable rather convincing evidence has been produced by Spratt, ’4-6, in the case of one bird, the Chick, in support of a fourth process. This will be discussed in some detail in our description of mesoderm formation in that form, but to make the record complete it must be briefly indicated here.

IV. The Method of “ Invagination.”—This is a term which it will be recalled was used in connection with gastmlation, and indeed the process as envisaged here is essentially the same as that which is sometimes employed to describe a similar activity in endoderm production. Again as in that case, however, the writer feels that involution would be a better word to use. In fact in this case the process may be even more accurately described as a sort of combination of involution and infiltration. What is said to happen is simply this: The epiblast cells from either side of the blastoderm which aggregate along a line to form what we have designated as the primitive streak, do not remain here. Instead many of them continually move ventrad through the streak and spread out on either hand to become mescderm (Fig. 44, C). Thus again this mesoderm might be called mesectodermal in origin.

THE SOURCES OF THE TISSUES

The three embryonic cell layers having thus been defined and their origin described, the subject may be concluded by indicating in a gem eral way the tissues to which each cell layer eventually gives rise.

1. The ectoderm produces the epidermal part of the skin, includ~ ing cutaneous glands, hair, feathers, nails, hoofs, and one type of horns and scales. It also gives rise to parts of the eye and of the internal ear, and the lining of the anus and oral cavities, including the enamel of

the teeth. It is the origin of the entire nervous system and a few muscles. .»

2. The mesoderrn gives rise to most muscles, as well as to adipose tissue and all other varieties of connective tissue including the dermis, certain types of scales and horns and the main portion (dentine) of the teeth. It also produces the skeletal system, the blood vascular system, and the greater part of the urinogenital system. It forms the coelomic epithelium, mesenteries, the outer layers of the alimentary tract, the Eustachian tube, and sometimes lines the middle ear.

3. The endoderm produces the lining of the alimentary tract and the epithelial parts of all the organs which arise as outgrowths from, it; i.e., the respiratory system, the thyroid and thymus glands, the liver, and the pancreas. It also lines the middle ear in some cases, and forms a small part of the urinogenital system 63 FERTILIZATION, EARLY DEVELOPMENT

THE NOTOCHORD

A characteristic feature of the embryos of all true Chordates is a rod of vacuolated tissue lying along the mid-dorsal line just above the gut. It is termed the notochord, and makes its appearance at about the same time at which the mesoderm starts to develop, or in some instances somewhat later. It is clearly derived in many cases from the dorsal wall of the archenteron, i.e., it is hypoblastic (Fig. 43, B, C, Di . In some instances, however, e.g., in Birds and Mammals, the origin of the notochord is apparently partially or entirely epiblastic. The position which the structure occupies is obviously that which is taken by the vertebrae of the higher adult Chordates, i.e., the genuine Vertebrates. As will appear, the bony structures which thus replace the notochord in the latter animals arise from certain of the mesodermal tissues which surround it, while the notochord itself is gradually absorbed.

Relation of Notochord to Germ Layers.—As has been indicated, in triblastic animals all tissues and structures are supposed to be derived from one of the three primary layers. The question frequently arises therefore as to just which layer the notochord belongs. As noted it is, like the mesoderm, derived from either epiblast or hypoblast. Yet it frequently originates, in some cases partly, and in other cases entirely, separately from the mesoderm. If one is to be consistent and stick to the three-layer idea, it is probably most logical to regard the notochord as a sort of specially derived mesoderm. Otherwise it becomes a kind of embryological orphan which no layer will own. A rather common method of avoiding this dilemma of nomenclature, however, is to refer to the third layer and notochord together as chorda.-mesoderm. Thus the intimate relation of notochord to mesoderm, as well as their semi-independent status, are both suggested in one compound term.

THE LATERAL PLATES, THE SOMITES AND THE NEPHROTOMESM

Among all the Chordates, except in the case of a few of the most primitive members of the group, there accompanies or immediately follows the development of the coelom, certain other fundamental differentiations of the mesoderm. These differentiations result in the formation of threemajor divisions of this substance, whose origin and character may be described in a general way as follows:

I. The Lateral Plates.——It has already been suggested that the main portion of the mesoderm upon each side of the animal gives origin LATERAL PLATES, SOMITES, NEPHROTOMES 69

to the coelom and its lining. It remains to state that each of these portions is frequently known as a lateral plate.

II. The Vertebral Plates. — The mesoderm which is not involved in the production of the lateral plates, nevertheless remains connected with them for a time, lying dorsally along either side of the notochord and nerve cord in the form of a relatively narrow band, a vertebral or segmental plate (Fig. 43, C). The major portion of each band (i.e., all of it, save a narrow strip connecting it ventrally with the respective lateral plate) then thickens somewhat, and soon begins to be transversely divided into a series of block-like masses termed somites. The more anterior members of the series usually appear first, and each one as it is formed proceeds to give rise to three fundamental elements: the dermatome, the myotome, and the sclerotome (Fig. 43, C, D). Of these elements the relatively thin dermatomes lie next to the ectoderm, and are concerned chiefly in‘ the production of the deeper layer of the skin, i.e., the dermis. The thicker myotomes come beneath and median to the dermatomes and give rise to the bulk of the voluntary muscles, While the sclerotomes, arising as proliferations of scattered cells, are nearest the notochord and produce the skeletogenous tissue of the axial skeleton.

It may be further remarked that in many instances at this period a small portion of the coelomic space extends up into each somite between the dermatome and myotome, and is there known as a myocoel. Like the connection between the somites and the lateral plates, however, it is of only temporary duration.

In Amphioxus, one of the very primitive Chordates referred to above, it should be noted that the term somite as used in the early history of this animal is somewhat more inclusive than in the foregoing description. Thus in this instance these bodies when newly formed, comprise not only the elements of the dermatomes, myotomes, and sclerotomes, but likewise those of the lateral plates. It may finally be added that since there are no bones in Amphioxus, the sclerotomes give rise only to connective tissue.

III. The Nephrotomes. —— It will be recalled that of each band of mesoderm lying between the lateral plate and the notochord, the major dorsal portion goes to form the somites. The remaining narrow strip, which for a time connects these bodies with the corresponding;-;lat)eral plate, is then designated as the nephrotome or internzpg§‘£dt£,cellqnjqé§, while its cavity, temporarily uniting the main coelomvanidghe myocoe"l~s,'L_,=,‘-\1

is the nephrocoel (Fig. 43, D). The nephrotomfisfsliter coptrihutej ca . \,_ _ “.s.'5“" chiefly to the formation of the excretory organs. ‘arr (K M... J L

J ," '‘‘‘*hm-mv-’‘ 70 FERTILIZATION, EARLY DEVELOPMENT

In Amphioxus and the other primitive Chordata no nephrotome exists, and the excretory organs are therefore of an entirely different

character and origin.

THE DORSAL NERVE CORD

The final fundamental feature of Chordate anatomy which appears in connection with these very early embryonic stages is the dorsal nerve cord or neural tube. The latter term is used not only because it indicates a characteristic of this structure which is peculiar to Chordatcs, but also because it suggests the method of its development, which is likewise peculiar to this group. This method is as follows.

Shortly following the processes of gastrulation, and more or less concurrent with the process of mesoderm formation. a broad strip of ectoderm along the future dorsal side of the animal becomes thickened. This thickened area is termed the medullary or neural plate (Fig. 43, A). The median portion of this plate then becomes depressed slightly to form a groove, the medullary or neural groove, while the sides are correspondingly elevated as the medullary or neural folds (Fig. 43, B). These folds gradually grow toward one another until their crests meet and fuse, and there is thus developed a tube, which presently becomes entirely separated from the ectoderm above it (Fig. 43, C, D) . This is the rudiment of the nerve cord or neural tube, while the canal which traverses its center is the neural canal or neurocoel.3 At its anterior end this canal opens to the exterior for a time through a small aperture, the neuropore. At the posterior end, on the contrary, the fusion of the medullary folds eliminates the external opening (except in some Sauropsids and Mammals) at an early stage, but preserves an internal passageway as follows. Instead of stopping dorsal or anterior to the nearly closed blastopore, the above folds extend slightly downward or backward upon either side of it. They then fuse above the latter orifice in such a way that through it, for a considerable time, the neurocoel communicates with the enteric cavity. The short bent portion of the neurocoel in this particular region, together with the remains of the blastepore, is then known as the neurenteric canal (Fig. 53) .

The process thus described has already been indicated as character 3 This method of nerve cord formation is, as noted, characteristic of most Vertebrates, but is modified somewhat in the case of the Lampreys and many of the Teleost Fishes. Thus in these animals no grove is formed in the thickening medullary plate. Instead the latter simply presses downward beneath the surface as a solid cord of tissue. The neural canal then arises later within this cord by the separation or disintegration of the cells along its axis (Fig. 144). REFERENCES TO LITERATURE 71

istic of all true Chordates, and as regards all fundamental points this is true. 11; should be stated, however, that once more in the case of

Amphioxus certain minor variations occur. These will be considered in connection with the development of that animal.

REFERENCES TO LITERATURE

Abbreviations for the names of periodical publications referred to in the literature cited at the ends of chapters are as follows:

Am. Anat. Mern. = American Anatomical Memoirs, Philadelphia.

Am. _l011X'- Aflat = American Journal of Anatomy, Baltimore and Philadelphia.

Am. Jour. Obstet. and Gynec. = American Journal of Obstetrics and Gynecology, St. Louis.

Am. Jour. Physiol. = American Journal of Physiology, Boston.

Anat. Anz. == Anatarnischer Anzeiger, Jena.

Anat. Hefte = Anatomische He/te, Wiesbaden.

Anat. Rec. = Anatomical Record, Philadelphia.

Arbeit. zool. Inst. Wien. = Arbeiten aus dcm zoologischen Institute zu Wicn.

Arch. Anat. u. Entw. = Archiv fiir Anatomic und Entwickclungsgeschichtc, Leipzig. { Same as Arch. Anat. u. Physiol.)

Arch. Anat. u. Physiol. = Archiv. filr Anatomic und Physiologic, Leipzig.

Arch. Biol. = Archives de Biologic, Leipzig and Paris.

Arch. d’Anat. Micr. = Archives d’Anatomie Microscopiquc, Paris.

Arch. Entw.—mech. = Archiv fiir Enzwickelungsmechanik der Organismen, Leipzig.

Arch. mikr. Anat. =Archiv fiir mikroskapischc Anatomic und Entwiclcelungsgeschichtc, Bonn.

Arch. Zellf. == Archiv fiir Zellforschung, Leipzig.

Arch. Z001. Exp. = Archives de Zoologic experimentalc ct gencrale, Paris.

Aust. J. Exp. and Med. Sci. = Australian Journal of Experimental Biology and Medical Science, Sydney.

Biol. Bull. = Biological Bulletin, Woods Hole, Mass.

Biol. Centr. = Biologisches Centralblatt, Leipzig.

B. M. C. Z. Harvard = Bulletin of the Museum of Comparative Zoology at Harvard College, Cambridge, Mass.

Bull. Soc. Impér. Moscou-=Bulletins dc la Societe Impériale de Natural—— de Moscou.

Carnegie Cont. to Emb. = Carnegie Institution. Contributions to Embryology, Washington.

Carnegie Inst. of Wash.=Carnegie Institution of Washington.

Cold Spring Harbor Symp. on Quant. Biol. Cold Spring Harbor Symposia on Quantitative Biology, Cold Spring Harbor.

C. I‘. Soc. Biol. Paris = Comptes rendus des séances ct mémoires de la Société de Biologic, Paris.

Deutsche Thieraerztliche Wochenschr.=Deutsche Thieraertzliche Wochenschrift, Karlsruhe.

Ergeb. Anat. u. Entw. = Ergebnisse cler Anatomic und Entwiclcelungsge schichtc, Wiesbaden.

Festsch. f. Gcgenbaur = F estschrift fur Gegenbaur, Leipzig.

Intern. Monatsschr.=Intcrnationale Monazsschrifz fiir Anatomic and Physiologic, Leipzig. 72 FERTILIZATION, EARLY DEVELOPMENT

Jena Zeitschr. = Jenaisclze Zeitschrift fz'ir‘NaturIz;isse_ns§l,l1léf¢, 1:113-B If r Johns Hopkins H°sp_ Rep_:]ahns Hopkins ospzta epor s, ‘a xmo_e.

A M d Assn. = Journal of the American Medical Association, Chicago. J°“" m‘ e ' - ' ,4 1 Plzvsiolo r London jour. Anat. Phys1o1.=JourII!I1 of 7"”0’"}’ ax’) -I ¥’i’5{_I'd 1 1:’ Joan Comp» New-=10“’"“’ "f 60"’-”""mzez ("lira ogii 1:11” E .p..1iid'Pi.z1ade1 Jour. Exp. Zo61.=Journal of Expenmenta oo 05)’. 3 H10“? 4 1' . . J°ml.3.n1l1/[O,ph_= foumgz of Morphology or Journal of Jlorphology and Pll_}‘5l 1 ~, Ph'l dl l'a. _ . . _ Jamil 05'lniv_ l1?01:;on.—= journal of the College of Science, Imperial University of Kg1T0S]:i::Ji1sk. Vet. Hand1.=Kongliga Svenska Vetenskapsokadenzie, Ablzantl»

lungen aus der Naturlehre, Leipzig: , ' ’ ' Mérn. Acad. Impér. St. P. =1lIémoLres cle l Acadernze Irrzperzale de St. PetersMenl.:ouAi‘ad_ 1.oy_ Be1g_ = Mémoires de l’Actzdemie royale tie Belgique. Mem: Boston Soc. Nat. Hist. = Memoirs of the Boston Society of Natural HisMéI'llJ.r}l,‘l. Y. Acad. Sci. = Ménzoirs of the New York Academy of Science?’ Mitt, zool. Stat. Neapel = Mzttezlungen aus der zoologzschen Station zu 1 cape], Berlin. Morph. Arbeiten. = .-llorphologisclte Arbeiten, Jena. Morph. Jahrb. = Morpholagisczes JahrBbui:h, Lexpzlg. Nat.-wiss. = Die Naturwissensc a. ten, er in. _ Phil. Trans. Roy. Soc.=PhilosophicIzl Transactions of the Royal Soczety of London. Physiol. Rev. = Physiological Reviezvs, Baltimore. . Physio}. Z061. =Physiological Zoology, Phllarlelphxa. ‘ Proc. Am. Acad. Arts and Sci. =Proceedzngs of the American Academy of Arts and Sciences, Boston. _ I . _ Proc. Am. Phil. .Soc.=l’roceedings of the American Philosophical Society, Philadelphia. d P d I I I _ 1

Proc. Internat. Cong. Z061. Camhri ge= rocee ings o (18 nternauona Congress of Zoiilogists, Cambridge. S I 1

Proc. Soc. Exp. Biol. and Med. =Proceedings of the ociety or Experimenta Biology and Medicine, New York.

Ptoc. Z061. Soc. = Proceedings of the Zoiilogical Society of London.

Q. J. M. S. = Quarterly Journal of Microscopical Science, London.

Quart. Rev. Biol. = Quarterly Review of Biology, Baltimore.

S. B. G. M. P. = Sitzungs-Berichte zler Gesellschaft fiir tllorphologie und Physiologie, Miinchen.

Sitzber. Ber. Akad. = Sitzungsberichte der Koeniglich Preussisclzen Alratlerizie tier Wissenschaft, Berlin.

Tijd. Nederl. dierk. Ver. ==Netlerlandsclze dierlcumlige Vereeniging, 7'1’/(L schrift, Leyden.

Trans. Am. Phil. Soc. = Transactions of the American Philosophical Society, Pliilaclelpliia.

Univ. Cal. Press. = University of California Press, Berkeley.

Verh. d. Anat. Gesell. = V erhandlungen der Anatomisclzerz Gesellschaft. Jena.

Ver. kon. Akad. Wetensch. = Verhandelingen lconirtklijke Al.-mlemie van Wetenscl-zappen, Amsterdam.

Verh. Phys.-Med. Ges. = Ferlzantlltzngen Physilmlisclze-Merlizinische Case!!schaft, Wurzburg. REFERENCES‘ TO LITERATURE 73

Zeit. Anat. Entvv. = Zeitschrift fiir Anatomic und Entwickelungsgeschicltte, Leipzig.

Zeit. ind. Abs. u. Vererb. = Zeitschrift fiir induktivc Abstammungs- und Vererbungslehre, Berlin. ‘

Zeit. Mikr.-Anat. Forsch.=Zeitschrifz fiir Mikroskopisch-Anaiomisc/1e F orschung, Leipzig.

Z001. Jahrb. = Zoologische Jahrbiicher, Jena.

CHAPTERS I AND II

Allen, B. M., “The Origin of the Sex-Cells of Chryemys,” Anar. Anz., XXIX, 1906.

Allen, Edgar, “ Ovogenesis during Sexual Maturity,” Am. Jour. Anat., XXXI, 1923.

Allen, E., Kountz, W. B. and Francis, B. F., “ Selective Elimination of the Ova in the Adult Ovary,” Am. Jour. Anat., XXXIV, 1925.

Babcock, E. B. and Clausen, R. E., Genetics in Relation 20 Agriculture, New York and London, 1918.

Benda. C., “ Die Mitochondria,” Ergeb. Anat. u. Entw., XII, 1903 (1902) . Bookhout, C. G., “ The Development of the Guinea Pig Ovary from Sexual Differentiation to Maturity," Jour. Morph., LXXVII, 1945. Boveri, Th., “ Die Entstehung des Gegansatzes zwischen den Geschlechtszellen und den somatischen Zellen bei Ascaris,” S.B.G.M.P., Miinchen, VIII, 1895. Bowen, R. H., “ Studies on Insect Sperrnatogenesis,” VI, “ Notes on the Formation ' of the Sperm in Coleoptera and Aptera, with a General Discussion of Flgellate Sperrns,” Jour. Morph. and Physiol., XXXIX, 1924. Biitschli, 0., Untersuchungen fiber mikroskopische Schaiime und das Protaplasma, Leipzig, 1392. Castle, W. E., Genetics and Eugenics, 2nd Ed., Harvard Univ. Press. 1920. Everett, N. B., “The Origin of Ova in the Adult Opossum,” Anat. Rec., LXXXII, 1942»-“ Observational and Experimental Evidences Relating to the Origin

and Differentiation of the Definitive Germ Cells in Mice,” Jour. Exp. Zool, LIXII, I94-3.

Flemming, W., Zellsubstanz, Kern und Zellteilung, Leipzig, 1882.

Geerts, J. M., “Cytologische Untersuchungen einiger Bastarde von Oenothera gigas,” Berichte Deutsche Botanisehe Cesellschaft, XXIX, 1911.

I Goldsmith, J. B., “The History of the Germ Cells in the Domestic Fowl,” Jour. Morph. and Physiol., XLVI, 1928. _ Goodrich, H. B., “ The Germ Cells in Ascaris,” Jour. Exp. Zo6l., XXI, 1, 1916. Hargitt, G. T., “ The Formation of the Sex Glands and Germ Cells of Mammals.” I. “The Origin of the Germ Cells in the Albino Rat,” Jour. Morph. and Physz'ol., XL, l92S.—-II. “ The History of the Male Germ Cells in the Albino Rat,” Iour. Morph. and Physiol., XLII, 1926.—-III. “ The History of the Female Germ Cells in the Albino Rat to the Time of Sexual Maturity.” -—- IV.

“ Continuous Origin and Degeneration of Germ Cells in the Female Albino Rat,” Jour. Morph. and Physial., XLIX, 1930.

Hertwig, A., Die Zelle und die Gewebe, Jena, I, 1893; II, 1898.

Holtfreter, 1., “ A Study of the Mechanics of Gastrulation,f' Part I, Jour. Exp. Zo6l., VIC, 1943.—-- Part II, Jour. Exp. Zool., VC, 1944.

Humphrey, R. R., “The Primordial Germ Cells of Hemidactylium and other Amphibia,” Jour. Morph. and Physiol., XLI, 1925. ~—“ Extirpation of the Primordial Germ Cells of Amblystoma: Its Effect_,,U_pg.n...the Development of the Gonad,” Jour. Exp. Zob'l., XLIX, I927.——i‘ The Early Position of the Pri mordial Germ Cells in_Urodeles: Evidence from Experimental Studies,” Anat. Rec., XLII, 1929. 74 FERTILIZAT ION, EARLY DEVELOPMENT

Jenkinson, J. W., “ Observations on the Maturation and Fertilization of the Egg of the Axolot ,” Q.J.M.S., XI, viii, 1904.-—Vertebrate Embryology, Oxford and London, 1913. ,

Jennings, H. B., “Paramecium hursaria. Life History. V. Some Relations of External Conditions, Past or Present, to Aging and to Mortality of Ex-conjugants, with Summary of Conclusions on Age and Death,” Jour. Exp. Zool., IC, 1945.

Kingsbury, B. F., “The Postpartum Formation of Egg Cells in the Cat,” Iour. Morph., LXIII, 1938.

Lewis, W. H., “ Mechanics of Invagination,” Anat. Rec., IIIC, 1947.

Lillie, F. R., Problems of Fertilization, Chicago, 1919.

McC1ung, C. E., “The Accessory Chromosome-— Sex Determinant?” Biol. Bull., III, 1902.

Meves, F., “Ueber Struktur und Histogenese der Samenfiiden von Salamandra,” Arch. milcr. Anat., I, 1897.

Moenkhaus, W. .l., “ The Development of the Hybrids between F tmdulus heterodirus and Mendidia notata, with Special Reference to the Behavior of the Maternal and the Paternal Chromatin,” Am. Jour. Anon, III, 1904.

Montgomery, T. H., In, “A Study of the Chromosomes of the Germ Cells of the Metazoa,” Trans. Am. Phil. Soc., XX, 1901.——“ On the Dimegalous Sperm and the Chromosomal Variation of Euschistus with Reference to Chromosomal Continuity,” Arch. Zellf , V, 1910.

Morgan, T. H., Heredity and Sex, New York, 1913. The Physical Basis of Heredity, Philadelphia, 1919. The Physical Basis of Heredity, Philadelphia and London, 1919. The Theory of the Gene, Yale Univ. Press, 1926.

Morgan, Sturtevant, Muller, and Bridges, The Mechanism of Mendelian Heredity, New York, 1915.

Oliver, J. R., “ The Spermiogenesis of the Pribilof Fur Seal (Callorhinus alascanus J. and C.),” Am. Jour. Anat., XIV, 1913.

Painter, T. S., “ Studies in Mammalian Spermatogenesis. II, The Spermatogenesis of Man," Jour. Exp. Zo6l., XXXVII, 1923.

Riddle, 0., “The Theory of Sex as Stated in Terms of Results of Studies on Pigeons,” Science, XLVI, 1917.

Rosenberg, 0., “Cytologische und morphologische Studien an Drosera longifolin X rotundifolia,” Kgl. Svenslc. Vet. Handl., 43, 1909.

Sharp, L. W., An Introduction to Cytology, New York, 1921.

Sinnott and Dunn, Principles of Genetics, New York, 1925.

Sneider, M. E., “Rhythms of Ovogenesis before Sexual Maturity in the Rat,” Am. Jour. Anat., LXVII, 194-0.

Strassburger, E., Zellbilzlung und Zellteilung (3rd ed), Jena, 1880.

Sutton, W. S., “On the Morphology of the Chromosome Group in Brachystola rnagna,” Biol. Bull., IV, 1902.

Van Beneden, E., “Recherches sur la Composition et la Signification de l’CEuf etc-a M97fl- 4604- 707- Belg», XXXIV, 1370.-—“ Recherches sur la Maturation de lfiluf et la F econdation,” Arch. Biol., IV, 1883.

Weismann, A., “Entstehung der Sexualzellen bei den Hydrornedusen,” Fischer Jena, 1885. ' Wilson, E. B., Atlas of Fertilization and Karyokinesis, New York. l895.——- The Cell in Development and Heredity (Columbia University Biological Series, IV,

}3g%I,e:’¢‘i;)i9l1‘I2¢=:w York, 1925.—-“Studies on Chromosomes,” four. Exp. Zool., HE EARLY DEVELOPMENT OF AMPHIOXUS

T H E early stages in the development of Amphioxus ( Branc/ziostomu. lanceolatum) are taken up because in this form these stages are thought to be as nearly primitive as those occurring in any other Chordate. This applies particularly to the method of segmentation, gastrulation, and formation of the mesoderm and coelom. Indeed the general resemblance of these processes to what occurs among Invertebrates, such as the Echinoderms, is so marked that their primitive character in Amphioxus can hardly be doubted. Also according to the most recent studies there is a marked and significant resemblance between the early stages of this animal and those forms sometimes designated as Protochordates, i.e., the Ascidians.

There are numerous accounts of the development of this classic form, some of the best known being those of Hatschek (1882, ’88), Wilson (1893), Cerfontaine (’O6) and the most recent that of Conklin (’32). The studies of the last named investigator, though agreeing in many respects with those of his predecessors, differ rather fundamentally in some of the earlier details. Since the work of Conklin is not only the most recent, but is supported both by elaborate observations of normal development, and by experimental procedures, it is believed to be the most accurate. It is therefore the one followed in this text except where otherwise indicated. It is assumed that the student has in mind a fair knowledge of the adult anatomy of the animal under discussion.

THE REPRODUCTIVE ORGANS THE OVARY

Since the work of Conklin does not cover very completely the character of the ovary and the process of oogenesisdthe following brief statements on these subjects are based on the account of Cerfontaine.

The ovaries are developed in each myocoel (Fig. 45) on both sides of the body from the tenth to the thirty-fifth or thirty-sixth sornite inclusive. Each originates as a proliferation of cells on the antero-ventral

5

{gs76 THE EARLY DEVELOPMENT OF AMPHIOXUS

Fig. 45.——Diagram of a section through the gonad of Amphioxus. From Kellicott (Chordate Development). After Cerfontaine. Right side adjacent to atrium.

b. Peribranchial (atrial) epithelium. c. Cicatrix. f. True follicular epithelium. fe. External layer of follicular epithelium. g. Gonocoel. ge. Germinal epithelium. 0;. Primary ovarian cavity. 02. Secondary ovarian cavity. pg. Parietal layer of gonocoel. v. Cardlinal vein. vg. Visceral layer of gonocoe .

wall of the myocoel. This proliferation then pushes forward as a small bud, covered by the portion of the myocoelic wall from which it arose. The bud of germ cells with its covering thus comes to project sac-like into the myocoel anterior to the one in which the proliferation started. The neck of the sac then forms a short stalk connecting it with the posterior myocoelic wall of the cavity into which the evagination has occurred. Thus in these animals each egg is not surrounded by its individual follicle, but is attached to the wall of the above sac, which acts as a general follicle for all the ova within it. As development proceeds, the most ventral part of each myocoel which contains the gonad is cut ofi” from the part above as the gonocoel. By the time a batch of ova is ripe, however, which occurs for the first time in animals about two centimeters in length, each ovary has grown so that it virtually obliterates all coelomic spaces surrounding it (Fig. 4-5) . These eggs are then extruded (see below),

while the ovary during the process almost disappears. It then develops anew in preparation for the next breeding season.

THE TESTIS

The development of the testes in Amphioxus is not so well known, but it appears to be similar in a general way to that of the ovary. The products are discharged to the outside as are the eggs. THE OVARY

Fig. 46.—The egg of Amphioxus. From Kellicott (Chardate Development). C. After Cerfontaine, others after Sobotta. A. The ovarian egg showing cortical plasm. The first polar body ls being pinched ofi, and the spindle for the second meiotic division is formed. B. The cortical layer forming the perivitelline membrane on the surface of the egg within the vitelline membrane. C. The fusion of the vitelline membrane and perivitelline membrane to form the fertilization membrane is complete, but the latter has not yet left the surface of the egg. D. The extruded and fertilized egg. The fertilization membrane is beginning to leave the surface of the egg.

c. Cortical layer. e. Endoplasm. m. Fused vitelline and perivitelline membranes, i.e., the fertilization membrane. p. Perivitelline space. s. Spermatozoiin. v. Vitelline membrane. I. First polar body. 11. Second polar spindle.

77 78 THE EARLY DEVELOPMENT OF AMPHIOXUS

THE HISTORY or THE OVUM To GASTRULATION

OGGENESIS

Multiplication and Growth.———After passing through a typical oogonial or multiplication stage the cells cease dividing and enter upon a period of growth. During this period the nucleus passes through the last processes prior to meiosis, while deutoplasm appears throughout the greater part of the cytoplasm. Inasmuch as this is a comparatively yolk-free egg the latter substance does not become very dense. It does become just abundant enough, however, so that the yolkless portion is clearly distinguishable. At the conclusion of growth and previous to the maturation divisions this portion apparently consists mainly of a thin vacuolated layer lying everywhere just beneath the Surface (Fig. 46, A). The germinal vesicle is in contact with this layer on one side, the animal pole, while the remainder of the egg cytoplasm is relatively full of yolk granules. Near the close of the growth period a thin vitelline membrane is formed.

MATURATION AND FERTILIZATION

The First Meiotic Division. —— When the egg has reached full size the first meiotic division takes place at the animal pole. It is preceded in this instance by the formation of tetrads (see page 20), and the spindle of this and the ensuing division are without centrosomes or asters. Immediately following this division, preparations for the second one begin, and proceed as far as the metaphase (Fig. 4-6, A} . The proc~ ess pauses in this stage until after fertilization. Meanwhile as the first polar body separates from the egg it pushes through the vitelline membrane, carrying a small portion of the latter with it. Hence it is entirely free and is often lost (Fig. 46, D). At the same time the egg bursts out into a portion of the gonocoel next to the atrium.

Spawning and Fertilization.———Spawning occurs throughout the spring and summer, and always toward evening, while the animals are swimming. At this time muscular contractions occur in the walls of the above gonocoel cavities and thus cause the eggs to burst through these walls, at certain points termed the cicatrices. The cutis wall of the atrium is also ruptured in these regions and the eggs thus reach the atrial cavity and from thence the exterior. As soon as the egg comes in contact with the sea water a second membrane is formed inside the first. It is called the perivitelline membrane, and is separated from the origiMATURATION AND FERTILIZATION 79

mil covering by a slight space} The new membrane seems to be formed from the outer part of the vacuolated cytoplasm (cortical plasm) at the surface of the ovum, with which for a short time it remains in close contact. It is at first of a fluid consistency, but after a brief exposure to the action of the water it begins to toughen. This process starts in the

dorsal future endoderm

anterior <-—


animal pole future ectoderm

ventral

Fig. 47.—A median sagittal section through the fertilized egg of Amphioxus, viewed from the left side. After Conklin. The egg is oriented in terms of the position of its parts relative to the future embryo. Actually, according to Conklin, it floats with the animal pole up at this time. The fertilization membrane is shown at some distance from the egg, and beneath it at the animal pole is the second polar body. The pronuclei are shown fusing in the midst of the clear hyaloplasm.

region of the animal pole, from where it soon spreads rapidly around the egg. '

Meanwhile the latter has become surrounded by sperm which have been shed into the water near the female. One or more of these sperm now penetrate the outer or vitelline membrane, cross the intervening space, pierce the inner membrane, and enter the egg. Such entrance is generally effected near the vegetal pole where the perivitelline covering remains longest in a fluid condition. As soon as the sperm have reached the egg itself, however, the toughening of this membrane is rapidly com 1 This space is literally perivitelline, and is often referred to as such. It differs from the space more usually so named, however, in that it exists previous to fertili zation, and also in that it is, at this time, separated from the egg by a separate covering, the pcrivllelline membrane.

ext 80 EARLY DEVELOPMENT OF AMPHIOXUS

pleted. Also it seems to fill the space between the egg and the original vitellineimembrane with which it apparently becomes fused (Fig. 4-6, B, C). The fused membranes thus form together what may be termed a fertilization membrane, and this presently becomes separated from the surface of the egg by the usual (“true”) perivitelline space (Fig. 46, D).

The Second Meiotic Division: Fusion of the Egg and Sperm Nuc1ei.—-The entrance of the sperm is a stimulus which causes the second meiotic division to become completed, and the second polar body is cut off. In this case, however, the body is retained beneath the fertilization membrane, thus helping to mark the animal pole, and so to orient the egg.

Meanwhile the sperm head (i.e., the sperm nucleus) enlarges so that it is equal in size to the egg nucleus. The two nuclei then meet and fuse in the usual manner. The point of this fusion is generally a little above the equator of the egg, and slightly toward the side which will eventually be the posterior of the embryo, as shown in Figure 4.7. As is indicated in this figure, the fused nuclei now lie within an area of clear cytoplasm lhyaloplasm) which, though it is mainly toward the animal pole, extends somewhat posteriorly. Cerfontaine represents it as a cone

as outlined in Figure 48, A, though Conklin ( Fig. 47) shows this shape .

less clearly. The source of this hyaloplasm is not quite clear, though Conklin seems to suggest that it arises from the breakdown of the germinal vesicle, at the maturation divisions. Whatever its source this clear material should be noted as the third differentiated substance in the unsegmented egg, the other two being the yolk filled cytoplasm, and the peripheral vacuolated layer. The further fate of these substances will be indicated presently. Any other sperm which may have gained entrance

degenerate without further activity and the process of fertilization may be said to be complete. EGG SYMMETRY AND SEGMENTATION

Symmetry and Orientation.-——-The polarity of the egg, i.e., the establishment of the animal and vegetal poles, is traceable to its point of attachment in the ovary; i.e., the vegetal pole is on the unattached side. This is a matter of considerable interest because, as Conklin has pointed out, in many Invertebrates it is the vegetal pole which is attached in the ovary. This writer then very pertinently suggests that this reversal may well mark the initiation of the later reversal in dorsaventral symmetry which places the nerve cord in Chordates on the dorEGG SYMMETRY AND SEGMENTATION 81

sal ‘instead of the ventral side. This seems reasonable, since such a profound and early appearing diflerence as this must certainly have its origin very far back in the ontological process.

VVhatever may be the conclusion with respect to this question, it is evident that the entrance of the sperm slightly to one side of the vegetal

futu re

future endodcrm ectoderm


Fig. 48.——Diagrarns illustrating the relations between the adult axes and the axes of the egg and early stages based on the accounts by Cerfontaine and Conklin. A. Fertilized egg. B. Fully formed blastula. C. Gastrulation begun. D. Fully formed gastrula. The arrow in each case indicates the future anterior-posterior ax-is, while the polar body marks the animal pole. In A the pronuclei are represented as fusing in the midst of the cone of yolk-free cytoplasm. (See ‘Fig. 47.) According to Conklin, the egg or embryo does not actually assume the position indicated until shortly after the closure of the blastopore (see text).

pole establishes a third point on the egg with reference to the two poles already present, and so determines a median plane. Not only is this true but, as later events prove, this median plane of the egg becomes the me‘diam plane of the future embryo, and the side toward which the sperm enters becomes the posterior side of the embryo. This is well to bear in mind since in the study of the Frog we shall find another case in which the sperm entrance point is significant in determining embryonic symmetry. , ‘

With respect to this matter of embryonic symmetry, a further word 82 THE EARLY DEVELOPMENT OF AMPHIOXUS

must now be said. Though the bilateral. and antero-posterior symme. tries of the future embryo have now been determined in the egg as indicated, the question arises as to how soon the floating egg or developing embryo actually becomes oriented with the antero-posterior and dorso-ventral parts in their definitive positions. It has been said that this occurs at the time of, or immediately following, fertilization so that the undivided egg actually assumes an orientation in the water such as indicated in Figures 47 and 4-8, A. As a matter of fact. however. this appears probably not true. Conklin does not refer to the point in his paper, but has been kind enough to inform the writer that in his opinion this definitive orientation probably does not occur until “ shortly after the closure of the blastopore.” In the meantime this investigator believes that the dividing egg probably floats like most other floating eggs, with the animal pole up. The lack of certainty in this connection is due, Conklin says, to the fact that “the polar bodies are minute and difficult to recognize,” while other means of orientation are also hard to discern in the living egg. His opinion under these circumstances is based on such observations as are possible, and on the fact that on the centrifuge the yolk pole always goes to the centrifugal position. However, in spite of this probable actual orientation of the animal and vegetal poles of the egg, it is convenient in describing development to assume a constant orientation from the very beginning. Hence in the ensuing description the terms dorsal, ventral, anterior, and posterior are used throughout with reference to the definitive position of these parts subsequent to gastrulation. This relation of the animal and vegetal poles of the egg to the orientation of the future embryo is indicated in Figure 43. On this basis it is evident that the anterior end of the future animal will lie about 30 degrees above the animal pole of the egg as here shown and the posterior of the animal about 30 degrees below the vegetal pole. It is to be borne in mind, however, that according to Conklin the developing ovum probably does not really assume this position until about the stage represented by Figure 51, F , or shortly thereafter.

In addition to the plane of symmetry‘ established by the mere entrance of thesperm and the position of the fusion nucleus, other significant reinforcements of the symmetry so initiated quickly ensue. As the sperm passes into the egg there is, according to Conklin, a flow of the superficial vacuolated layer of cytoplasm from the animal pole to the region where the spermatozoon entered. Here it forms a crescent of material across the future ‘posterior surface of the egg, as above deEGG SYMMETRY AND SEGMENTATION 83

fined, with the horns of the crescent extending somewhat anteriorly. This, Conlclin emphasizes, is exactly comparable to the mesodermal crescent similarly formed in the Ascidians, and it has exactly the same fate, i.e., it gives rise to all the future mesoderm. This conclusion is based on a study of sections of successive stages, the flow not being actually observed in the case of Amphioxus. Aside from the potential


Fig. 49.—Prophase of first cleavage figure in Amphioxus. From Kellicott (Chordate Development). After Sobotta. Inner and outer membranes fused and separated from the egg by a wide space called the perivitelline space.

mesodermal material which Conklin thus finds preformed in the egg, this investigator also noted that the future endodermal substance consists of the yolk-filled cytoplasm now located dorso-anteriorly to that destined to be mesoderm. The remaining yolk-free cytoplasm or hyaloplasm, containing the cleavage nucleus then lies, as noted, largely toward the antero-ventral side, and is destined to become ectoderm and notochord (Figs. 4-7, 48) . We are now prepared to describe the process of segmentation, keeping always in mind the sense in which dorsal, ventral, anterior, and posterior are being used. Segmentation.——Segmentation in Amphioxus is of the total or holoblastic type, but is not quite equal. The first division occurs about 84 THE EARLY DEVELOPMENT OF AMPHIOXUS

an hour after fertilization, and the second about an hour after the first. Subsequent divisions follow each other at intervals of fifteen or twenty minutes. 4

First Cleavage. —- The first cleavage spindle becomes situated within the cone of clear protoplasm, where its position is such that its center is cut at right angles by the median plane of the egg. The line of cleavage, therefore, coincides with that plane, and divides the ovum, including its three preformed substances, into equal right and left halves (Fig. 49).

Second Cle¢wa.ge.—The second cleavage is at right angles to the first, and is also approximately meridional. It is not exactly so, however, since its plane lies a little postero-ventral to the animal and vegetal poles, thus causing the antero-dorsal pair of blastomeres to be slightly larger than the postero-ventral pair (Fig. 50, A). This is the interpretation of Conklin, and is exactly the opposite of that of Cerfontaine and others. It is significant because it carries through the entire early development, and is necessary in order to locate the potential mesoderm in the ventro-lateral lips of the early gastrula where Conklin insists it is. We shall follow Conklin’s interpretation. This writer also calls attention to a slight spiral tendency in this cleavage comparable to what regularly occurs in Annelids and Gastropods. He maintains that usually two of the four blastomeres are sufficiently in apposition so that when viewed from the animal pole the line ofcontact at that pole appears as a short furrow turning to the left. From the same viewpoint the furrow at the vegetal pole turns to the right. This feature, however, does not have the constancy which is characteristic of the Invertebrate forms referred to.

Third Cleavage. —The third cleavage plane is at right angles to the first two; i.e., it is latitudinal with respect to the animal and vegetal poles of the egg. It is not quite equatorial, however, since it is situated slightly nearer the animal pole. The result is the production of four pairs of cells, the two at the animal pole being termed micromeres, and the two at the vegetal pole macromeres (Fig. 50, B, C }. As regards the orientation of these cells relative to the future embryo, the upper pair of micromeres are anterior, and the lower pair ventral, while the upper pair of macromeres are dorsal and the lower pair posterior. From the account of the preceding cleavage also it is evident that the anterior pair of micromeres and the dorsal pair of ma_cromeres are respectively slightly larger than the other pair of the same type. Likewise it is to be noted that the potential mesodermal material is largely located in EGG SYMMETRY AND SEGMENTATION 85

Fig. 50.———Cleavage in Amphioxus. After Conklin. A. Four-cell stage viewed from the animal pole. B. Eight-cell stage viewed from the animal pole, showing the four sizes of the cells. C. Eight-cell stage viewed from the left side. The arrow indicates the anterior-posterior axis. Again note the relative sizes of the cells, the anterior micromeres being slightly larger than the ventral ones, and the dorsal macromeres slightly larger than the posterior ones which contain the mesodertnal substance. D. Eight-cell stage going into sixteen viewed from the animal pole. E. Thirty-two cell stage viewed from the left side with many of the cells about to divide again. The arrow indicates the anterior-posterior axis. E. About the 128-cell stage, four hours after fertilization, viewed from the left side. The arrow indicates the anterior-posterior axis. Note that at this time the largest of all the cells are at the future dorsal blastoporal lip, and represent the endoderm. (See Fig. 51, A.) 86 THE EARLY DEVELOPMENT OF AMPHIOXUS

the two posterior (smaller) macromeres, the potential endodermal material in the dorsal macromeres and the dorsal parts of the posterior macromeires, and the potential ectodermal substance chiefly in the micromeres (Fig. 50, C).

Fourth Cleavage.-——The planes of this cleavage are again approximately longitudinal or meridional with respect to the poles of the egg. The cleavage is not precisely meridional, however, in all the blastemeres but very slightly bilateral. Thus in the four micromeres each of the new planes runs not exactly toward the center of the egg, but a little toward the plane of the first cleavage, while in the macromeres the inclination of these fourth cleavage planes is a little toward the plane of the second cleavage. This may be noted to some extent in Figure 50, D, although the incipient planes of the macromeres in this case appear to be essentially meridional.

Fifth Cleavage. —-—- This division is typically again latitudinal, so that there result two layers of micromeres and two of macromeres. Thus, in all, there are thirty-two cells arranged in eight meridional rows with respect to the original animal and vegetal poles, the micromeres toward the former and the macromeres toward the latter. It should be added, however, that the arrangement of the cells following this cleavage is seldom entirely regular so that a strictly meridional appearance such as shown in Figure 50, E is not often seen.

The Blastula. — The sixth cleavage is more or less meridional, giving rise to sixty-four cells. The arrangement is even more irregular than in the lastcase, however, and it is impossible to identify exactly the various cells in terms of their origins. Although the seventh cleavage is more irregular than the sixth, about one hundred twenty-eight cells are produced, and by the eighth cleavage the synchronous character of the divisions is also lost.‘ This dividing mass of cells may now be termed a blaszula (Fig; 50, F). From the figure just referred to it will be evident that this blastula is not round. Instead it is somewhat pear-shaped with the small end of the pear posterior. Also, as might be assumed, it is not a solid mass of cells, but as usual contains a cavity or blastocoel. This indeed has existed from the four cell stage since the cells are rounded and hence not in complete contact. The space in question is filled with a gelatinous material which Conklin calls blastocoel jelly, and at first communicates with the outside through spaces between the rounded cells. As cleavage continues, however, the cells establish contacts except at their inner ends, and thus close the openings into the blastocoel, the ones at the poles persisting longest. Meanwhile the jelly GASTRULATION 37

in the hlastocoel is absorbing water, so that it greatly increases in volume, and becomes quite fluid. As a result of this increase in volume the size of the completed blastula is about one third greater than that of the unsegmented egg.

The fact that the cells of the blastula are somewhat irregularly arranged makes it, as noted, almost impossible to identify each one precisely in terms of its source. Nevertheless this relationship can be approximately determined by the positions of the cells with respect to

the polar body, and by their relative sizes. Thus it appears that the‘

smallest and most rapidly dividing cells of the blastula are located posteriorly. Hence they are derived from the two posterior macromeres of the eight cell stage, and represent potential mesoderm. The somewhat larger and slightly more slowly dividing cells located in the anteroventral region are derived from the four micromeres of the eight cell stage, and are potential ectoderm. Finally the largest and most slowly dividing cells in the postero-dorsal region are derived mostly from the

dorsal pair of macromeres of the eight cell stage and are potential endoderm (Fig. 50, F).

GASTRULATION, FORMATION OF CENTRAL NERVOUS SYSTEM, MESODERM, NOTOCHORD, AND COELOM

GASTRULATION

The exact nature of the process of gastrulation in Amphioxus has been the subject of much dispute. This is owing partly at least to the minute size of the larva at this time, and the consequent difliculty of determining just what occurs. As before, the account which will be followed here is that of Conklin, according to whom the main processes are invagination, involution and a kind of epiboly. It should be stated, however, that Conklin does not himself employ the last named term. Concrescence, which is said to occur hy Cerfontaine and other writers, is. according to this investigator entirely lacking. Conklin indeed does not even refer to convergence.

Invagination and Involution. —-—As noted the completed hlastula consists of a hollow pear-shaped mass of cells the wall of which is everywhere a single cell layer in thickness. Antero-dorsally from the smaller posterior end of this pear shaped structure, the hypohlastie wall, consisting of potential endodermal cells, is already somewhat flattened (Fig. 51, A), and this process soon involves the whole postero-dorsal ventral blastuporal

lip

notochordal primardium future esoderm

blaxtopore

3 «E E

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Fig. 51.-—~Gastrulation in Amphioxus. After Conklin. Arrows indicate anteriorposterior axis. A. Hemisected blastula from left (cut) side. Note flattened vegetal pole preliminary to gastrulation, also position of future endoderm and mesotlernt. B. Moderately early hemisected gastrula from left side with epiblast of right side removed, permitting view through remains of blastocoel. C. Slightly later gastrula. Same view and treatment as in B. Note position of future mesoderm. D. Still later gastrula. Same view and treatment as in B and C. Note posterior movement of dorsal lip and dorsal movement of ventral lip, thus bringing mesoderm nearer to dorsal lip. E. Posterior view of total gastrula slightly later than D. Future mesoderm apparent in both lateral lips, but not in ventral lip, though it is there. (See text) F. Much later hemisected gastrula, again viewed from left (cut) side. 0. Completed hemisected gastrula from left side. Mesoderm, except at blastopore, is in enterocoelic fold and mostly invisible. (See text.) H. Young hemisected embryo from left side. Neural folds forming and covering blastopore to form netttenteric canal. Only one layer in “fold” at this stage. (See text.)

88 ' one choses to regard the angle where these last named lips meet as such.

as being the probable immediate cause of the process of involution.

[of the gastrulation of Amphioxus. This is probably because of the effort of this

GASTRULATION ' T I 39

side. Because of the general form of the blastula this flattening wall or plate when viewed from the future posterior, has the shape of a triangle with slightly curved sides. The widest side of this triangle is anterodorsal toward the larger end of the pear. The other two sides extend postero-ventrally until they meet at the smaller end. As will presently appear, the broad transverse antero-dorsal edge of the plate will constitute the dorsal lip of the blastopore. The other two edges will constitute the ventro-lateral lips, there being no strictly ventral lip unless

Hence the blastopore as it develops will, for a time at least, be triangular rather than round (Fig. 51, E).

The flattening of the hypoblastic plate is further accentuated, and presently the cells so affected begin to move inward somewhat as in the typical illustration of the invagination process." In this case, however, the movement is not equal on all sides. Instead it is greatest at the broad transverse dorsal lip, becoming less as one goes posteriorly along the postero-lateral lips. It is somewhat as though the hypoblastic plate were a door swinging inward, with the more posterior part of the postero-ivem tral lips acting as the hinge. It is clear, however, that the swinging in movement cannot occur to the exclusion of other processes. If it did a break would necessarily take place between the plate and the part of the lip from which it is moving away. That such a break does not occur is 2 apparently due in part to the involution or inflection of cells at these regions of the lips, particularly at the dorsal lip. This in turn is made possible by active cell division. It may also be noted in this connection that the cells of the hypoblastic plate whose inner ends are distinctly rounded have become more columnar in shape, while those of the epiblast have become less columnar and more cubical (Fig. 51, A). These and other changes in shape of gastrular cells have already been noted

Another feature to be mentioned at this point is the fate of approximately six transverse rows of cells just at, and immediately anterior to, the dorsal blastoporal lip. As involution proceeds three of these rows

9 The terms epiblast and hypoblast are not used by Conklin in his description

author to emphasize the fact that the materials for all three germ lay_¢;rs;;;arre,‘d;istinguishable, as noted, from the very beginning. However, it seems ber@fto‘be 'cjgg‘h-‘. V_ sistent in our use of these terms. Therefore, we shall apply the nz'rt‘§les,«ec”toderrr‘i’,*.:-'

endoderm and mesoderm to these layers only after they have actuall§'v'bo€n set aside ‘‘ -,,’t as definitive cellular sheets. Previous to that we have referred to « " rfiaterials con-vi , ...."* cerned as “ potentially” this or that. During gastrulation the ‘ unéy intierl"aii{'l“ -l outer layers will be designated as usual as hypoblast and epiblas 7; K . ,7’ :~ «.4 . x. V t;,: 90 EARLY DEVELOPMENT OF AMPHIOXUS

are turned over the edge of the lip and into the growing archenteric roof. These turned-in rows thus become a part of the hypoblast, while the other three remain outside as part of the dorsal epiblast. The former cells will eventually be the source of the notochord, while the latter, i.e., those not involuted, will furnish material for the neural tube. This will be referred to again when the origin of these structures is described.

Epiboly.———This process is typically thought of in connection with large yolked eggs in which a layer or layers of cells overgrow a mass of yolk. There is of course ‘no such mass in the case of Amphioxus. Nevertheless part of the gastrulation process here is essentially epibolic, the gastrular cavity taking the place of solid nutrient material. This epiboly is accomplished initially in the following manner: The ventrolateral lips tend to become continuous and begin to grow dorsally while at the same time the dorsal lip becomes more arched. In this way the originally triangular blastopore loses its angles and becomes more or less of a transversely placed oval. The dorsal side of this oval now constitutes the dorso-lateral lip of the blastopore, and the ventral side the ventro-lateral lip. All parts of the oval then grow toward one another with the lateral parts moving relatively more rapidly than the dorsal and ventral. As a result of these activities the oval blastopore presently becomes a small circular opening. Thus an essentially epibolic process is responsible for covering over the gastrular cavity. At the same time there is also occurring a gradual lengthening of the entire gastrula owing to active cell division in the blastoporal lips and elsewhere. In this manner what. might be described as a double walled tube-shaped sac is formed, the outer layer of the wall being epiblast, and the inner wall hypoblast (Fig. 51, D, F , G, H). Henceforth this sac-like structure may be referred to as a larva or embryo. Accompanying these movements there has necessarily been a redistribution of the material of the mesodermal crescent which Yaccording to Conklin lies in the two original ventro-lateral lips. The details of this rearrangement will be taken up in connection with the history of the definitive mesoderm.

Convergence. —— According to previous accounts gastrulation in this animal has also involved a distinct process of concrescence or flowing together of the material from the two sides of the dorsal lip along a median line. Conklin, however, asserts very positively that nothing of this sort occurs. Nevertheless, he does admit that regions in and about the lip do contribute substances to the embryo by a process which is THE CENTRAL NERVOUS SYSTEM 91

essentially convergence as described below. In correlation with this view Conklin never refers to the lips as the germ ring.

During the above processes the ectoderm cells develop cilia which vibrate and thus cause the embryo to rotate slowly within the egg mem -brane.

THE CENTRAL NERVOUS SYSTEM

The early development of the nervous system occurs more or less simultaneously with the differentiation of the notochord and the mesodermal somites. It is convenient, however, to describe these three proc esses separately, and we shall therefore begin with the‘ nervous system. ..

The Neural Plate and the Neural Fo1ds.—As previously suggested there exist in the early gastrula about six rows of approximately ten to twelve cells, each extending transversely across the embryo just at, and anterior to, the dorsal lip of the blastopore. As indicated the row immediately adjacent to the lip, and the two next anterior rows, are presently involuted to the roof of the archenteron. Here they will be referred to later in connection with the development of the notochord. The three cell rows which remain outside, together with all the rest of the epiblast, may henceforth be called ectoderm. These three rows (Fig. 51, E) then give rise to the nervous system in the following manner: As the embryo increases in length the cells in these rows divide, along with the others, and so continue to extend from the margin of the dorsal blastoporal lip very nearly to the anterior end of the embryo. They thus constitute an elongated‘ band of material about twelve cells in width. It is the neural or medullary plate. At the same time the ectoderm along each side of this plate becomes slightly elevated, and these elevations then begin to grow toward one another above the plate. As this process continues the ectoderm constituting the elevations becomes separated from that at the margins of the plate, and the former gradually approach each other until they meet and fuse along the median line (Fig. 52, A, B, C). Thus the medullary plate itself is entirely roofed over, and during the process it is customary to speak of the free edges of the two approaching layers of ectoderm as the medullary or neural folds. As a matter of fact, however, these layers obviously involve none of the actual medullary plate, and constitute only the outer half of a true fold (Fig. 52)‘. Hence the neural folds, as here indicated, are but partly homologous with the similarly named structures in most higher forms (see below). It should now be added that the phenomena just described do not occur everywhere’ simultaneously. The depression of the neural

/figs»: 92 THE EARLY DEVELOPMENT OF AMPHIOXUS

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showing formation of nerve cord, notochord and mesoderm. From Kellicott (Chordate Development). After Cerfontaine. A. Commencement of-the growth of the superficial ectoderrn (neural folds) above the neural or medullary plate. B. Continued growth of the ectoderm over the neural plate. Dilferentiation of the notochord, and first indications of the mesoderm and enterocoelic cavities. C. Section through middle of larva with two somites. Neural plate folding into a tube. D. Section through first pair ‘of mesodermal somites, now completely constricted oil. E. Section through middle of larva with nine pairs of somites. Neural plate folded into a tube. Notochord completely separated. In the inner cells of the somites, muscle fibrillae are forming. ‘ c. Enterocoel. ch._ Notochord. ec. Ectoderm. en. Endoderm. f. Muscle fihrillae. g. Gut cavity. m. Unsegrnented mesoderm fold. ms. Mesodermal somite. nc. Neurocoel. nf. Neural fold. np. Neural plate. nt. Neural tube.

Fig. 52.———Transverse sections through young embryos of Amphioxus, ‘ THE CENTRAL NERVOUS SYSTEM 93

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,'.a 1;.“ I! Fig. 53.—-—Optical sections of young embryos of Amphioxus. From Kellicott (Chordate Development). After’ Hatschek. The cilia are omitted. A. Two-somite stage, approximately at the time of hatching, showing relation of neuropore and neurenteric canal. B. Ninesomite stage, showing origin of anterior gut diverticula. C. Fifteen-somite stage. End of the embryonic period. ‘

ap. Anterior process of first somite. According to Conklin the existence of this is doubtful. c. Neurenteric canal. ch. Notochord (or its rudiment, in A). cg. Clubshaped gland (or its rudiment in 8?. ago. External opening of club-shaped gland. co. Coelomic cavity of somite. cu. Cerebral vesicle. g. Gut cavity (enteron, mesenteron). gs. Rudiment of first gill slit. 1'. Intestine. ld. Left anterior gut diverticulum (preoral pit in C). In. Mouth. mes. Unsegmented mesoderm. n. Nerve cord (or its

V rudiment, in A). no. Neurocoel. nip. Neuropore. p. Pigment spot in nerve cord. rd.

Right anterior gut diverticulum (preoral head cavity in C). st 52. First and second mesodermal somites. spc. Splanchnocoel (body cavity). 94 THE EARLY DEVELOPMENT OF AMPHIOXUS

plate begins just in front of the blastopore, and extends anteriorly, while the fusion of the neural folds begins slightly further forward and extends both ways. The latter process is further augmented, according to Conklin, by the continued upgrowth of the ventral lip of the blastopore over the dorsal side. Insofar as this occurs the layer so arising simply fuses with that of the lateral neural folds as described above (Fig. 51, H). As a result of these processes the blastopore is presently entirely roofed over.

The Neuropore. —— Although the blastopore has been covered in the manner just indicated, the archenteron still communicates with the exterior. This is accomplished by means of the space extending along the back of the embryo between the neural folds above and the medullary plate beneath. This space leads from the blastopore forward to the point where the folds are still in the process of uniting, and here opens to the outside. This opening is termed the neuropore, and is constantly advancing as the meeting of the folds continues. At the time of hatching, which occurs eight to fifteen hours after fertilization, this point is generally somewhat anterior to the middle of the embryo (Fig. 53) .

The Neurocoel and the Neurenteric Canal. —When in approximately this condition as regards the nervous system, the young embryo breaks out of the egg membranes. Further development of this system then proceeds as follows. The process of roofing over the medullary plate is completed so that the neuropore is carried almost to the anterior end of the animal. The center of the neural plate is then some _ what further depressed, while its edges 3 are bent upward and inward

until they meet (Fig. 52, C, D, E). There is thus formed within the old space between the archenteric roof and the fused neural folds, a new tube—- the neural tube, containing a canal, the neural canal or neurocoel (Fig. 53, B). The inner surface of this canal is evidently that of the original neural plate, and hence as might be expected, is lined with cilia. From the method of its formation also, it is clear that anteriorly the neurocoel will open to the exterior at the neuropore and that posteriorly it will still communicate with the archenteron through the blastopore. This posterior passageway through the blastopore into the neurocoel is now termed the neurenteric canal. Both neurenteric canal and neuropore remain open throughout the embryonic period, i.e., until the mouth is formed.

Later, the anterior portion of the neural tube widens somewhat to

3 These edges are mostly homologous with the inner or nervous portion of the neural folds as described in the Frog (see below). DEVELOPMENT OF THE NOTOCHORD 95

form the rudiment of a brain while within the tube at this and other points, pigment spots appear. These, or the tissues externally adjacent to them, are probably light receptors.

This is as far as it is necessary to consider the development of 'the nervous system in Amphioxus. In comparing this development with that of most higher Chordates there will be found a fundamental similarity. There is one variation in detail, however, which, though it has already. been indicated, deserves a further word of emphasis. In all those cases where the neural tube is formed by so-called neural folds it is only in Amphioxus that the completion of the real tube occurs later than,..and hence separately from, the overgrowth and fusion of the folds. Indeed, as will appear from reference to Fig. 43, in all true Vertebrates in which the tube arises by fold formation, the edge of the plate remains united to the edge of the outer layer of overgrowing ectoderm until the folds from opposite sides meet. Thus in these latter cases the structures

so named are truly folds, instead of being only the outer half of a fold as in Amphioxusf

THE DEVELOPMENT OF THE NOTOCHORD, MESODERMAL SOMITES AND COELOM

The Notochord. —-It will be recalled that in connection with the development of the nervous system reference was made to the occurrence in the early gastrula of three transverse rows of cells immediately adjacent to the dorsal lip of the" blastopore. It was indicated that these cells are involuted into the roof of the archenteron. As the gastrula increases in length the hypoblast cells of these inturned rows multiply along with the outer epiblastic cells which are to give rise to the neural plate. Thus like those of the latter structure they produce a lengthening band ten or twelve cells wide which forms the archenteric roof. As in the case of the neural plate, this hand then begins to fold, but in this instance the edges are directed downward instead of upward. Also as the sides of the fold come together the cells tend to interdigitate (Fig-. 52, B, C, D, E). In this manner a solid rod of tissue is formed, the notochord, lying immediately beneath the neural tube. Although at first the notochordal cells are wedge shaped and interdigitated, they eventually become disc-shaped and in a cross sectional View appear, as Conic 4 The peculiar method by which the neural tube is formed in Amphioxus must probably be regarded as specialized rather than primitive. Upon this same basis

some authorities do not hornologize the overgrowing ectoderm with any part of a true neural fold. 96 THE EARLY DEVELOPMENT OF AMPHIOXUS_

lin says, to be piled like a stack of coins. Finally their nuclei and protoplasm disappear, leaving a clear substance, presumably possessing a turgor which helps give rigidity to the entire structure. Posteriorly the

notochord ceases at the neurenteric canal, while anteriorly it eventually

reaches to the extreme anterior end of the embryo in front of the brain (Fig. 53, C). In this last respect Amphioxus differs from other Chordates in which the notochord always stops beneath the mid-brain.

The Mesodermal Somites and Coelom. — It will be recalled that according to Conklin the material destined to be mesoderm, like that destined to form ectoderm and endoderm, is differentiated and visible

clear back in the fertilized egg. Here the potential mesodermal sub- —

stance is gathered in the form of a crescent across what will presently be the posterior side of the larva. As segmentation occurs this crescent, as was noted, retains its position, and thus in the early gastrula comes to lie just inside the ventro-lateral lips of the triangular blastopore. Its middle section is at the median and ventral-most region where the two

' lips may be said to meet one another, while the two horns of the cres cent extend antero-dorsally to the angles made by the junction of the ventro-lateral lips with the dorsal lip (Fig. 51, E, F). It will now be recalled that the two ventro-lateral lips presently become one, the angle between them never having been a very acute one. Thus as previously noted the entire‘ blastopore takes on the shape of a transversely placed oval, the lower lip of which becomes identical with the posterior border of the crescent. As already indicated, as this ventral lip then moves upwiard, the middle part of the crescent is likewise raised, and the sides or horns assume an almost horizontal antero-posterior position; Meanwhile the cells of this potential mesodermal region have become the most actively dividing in the embryo, and hence the smallest. With the ensuing drawing together of the blastoporal lips and the lengthening of the embryo, the material in the former mesodermal crescent suffers a, further redistribution as follows: The posterior part of this potential mesodennal material, i.e., the part which has formed the middle of the crescent, now passes around the ventral and lateral side of the contracted blastopore just within its margin. As a result of the lengthening process, the former horns then proceed forward in two bands, each of which is six to nine cells in width. Each band is immediately adjacent to the edges of the rather flat archenteric roof which is about to fold downward in the manner indicated to form the notochord. Thus the

hypoblastic bands of potential mesoderm occupy the angles uniting the _

roof of the archenteron with its sides. Before-proceeding further with DEVELOPMENT OF THE NOTOCHORDE 97’

the fate of these bands it is necessary to pause a moment to consider one or two theoretical matters.

It will be recalled that under the general discussion of the processes of gastrulation in the preceding chapter it was indicated that the lip of the blastopore is sometimes referred to as the germ ring. This is done, it was said, on the ground that this lip or ring comprises the “ germ” of the embryo in that each side of it contains half of the embryonic anlage which is then brought into contact with the other half by commacence of the blastoporal lips to form a whole. It was suggested, how-. ever, that this is scarcely true in the sense originally conceived, and the present case alfords a good instance of the ways in which the original conception has had to be modified. First, it is quite evident that only in the vaguest sense can a half embryonic anlage be said to lie in the lateral blastoporal lips. All that can be said is that certain materials for the embryo do pass into it from within or near the lips of the blastopore, the potential mesoderm from the ventro-lateral lips, and potential neural and notochordal material from the dorsal lip. Secondly, as we have seen, these materials do not assume their definitive positions by a simple process of the concrescence of two sides, though the process may be thought of as a kind of convergence or confluence. If the term germ ring is to continue to be employed at all therefore it can only be with a considerably modified significance as indicated in this instance.

Returning now to the further history of the potential mesoderm it soon appears that the hypoblastic bands on either side of the notochordal region very shortly become folded so as to form grooves with the grooved side of the fold facing the archenteron (Fig. 52, B, C). In this manner this part of the hypoblast becomes cut olf from the archenteron, and thus becomes definitive mcsoderm. At the same time the hypoblast to the lateral side of each groove is drawn toward the midline. Here, as the notochord is also becoming folded off, it is finally drawn completely together so as entirely to line the archenteric cavity as definitive endoderm. In both these situations it may be noted that the folding process is accompanied by, and probably dependent upon, a change in the shape of cells, causing them to roll over a lip. The gen~ eral significance and widespread occurrence of this mechanism for cell rearrangement was pointed out in connection with the general discus? sion of involution as a method of gastrulation.

Meanwhile as the folding process is taking place the mesoderm forming each lateral groove is becoming distinctly moniliform, i.e., transverse constrictions are developing in it particularly at the anterior end. 98 THE EARLY DEVELOPMENT OF AMPHIOXUS

In this way there are soon produced anteriorly definitely separate mesodermal blocks, each with a small cavity within it. These blocks are termed mesoclermal somites, and it is to be noted that they are formed essentially as enterocoelic pouches by a process of folding off from the archenteron in the presumablyaprimitive manner. Only the first two or three somites thus formed, however, have actual cavities at this time. Posteriorly the groove closes as it forms, and the cavities within the constricted blocks of mesoderm form later. Whenever formed such cavities represent the beginnings of the coelom, and certain other spaces to be described presently. Eventually as many as sixty-one pairs of somites are thus produced. In this connection it must be clearly noted that the tem somite as used with respect to Amphioxus applies both to the myotomal region (segmental plate in true Vertebrates), and to the lateral plate, instead of only to the former. This will become apparent from what follows. ,

Before proceeding with the further development of the somites a word should be said concerning a certain classification of mesoderm which is sometimes made on the basis of the method of its setting aside as such. Thus it has been seen that the rnesoderm of the first eight or ten somites arises by the folding off of material which just preceding this

' process lies within the archenteric wall. Later somites, however, arise

more directly from material which is paid into the dorso-lateral regions from the lips of the blastopore as the embryo elongates. Hence the somite material (mesoderm) which is set aside in the former manner has been called gastral, while the "latter arising more directly from the lips of the blastopore is called peristomial. In view of the fact, however, as brought out by Conklin, that apparently all the mesoderm has its origin from material at first lying within the -blastoporal lips, such a distinction as the above largely breaks down. All of it is really peristomial.

THE FURTHER DEVELOPMENT OF SOMITES AND COELOM

By the time seven or eight pairs of somites have been formed, it becomes evident that only the members of the first pair and the upper parts of the second are exactly opposite one another. Posterior to this the somites of the left side are more and more in advance of their mates on the right, until soon they alternate. This is a feature peculiarly characteristic of Amphioxus.

The Lateral Plate.——At the stage of fourteen or fifteen somites certain further changes begin to appear in the more anterior pairs. In each somite the enterocoel becomes larger, while the walls of the venSOMITES AND COELOM 99

tral portion below the level of the notochord become thinner. At the same time this portion begins to lengthen in a postero-ventral direction, the region thus affected being known as the lateral plate.

The outer wall of this plate next to the ectoderm is called the somatic or parietal mesoderm, while the inner wall next to the enteron is splanchnic or visceral mesoderm. The part of the enterocoel which lies between them is the splanchnocoel or true coelom. The lateral plates on each side of the embryo continue to grow ventrally until they finally meet. Presently the ventro-median wall which at first separates the splanchnocoels of the two sides largely disappears, as well as the walls sepa rating the successive

splanchnocoels of the F_ 54 D, f tr _ . 1 . .— a

Sarne 5! ae. Thus the g 1 grams 0 KIISVCISG SCCUOTIS

through Amphioxus larvae. From Kellicott (Chor


splanchnocoel or coelom becomes completely continuous throughout the entire lateral and ventral region of the animal. The Myotomal Region. ——While this is go date Development). A. Through the body region of a larva with five gill slits, showing separation of mycococl and splanchnocoel (coelom). B. Through the region between atriopore and anus of young individual, shortly after metamorphosis, showing relations of sclerotome. After Hatschek. a. Dorsal aorta. c. Coelom (splanchnocoel). ch. Notochord. d. Dermatome. df. Dorsal fin cavity. :3. Epidermis. i. Intestine. mc. Myocoel. mp. Muscle plate (myotome). n. Nerve cord. s. Sclerotomc. v. Subintestinal vein. 1:)‘. Ventral fin cavity.

ing on in the lower portion of each somite, the upper portion on a level with the notochord is assuming the < shape characteristic of the adult. It is also becoming thicker, largely as a result of the horizontal flattening of its cells in the wall adjacent to the notochord. These cells presently become differentiated as muscle cells, extend throughout the length of the somite, and nearly obliterate the enterocoel in this upper region. The thickened muscular tissue of each somite is then called a myotome, while the slight entero coelic space still remaining between the latter and the outer unthickened . 100‘ THE EARLY DEVELOPMENT OF AMPHIOXUS


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Fig. 5$.—-Sections through young Amphioxus embryos showing the origin of the anterior gut diverticula. From Kellicott (Chordate Development). After Hatschek. The cilia are omitted. A. Frontal section through embryo with nine pairs of somites. (See Fig. 53, B.) The dotted line marks the course of the gut wall ventral to the level of the section. B. Optical sagittal section through anterior end of embryo with thirteen pairs of somites, showing position of right anterior gut diverticulum. C. Same in ventral view. c. Coelomié cavity of somite. ch. Notochord. csg.

' Rudiment of club-shaped gland. cl. Rudiment of an terior gut diverticula. ec. Ectoderm. en. Endoderm. g. 'Gut cavity (enteron, znesenteron). gsl. Rudiment of _iirst gill slit. ld. Left anterior gut diverticulum. n. Nerve cord. np. Neuropore. rd. Right anterior gut

diverticulum. $15259, First, second and ninth mesodermal somites.



wall is termed a myocoel (Fig. 54). Later, between the myotome and the lateral plate there develops a horizontal partition which acts as a boundary between the two regions. Eventually also there grows out from the ventral region of the myotomal portion of each somite a fold of tissue which presently becomes divided into two parts. One part then extends upward between the myotome and the noto_chord and nerve cord as the sclerotome. The inner layer of this sclerotomal part finally forms the skeletogenous sheath for the latter structures, while its outer layer forms the covering or fasciae for the inner sides of the myotomes themselves; the latter have no fasciae on their outer sides, as they do in the Craniates. The

other portion of the original fold meanwhile extends outward and downward between the somatic layer of the lateral plate and the ectoderm. This fold, together with the outer unthickened wall of the upper or myo,-tomal region, is known as the dermatome. The upper myotomal portion of the dermatome gives rise to the cutie layer of the integument in the dorsal part of the animal, while the fused inner and outer sheets of the

I dermatomal fold constitute the same layer ventrally. These points should be kept in mind, in connection with the development of homologous parts in the higher Vertebrates.

The Anterior Gut Diverticula. ——Although it is not strictly connected with the formation of the somites, we may mention in closing the appearance of certain diverticula of the archenteron, which in their early stages are not unlike enterocoels.5 When about seven pairs of somites have been formed, there develops from the dorsal wall of the gut in front of the most anterior somites a transverse ridge. This ridge thus produces a sort of dorsal bay or pouch at the anterior extremity of the gut beneath the notochord (Fig. 53, B). The sides of this bay then push upward on either side of the notochord, thus forming two dorsalateral pouches. The ventral edge of the transverse ridge now grows anteriorly cutting off these two pouches ventrally from the anterior extremity of the gut beneath them. Each then develops in its own peculiar fashion (Figs. 53, 55). The right one becomes greatly enlarged, assumes a median position, and occupies the whole of the space beneath the chorda and in front of the enteron. The left remains smaller «and finally acquires an opening to the outside of the head known as the preoral pit (Fig. 53, C).

The later development of Amphioxus is too highly specialized to help us much in an understanding of the higher and more typical Chordates. It will therefore be omitted. Those students who are interested in the further history of this animal, however, will find a good brief account with references to original papers in Kellicott’s Chordate Development. They should also note the references at the conclusion of this chapter.

References to Literature

Cerfontaine, P., “'Recherches sur le développement de l’Amphioxus,” Arch. Biol., XXII, 1906.

Conklin, E. C., “ The Embryology of Amphioxus,” Jlaur. Morph, LIV, l932.—“The Development oi Isolated and Partially Separated Blastomeres of Amphioxus," Jour. Exp. Zob'l., LXIV, 1933.

Garbowski, T., “ Amphioxus als Grundlage der Mesodermtheorie,” Anat. Anz., XIV, 1898.

Hatschek, B., “ Studien iiber Entwickelung des Amphioxus,” Arbeit. zool. Inst. Wien. IV, 1882. “Ueber den Schichtenbau von Amphioxus” (Verhand. d. Anal. Gesell., II), Anat. Anz., Ill, 1888.

Klaatsch, H., “Bemerkung iiher die Gastrula des Amphioxus,” Morph. Jahrb., XXV, 1897.

Kowalewsky, A., “Entwickelungsgeschichte des Amphioxus lanceolatus,” Mém. Acad. Impér. St. P., VII, 11, 1867.—“Weitere Studien fiber die Entwicl:e 5 By some authorities (Hatschek, MacBride) these structures are regarded as actual, though modified, mesodermal soxnites. 102 THE EARLY DEVELOPMENT OF AMPHIOXUS

Iungsgeschichte des Amphioxus lanceolatus, nebst einem Beitrage zur Homologie des Nervensystems der Wiirmer und Wirbelthiere,” Arch. mikr. Anat., XIII, 1877.

Legros, R., “ Sur quelques cas d’asyntaxie blastoporale chez l’Amphioxus,” Mitt. Zool. Stat. Neapel, XVIII, 1907.—-“Sur le développement des fentes branchiales et des canicules de Weiss-Boveri Chez l’Amphioxus,” Anat. Anz., XXXIV, 1909. — (Published anonymously.) “ Sur quelques points de l’anatomie et du développement de l’Amphioxus: Notes préliminaires. 1. Sur le néphridium de Hatschek,” Anat. Anz., XXXV, 1910.

Lwofi, B., “ Ueher einige wichtige Punkte in der Entwickelung des Amphioxus,” Biol. Centr., XII, 1892.—“ Die Bildung der primiiren Keimbléitter und die Entstehung der Chords. und des Mesoderms bei den Wirbelthieren,” Bull. Soc. Impér. Mascou, II, 8, 1894. ‘,

MacBride, E. W., “The Early Development of Amphioxus,” Q. J’. M. S., XL, 1898.-——-“ Further Remarks on the Development of Amphioxus,” Q. J. M. S., XLIII, 1900.—“ The Formation of the Layers in Amphioxus and its Bearing on the Interpretation of the Early Ontogenetic Processes in Other Vertebrates,” Q. J. M. S., LIV, 1909.

Morgan, T. H. and Hazen, A. P., “ The Gastrulation of Amphioxus,” Jour. Morph., XVI, 1900.

Samassa, P., “ Studien fiber den Einfiuss des Dotters auf die Gastrulation und die Bildung der primiiren Keimhléitter de Wirbelthiere, IV. Amphioxus,” Arch. Entw.-mech., VII, I898.

Sobotta, .I., “ Die Reifung und Befruchtung des Eies yon Amphioxus lanceolatus,”

~ Arch. mikr. Aruzt., L, 1897.

Willey, A., “The Later Larval Development of Amphioxus,” Q. I. M. S., XXXII, 1891. -—Amphioxus and the Ancestry of the Vertebrates (Columbia University Biological Series II), New York, 1894.

Wilson, E. B., “ Amphioxus and the Mosaic Theory of Development," four. Morph., VIII, 1893. I

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