Text-Book of Embryology 2-1 (1919)

Chapter I - Segmentation, Gastrulation, and the Formation of the Germ Layers

THE Vertebrate begins its individual existence in the form of a single cell, the Zygote or fertilized egg, which in turn originates in the process of fertilization by the fusion or conjugation of two gametes.‘ Of these the microgamete or spermatozoon, derived from the male parent, is of relatively insignificant bulk as compared with the macrogamete or unfertilized egg. As a consequence the more obvious features of the Zygote, such.as shape, size, and so on, are simply taken over from the macrogamete-in other words, they are of maternal origin. Such maternal features may remain obvious for some time during early stages of development, so long in fact as the maternal protoplasm remains predominant in bulk as compared with that elaborated under the control of the Zygote nucleus, but it seems unnecessary to assume that this fact has the important bearing upon questions connected with Heredity which has been claimed for it by some workers on Invertebrates.

The Zygote is a typical cell, composed, so far as its living substance is concerned, of cytoplasm and nucleus, the cytoplasm containing a lesser or greater amount of food-material or yolk. In shape it is in the vast majority of cases approximately spherical. In the Myxinoids it is elongated, almost sausage-shaped, and in a certain number of cases, for example Amia, its sh e is literally “ oval.”

The macr0gamete——-and fore the Zygotemdiifers much in size in different Vertebrates, ranging from about '1 mm. in diameter in Amphioacus to as much as 85 mm., or more, in the case of the African Ostrich. In some of the Sharks the size of the Zygote is also very great and this was doubtless the case too, with that of‘ such extinct birds as Aepyozmls.‘-3 Such relatively huge Zygotes are of

1 A general account of the processes of gametogenesis and fertilization has already been given in Vol. I. and they are not further dealt with in this volume. I

2 Assuming that the Zygote of Aeyyormis bore the same ratio in size to its protective envelopes as does that of the Ostrich it would measure about 160 mm. in diameter.

VOL. II B 2 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

interest as being in bulk the largest single cells known in either the

Animal or the Vegetable Kingdom. The adjoining list based upon data obtained by various observers

-—M‘Intosh and Masterman, Bashford Dean, Boulenger, Budgett, Bles, Semon, Salensky and others~—-brings out in more detail the differences in the size of the Zygote amongst the lower Vertebrates.

APPROXIMATE DIAMETER 01*‘ EGG (lN M1LLlMETRES) OF VARIOUS FISHES AND AMPHIBIANS

Amph/io:1:u.s' -1. Botlms ma.'r'i'nm.s 1. Petro1nyr:on 1. Pleummectes platessa. 1-8. Bdellosfnmu. 14 -— 29 x 7 - 10-5. P. 7m7('rocephalw.s 1-3. P'I"ist't'm‘u,s melnxnustomus 16. P. limamla -75. A(:¢mth'ias 35 - 40. Salsa, 'vu.l_qm"z's 1-2.

J apanese L.-imnid '1 (1 )oflein) 220. C'l'u.pea hctreugus -9 — 1. Torpedo m:¢:ll(I.fa 20 — 25. 0. sprattws -1. P0lypf.m'ns 1-1. (7m'wt0du.s 2-8.

A ¢'v'penser 2 —— 2-8. I"r0t0pfM'u.s 3-5 — 4. I.ep1Idosteu.s 3. ' Lep'i¢l0s'i7-en 6-5 ~ 7. A'rmTa, 2-5 —- 3 x 2 -—- 2-5. Axolotl 2.

Boccus lalmuv 1-4. Salmncmtlra. wmculosa 4. M wllus .su/r'rm1.la:t'u.s -9. T'rz'(o'n 1-8.

Cottus scorgnlus 1-75. Nectums 6.

Tr'igla gumardus 1-5. Hypogeophols altcmans 4 -— 5. Agonus catwphractws 1-8. - H. 'rostrams 7 — 8. Tmchxinus vipera 1-3. ' Xenopus laemls 1-5. Scomber soombrus ]-2. Pipu; 6 —- 7.

’ Gobius mrinutus I — 1-4. Alytes obstemfcans 3 - 5. Oyclopterus lumpus 2-5. Pelobates fuscus 1-5. Anarrhichas lupus 5-5 — 6. Bufo lent1'g7I'nosu.s 1.

Pholis guomellus 1-7. Hyla. goeldz"i 4.

Garlus collcwis 1-4. Nototrema fissvipes 10.

0. aeglefimos 1-4. Paludvlcola fwscomacmlata 1. G. m'reus 1-l. .Engg.s-towwr, ovale 1-25. Motella mustela -7. Oomufer salomom's 5. Brosmz'us brosme 1-3. Rhacophorus re1Inwcmltz"i 3. Ammodytes lcmceolatus -76. Rana. temporarta 2. Hippoglossus mtlgaris 3 — 3-8. R. opisthodon 6 — 10.

Within the limits of a single genus different species may show marked differences in the size of their eggs, (2.9. the Teleostean fish Arius austmlis has eggs a little over 3 mm. in diameter (Semen) while in the case of A. boekii they measure over 10 mm. in diameter and in A. commersom"i as much as 18 mm.

Even within the limits of a single species quite measurable, though less conspicuous, differences in size exist between the eggs of different females, and the same holds also, though to a far less extent, for the individual eggs laid by a single female.

The differences in size which have just been alluded to are correlated with the fact that the egg of the Vertebrate carries in its cytoplasm a less or greater amount of reserve food-material or yolk The presence of a readily available supply of food within the egg carries with it the immense .adva‘i1tage of freeing the young I YOLK V 3

animal, during the early stages of its development, from the need of having to fend for itself. And, correlated with this, the necessity of developing more or less complicated adaptive features to fit it for survival as an independent freeliving creature in these early stages is avoided. The yolk consists occasionally of but more usually of rounded or { sub - 0 s ‘ highly nutritious material. The yolk granules are frequently of a characteristic co r salmon colour or «rreenis and these impart their colour to the egg as a whole. vvhere however the yolk A.-—Section through egg of A7Ii—p/I.io.-:.'u...s'. becomes very finely subdi- After Cerfontaine, 1906.) i

vided W8 filld, 3.3 ill the (53.89 The small crosses mark the position of apical and abapical

0 ' col 1. d 1 roun poles. Near the apical pole is seen the second polar body t on e g ass g d adherent to the surface of the egg. The egg- and sperm into Powder: that the char‘ nuclei have not yet fused, and are seen in close proximityito acteristic colour is replaced one another.

by white. ' ‘his finesubdivision of th yolk with its acconi- ‘s paiiying White colour is commonly found in parts of the egg { where metabolism is particularly active, for example those p_ortions in which active growth or cell division is about to take place, the fine subdivision making the yolk readily assimilable and so available for 1ngt',a_ B.———-Section through egg of 1'...-',,-',».'» . .-..~.-, showing more marked

- _ tendency for the yolk granules to concentrate towards b°11° needS- Where the abapical pole (Teloleeithal condition).

the yolk is com- y . l

, _ . n, lint-leIi.~. 1;, pigmi-iii. pa-I'al':1V61y 3131311 111 'l‘lu~ yolk gi‘:inule.s' :m- lll|ll(':I.lI-Il in lmth li;_:un_-s by dark dots. amount, as in Amphilomus (Fig. 1, A), it may be distributed nearly equally throughout the egg substanoe; in other words there is an 3 approach

Fin. 1. 4 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

to the isolecithal condition: but as a rule in the Vertebrate the yolk is large in amount and is concentrated towards the lower or abapical pole of the egg, the protoplasm towards the upper or apical pole being comparatively poor in yolk (Telolecithal condition).

This segregation of the dead yolk and the living protoplasm towards opposite poles of the egg is well seen in the relatively huge egg of the bird where the protoplasm is concentrated in a germinal disc containing practically no yolk and forming a cap at the apical pole of an enormous mass of yolk practically free from protoplasm.

' It has already been indicated that the egg may have a characteristic coloration due to the colour of the yolk. Such yolk coloration may be looked upon as accidental and without any special biological significance in itself. Many eggs on the other hand especially amongst the Ganoid fishes and the Amphibians are given a dark colour by the presence within them of brownish-black pigments belonging to the melanin group. Such pigment appears to be of definite biological significance, providing as it does an opaque coat which protects the living protoplasm from the harmful influence of light. Eggs in which it occurs develop, as a rule, under conditions where they are exposed to intense daylight. The eggs of ordinary Frogs and Toads for example which are surrounded by clear transparent jelly have a well-developed pigment coat. On the other hand in the case of Frogs and Toads whose eggs are surrounded by lightproof foam (see Chapter VIII.) or are deposited in burrows underground‘ they are commonly without pigment.

In all probability this deposition of melanin pigment in the superficial protoplasm of the egg (normally in its w.ppe'r portion) is to be interpreted as having been originally a direct reaction to the influence of light, the metabolism being so affected as to bring about the formation of this particular iron-containing excretory pigment.

It may be objected that the pigment is produced before the egg is laid (eg. the (lommon Frog) and therefore before it is exposed to the action of light, but as a matter of fact the body-wall of the adult is by no means opaque to light rays and even while still in the ovary the eggs are exposed to the influence of faint light. If we may take it, as seems probable, that the influence of natural selection has gradually developed in such cases the particular type of sensitiveness to light which leads to the formation of melanin, on account of its protective value, then there is nothing surprising in the developing of this sensitiveness at earlier and earlier periods until at last it has resulted in the pigmentation of the still intra-ovarian egg in response to the feeble light rays which penetrate the body-wall.

The other possible explanation of this precocious pigment formation is that the production of the pigment though originally taking place as a direct reaction to light in the laid egg, has become so engrained in the constitution of the species that it now comes about even in the absence of the original stimulus. The objection to this explanation is that it postulates the inheritance of an “acquired I SEGMENTATION 5

character,” and that is unfortunately not justified by our knowledge so far as it goes at present.

SEGMENTATION

The first important steps in the evolution of the unicellular Zygote into the multicellular adult are seen in the» process of Segmentation which is, in fact, a process of mitotic cell division showing special peculiarities in difl'eren_t groups of the Vertebrata. During this process there appear in succession on the surface of the egg grooves which gradually deepen and eventually divide the egg incompletely or completely into distinct segments or Blastomeres. Before entering into the details of this process it will be convenient to describe it in outline and define the various technical terms used in its description.

The first phase of segmentation is commonly marked by the appearance of a superficial groove which may conveniently be designated by the letter a, passing through both the upper and lower poles of the egg. Such a groove or furrow is termed meridional, as it marks a great circle on the surface of the egg corresponding to a meridian of longltlide on 3’ terresbrlal globe’ Fm. ‘2.——-Diagram to illustrate technical The Slllgle nucleus Of the 80139 terms used in describing the process of meanwhile divides by mitosis -— a segmentationdallghter nucleus passing into each am, apical pole; ab.p, abapical pole; c, equahernisphere. From the known facts §‘i’(‘;:f‘;1.f,}1‘l‘I‘_’;‘(’)““’,‘, '1 3 ’"” ‘""" of fertilization we have reason to ' A‘ ' believe that the Zygote nucleus contains exactly equivalent amounts of chromatin from each of the two parents. In the process of mitosis this maternal and paternal chromatin is again shared equally between the two daughter nuclei. L

The first meridional furrow gradually deepens so that the egg becomes completely divided into two blastomeres or segments each representing a hemisphere of the Zygote. A second meridional furrow (/3) now appears in a plane perpendicular to that of the first and by the deepening of this the egg becomes divided into four equal blastomeres. s

The next furrow to appear may be one running round the equator of the egg (equatorial). In eggs, however, which are not absolutely isolecithal-—~and this holds for all the lower Vertebrates-— 6 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

the third furrow appears, not at the equator but at a level nearer the apical pole, and is termed a latitudinal furrow, corresponding as it does with a parallel of latitude upon a terrestrial globe. The distance of this furrow from the equator, its degree of latitude so to speak, is roughly proportional to the degree of telolecithality of the particular egg, suggesting that the volume of living protoplasm may be roughly equivalent in amount upon the two sides of the division plane to which this furrow gives rise.

When this third division is completed the egg consists of eight blastomcres, the four on the apical side of the division plane being smaller (micromeres) than those on the abapical side (macromeres).

The next furrows to appear are two in number and in the simplest condition they are meridional, bisectin g the angles between the two first furrows. More frequently however these furrows instead of traversing the pole of the egg are discontinuous at this point and each is displaced somewhat so as to join the first or second meridional furrow at a less or greater distance away from the pole. To such a furrow we apply the term vertical (Fig. 2, '12., cf. also Figs. 14 and 16, C).

It is as a rule noticeable that meridional or vertical furrows tend to become apparent first in their portions nearest to the upper or apical pole of the egg, their lower ends gradually extending downwards towards the abapical pole. This phenomenon appears to be due to the retarding influence of the dead and inert yolk. The proportion of this to the living protoplasm becomes greater and greater as the distance from the apical pole is greater, and in correlation with this the retarding effect becomes more and more pronounced.

After segmentation has reached the stage indicated its further progress tends to become irregular. New furrows make their appearance—~latitudina1, and vertical or meridiona1—and the surface of the egg takes on the appearance of a mosaic-work, while its substance becomes cleaved or split apart into corresponding blastemeres as the superficial furrows gradually deepen into slits.

At somewhere about this period there begins a new type of mitotic division in which the individual blastomere becomes split in a direction parallel to a plane tangential to its outer surface, so that it divides into an outer blastomere visible in surface view and an inner one concealed in the interior of the egg.

With the further progress of segmentation the blastomeres divide over and over again, so that eventually the egg is converted into a very large number of small cellular elements. The rapidity with which the cells divide bears a rough inverse relation to the richness ‘of their contents in yolk. Dead inert yolk tends to cause the cell to lag behind in the process of division, and the result of this in telolecithal eggs is that the difference in size between inieromeres _and macromeres becomes more and more marked as segmentation goes on—-—the lower and more richly yolked segments 1 SEGMENTATION 7

tending to lag, in their mitotic division, more and more behind the less yolky upper elements. This inequality is found at its maximum in the large eggs of Elasmobranchs, Reptiles, and Birds, where the main mass of the egg has its proportion of protoplasm reduced so nearly to vanishing point that it does not divide at all. It is only a small portion of the egg in the neighbourhood of the apical pole that is rich enough in protoplasm to carry out the process of segmentation into separate cells. This is known as the germinal disc or, later on, when it has segmented into a mass of cells, blastoderm. An egg of such a type, showing partial or incomplete segmentation, is termed meroblastic in contrast with the more primitive holoblastic type in which the egg segments as a whole.

The blastomeres into which the egg divides being composed of protoplasm——a somewhat viscous fluid——-tend under the physical laws of surface tension to assume a spherical shape except when flattened by pressure against their neighbours. There thus exist normally chinks between the blastomeres filled with wateryfluid.

As the process of segmentation proceeds this intercellular fluid increases 'in amount and the process normally culminates in the stage known as the blastula. The blastula consists of a more or less spherical mass of cells surrounding a relatively considerable volume of fluid which is for the most part no longer distributed in small chinks but collected together into a large space——the blastocoele or segmentation cavity.

In the simplest case, that of Amphiowus, the wall of the blastula is composed of a single layer of cells—the cells towards one pole being larger and containing fine granules of yolk or food material. In holoblastic Vertebrates above Fishes it is however, as a rule, no longer composed of a single layer, the roof of the segmentation cavity being frequently composed of two layers while the floor is composed of a thick mass of large heavily yolk-laden cells.

The details of the segmentation process may now be followed out as it occurs in the various types of lower Vertebrates.

AMPHIOXUS

Amphioxus is, of all the lower Vertebrates, that in which developmental processes are least interfered with by the presence of yolk, and for this reason the phenomena shown during its segmentation must form the basis for the comparative study of the corresponding phenomena in the Vertebrata in general.

The process of segmentation in Amp}:/iowus was described first in two works which are now amongst the classics of morphological science: the first by A. Kowalevsky (1867) and the second by B. Hatschek (1881).

The process begins (Fig. 3) with the appearance of a depression of the surface in the region of the apical pole. This depression takes an elongated groove-like form and extends outwards at each -8 l<‘..\-all-’._l=€\r_"(_) "H" (f)l<‘ 'llll'|C l.()V\’|<ll{ \~’F.l{.'|‘l*}l-‘.lcl.-'\PICS cu.

F" end until linal .‘ ‘it forms a wide Ill(:1‘.i(.llull:.l.l vulle_y encircling the entire egg (}'_4"ir_f. 3, This valley g1‘aLl11ally deepens dividing the egg into two halves. Finally after about 5 minutes from the commencetm-nt of the process the protoplasmic bridge connecting

the two lmlves srmps across and the egg is now completely divided

Fro. 3.»--Illustrating the process ofsegI'n'1cntat.i«)n1 o: (_After Hnt.~'«_-.h(-.k, 188].)

'I'}'w::pi(e:11 pole is a‘.»n\'u- in 4-.:u-h Iigm-r-.. The serum! pnlm ljmcly is sc-en in proximity to it.

into two blastomeres, each of which assumes a spherical shape in response to surface tension. The two hlastomeres become slightly flattened where they are in contact 71.6. in the plane of the first meridional furrow (Fig. 3, C). The future course of development shows that this plane corresponds to the sagittal plane of the embryo (Cerfontaine, 1906): in other words the two blastomeres represent the right and left halves of the developing individual. I SEGMENTATION 9

After a resting period of about an hour a second meridional furrow develops in a manner similar to the first and in a plane perpendicular to the plane of the first furrow. This gradually deepens and each hemisphere becomes divided into two blastomeres, each of which as before assumes a spherical shape and then becomes flattened o11t slightly against the other. Of the four blastomeres which are now present two, shown by subsequent development to be anterodorsal in position, are according to Cerfontaine normally smaller than the other two. .

The two meridional furrows (a. and /3) are followed after an interval of about a quarter of an hour by a latitudinal furrow slightly above the equator and this divides each of the four segments into two. The egg now consists of eight blastomeres———four smaller micromeres on the apical side of the latitudinal division plane, and

Fm. 4.—-—Apioal view of Am_phio.\'us eggs at the oiglnt-l)lastome1'c stage. (After E. B. Wilson, 1893.)

A, “Radial” type; B, “Spiral” type; and C, “ Bilateral ” typo.

four larger macromeres upon its abapical side. Each microinere lies, according to Hatschek, exactly over the corresponding macromere so that the apical side of the egg as seen from above looks like A in Fig. 4.

Wilson (1893), followed by Samassa (1898), has however drawn attention to the fact that in a considerable proportion of cases the cap of four micromeres is, as seen from above, rotated in a clockwise direction through an angle varying from 0° to 45° (Fig. 4, B) thus conforming to Wilson’s “spiral” type of segmentation or cleavage. Again in a still smaller percentage of eggs at this stage the blastemeres are arranged according to Wilson’s “bilateral” type (Fig. 4, C) the eight blastomeres being arranged symmetrically on each side of the first division-plane but either two or all four macromeres being displaced outwards somewhat from this plane.

Fourth division. After another short interval (less than a quarter of an hour) a new set of furrows appear bisecting each of the already existing blastomeres so that the embryo comes to consist of sixteen blastomeres arranged in two tiers, eight micronieres above and eight macromeres below (Fig. 3, F). Hatschek described this fourth set of furrows as being meridional (Fig. 3, F) while according to Cerfontaine (1906) the division planes are when first 10 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

indicated meridional in position but become displaced somew-hat so as to be in the case of the micromeres perpendicular to the first (sagittal) division-plane or in that of the macromeres slightly oblique.

Fifth division. Each blastomere divides again} the smaller blastomeres towards the apical pole dividing rather earlier than, the others, and the result is that there are now thirty-two blastomeres in all, arranged in eight meridional rows of four cells each, the cell at the lower (abapical) end being decidedly larger than the others. Between these four large elements a wide opening is present (Fig. 3, G) leading into a space which made its appearance as a little chink between the blastomeres of the four-cell stage but which has since then increased greatly in size. This space in the interior of the egg is the blastoeoele or segmentation cavity.

From this period onwards the segmentation process becomes less regular. There has already shown itself a tendency for the larger blastomeres towards the lower pole to lag behind somewhat. And the arrangement of the blastomeres becomes less regular as they become smaller and fit more closely together. In particular the bilateral symmetry in the arrangement of the blastomeres which is conspicuous in most of the eggs during the earlier stages (Cerfontaine) ceases to be apparent.

To summarize the later phases of segmentation it may simply be said that the blastomeres go on dividing, the segmentation cavity increases in size, its communication with the exterior closes up, and there is formed eventually a blastula of approximately spherical shape. The wall of the blastula is composed of a single layer of cells those towards the apical pole being smaller and less rich in yolk than those on the opposite side (Fig. 3, I).

RAN A

In the case of the Frog we have an egg in which as compared with that of Amphiowus there is present a much greater proportional amount of yolk and which in consequence serves well to illustrate the nature of the influence of yolk upon segmentation.

The process of segmentation begins with the appearance, in the region of the apical pole, of a small dimple on the surface of the egg which gradually lengthens out to form the first meridional furrow (a). The furrow gradually extends downwards over the surface of the egg (Fig. 5, A) and becomes completed by reaching the lower pole after about an hour and a quarter.” It also extends inwards from the surface and finally bisects the egg into two

hemispheres. The second furrow (,8) is also meridional and is in a plane

1 As there are marked discre ancies between the accounts given by different observers we may take it as pro able that there is considerable variability in the details of segmentation about this stage.

9 See, however, later for caution in reference to the time factor in development. noticed further that the egg as

I SEGMEN TATION 11

peI'pem‘liuul.a1' to that of the first. It appears about three-quarters of an hour after the latter and, like it, extends downwards and inwards so that the egg becomes divided into four approximately equal segments.

The third furrow is latitudinal in position being situated (Fig. 5, C) roughly about 20° above the equator. It extends inwards and the egg is now converted into eight blastomerr-s, four micromeres towards the apical pole and four macromeres towards the lower pole. ‘ ‘

Closer study of these first three cleavages in the ease of the Frog brings out a number of im-. portant points. It will be noticed in Fig. 5 that the circular area of the egg-surface which is free from pigment is placed somewhat eccentrically so that at one edge it approaches the equator of the egg much more nearly than it does at the opposite edge. It will be

judged by the distribution of pigment is arranged symmetrieally about the plane of the first furrow. This furrow seems to correspond, under normal conditions, with the sagittal plane of the embryo, and therefore the two hemispheres separated by the first furrow correspond to the right and left halves of the embryo. The study of later stages will bring out the fact tha'.t the point: In the Flu. 5.——lllu.stratiug segnwulationofl~‘rog's boundary of the unpigmented egg_ (Mm sc1,i.m.., 1.s9o,) portion which lies nearest to the equator marks what will become the posterior end of the embryo.

From tlw time of appearance of l".-lll‘. third furrow onwards wide tlilh-I.'m1(:<~.:-i ooour lwtween different Occasionally one may be found in which matters proceed with diagrammatic regularity. Two new 'lIH_']‘l(ll()l1al furrows appeai.- i11tei'seeting the angle between a and ,8 and like the latter tlwy gradually extend downwards, halving each of the existing lvlastonieres and thus giving rise to sixteen blastomeres—-in two tiers of eight, .micro_1nere..s above, macromeres below. Then a latitudinal furrow e'tpp(’.a.1.‘H dividing the micromeres, and later a similar furrow dividing the macromeres; so that there are new four tiers of eight blastomeres each.

Commonly however there is no such regularity either in the arrangement or in the time of appearance of the furrows. The meridional furrows in particular tend to la‘ roplzuizcd by vertical 12

furrows which intersect u or /3 at some distance froin the poles.

l*}MP.RY(_)L()GY 011‘ THE LOWER V ERTEBRATES

CH.

As

regards the variation in order of development of the various furrows a good idea will be got from Fig. 6. Whatever be the case with the divisions immediately succeeding

D .

Fro. 6. «Illustrating the variation in the order of appearance of the first cleavage furrows in Rmm p¢¢lu.s'trz'.s'. (After Jordan and Eyelashyincr, 1894.)

The .s4‘qllt*l1('e, in time, of the appearance of the furrows is in. «heated as follows:—-1, ; -3, _.._-; 3, ..... .. ; 5, .

4, -1-mu‘

7

several cells thick (Fig

the eight cell stage, from now onwards there is little regularity. All that can be said is that each individual blastomere goes on dividing over and over again, the length of time elapsing between successive divisions bearing a rough relation to the amount of yolk present in the particular blastemere.

Already at the third cleavage the eight blastomeres have a distinct chink——the commencing b1astocoele— between their inner ends and as segmentation goes on this space becomes larger. The thirty-two-cell stage is a blastula which in a meridional section (Fig. '7, A) is seen to correspond in its general character with the blastula of A mp/mloazus but to diifer from it in three features: (1) it is of larger size, (2) it is co1nposed.oi' fewer cells and (3) the difference in size between the less richly yolkcd cells towards the apical pole and the more heavily yolked cells towards the opposite pole is more marked.

As development proceeds a farther difference becomes apparent. In the various mitotic divisions during the preceding phases of segmentation the axis of the spindle has been arranged more or less tangentially but now divisions begin to take place in which the spindle axes are arranged radially and the division-planes tangentially. When this happens one of the two resulting daughter cells is nearer the centre, the other nearer to the surface of the blastula and the effect of repeated divisions of this type is that the blastula-wall loses its original character of being composed only of a single layer of cells and becomes

. '7, B).

ELASMOBRANCHS

The egg of any ordinary Elasmobranch such as a Dogfish, Skate, or Torpedo, illustrates the type of segmentation that takes place I i SEGMENTATION . 13

in an egg in which the proportion of yolk present approaches the maximum. In this case the zygote nucleus commonly undergoes two mitotic divisions before there is any external symptom of segmentation. of the cytoplasm. Usually a single furrow makes its appearance first, incising the surface of the germinal disc but not extending to its periphery (Fig. 8, A). Occasionally a second regular furrow makes its appearance intersecting the first at right angles

' .31 0' ¢‘;“*"'I’~70p' -- «ms--.: ,_\__.',_:_‘..‘_1§T.__..‘_ _

\ FIG. 7.-—-Vertical (meridional) sections through hlastulae of Frog. (From Morgan,- 1897.) .1 R, commencing invaginatlon ; SG, segmentation cavity.

and it is a curious point that it is sometimes this second furrow

which corresponds to the first nuclear division.

These first two furrows apparently represent the first two meridional furrows of the holoblastic egg though in the Elasmobranch the first to appear may be either or. or ,8. More usually, in place of a second regular furrow developing, irregular branches of the first furrow, or even independent furrows, appear and an arrangement of

somewhat radiating furrows is brought about which gradually becomes converted into a network (Fig. 8, B, C, D).

It should be noticed in regard to these segmentation furrows 14 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

that the first latitudinal furrow cannot be identified in the Elasmobranch and further that the study of sections shows that the furrows sometimes out into the germinal disc obliquely instead of being perpendicular to the egg surface.

The nuclei of the blastoderm divide synchronously and after four divisions have taken place, when there are sixteen nuclei in place of the original single zygote nucleus, the segmentation furrows (Fig. 8, 0) form a network dividing up the blastoderm into smaller central and larger peripheral blastomeres. These hlastomercs are, however, not completely isolated from one another but are still in continuity

A 3 B‘

" ‘- 7- ‘*—

‘v \

a '/’F'I_Lr.§{ \ K“

1' \ I \ I I '. I‘ I , I i I

s‘ ‘ J \ ’ ’

' ‘- __ .r

Fm. 8.--Surface views of the blastoderm of Elasmobranehs illustrating the process of segmentation. (After Ruckert, 1899.) .

A, 13, U, Torpedo; B*, 1’rist'£m'us; I), E, .\'cyllium.. Fig. 13* shows an abortive segmentation which may often be observed transitorily round the margin of the germinal disc [Merocyte segments of ltuckert].

at their bases, and, in the case of the peripheral blastomeres, at their outer ends.

Up to the fifth mitotic division the axes of the mitotic spindles have been approximately parallel to the surface but now blastomeres begin to divide with their spindle axes perpendicular to the surface so that a set of superficial segments becomes separated off. Beneath these fluid accumulates intercellularly and a segmentation cavity arises (Fig. 9, B).

During the sixth division some of the blastomeres forming the floor of the segmentation cavity become. separated off from the 1 6 SEGMENTATION 15

underlying, unseglnented, yolk (Fig. 9, C) and in surface View the

blastoderm assumes the appearance shown in Fig 8, D. Up to and including the seventh division mitosis takes place ?‘K'g£? ». -4 r u "

.u -I-rvpln n ‘r ._,’ ‘iI\~“_' F n V“ "I

FIG. 9.——Vertical sections through Elasmobranch blastoderms illustrating the process of segmentation. (After Riickert, 1899_.)

A, U, D, E, Torpedo; B, Scyllium; F, G, P-ristiums. E, F, and G are sagittal sections with posterior edge of blastoderm to the right.

practically synchronously throughout the blastoderm. In Torpedo Riickert (1899) found that even in the ninth division the majority of the nuclei still divided synchronously and that in some eggs the same was the case with the tenth division but in any case approxi16 EMBRYOLOGY OF THE LOWER VERTEBRATES C11.

mately about this period individual nuclei lag behind others and the regular rhythm becomes lost.

This rhythm of nuclear division is of interest in relation to the size of the individual blastomeres. It is often noticeable in an Elasmobranch blastoderm that the blastomeres are somewhat smaller in what is shown by later development to be its posterior half 'i.('. the half next the side on which the embryonic rudiment makes its appearance later. It would be natural to suppose that the smaller size of the blastomeres is due to their having gone through a greater number of divisions but this explanation is rendered less satisfactory by the synchronism of the mitotic divisions. Apparently the inequality is at least to some extent due to the zygote nucleus, and, later on, the first segmentation furrows, being not quite central in position in the germinal disc but situated slightly towards its posterior edge (Riickert).

The stage up to which mitosis remains synchronous varies amongst individuals of one species and an fortioml amongst those of difierent species ‘and genera. Thus in Prristvlurus it is, commonly, regular only up to the fifth mitosis according to Riickert.

While segmentation has been proceeding, important changes have been taking place in the segmentation cavity. About the time of the seventh division the rounded inner blastomeres fill up most of the cavity so that it becomes reduced to chinks between the individual blastomeres. These chinks are filled with fluid secreted by the egg substance, and in the yolk beneath the blastoderm the activity of this process of secretion is indicated by the appearance of fluid vacuoles.

As development goes on the amount of fluid increases greatly and about the tenth division it begins to collect especially between the blastoderm and the yolk, forming the “germ cavity” of Riickert (Fig. 9, D, E, F). This cavity is best marked towards the posterior side of the blastoderm and in ground-plan is crescentic in shape. It varies greatly in its degree of development in different individuals.

Whether it is advisable to use a separate name for this cavity is very questionable. When a broad View is taken of the relations of blastomeres and segmentation cavity in the Elasmobranchs these seem to be similar in kind to those which hold iii the case of the Lung fishes. In these fishes, as will be shown later, the blastomeres which originally formed the floor of the segmentation cavity become later on shifted in position towards its roof but the resultant change in the topographical relations and form of the segmentation cavity would clearly afford no valid reason for giving it a new name.

The Yolk Syncytium.——- The layer of substance immediately underlying the blastoderm and segmcntation- or‘ germ-cavity is distinguished from the main mass of yolk upon which it in turn rests by the finer grained character of its yolk granules, and by its I SEGMENTATION 17

greater richness in protoplasm. This layer shows no division into cells and is therefore termed the yolk-syncytium‘ (II. Virchow: R1'ickert’s term “merocytes ” is synonymous). '1‘l1e marginal portion round the edge of the blastoderm is sometimes termed the germ-wall.

Functionally the yolk-syncytium is apparently concerned especially With the digestion and assimilation of the yolk. Scattered about in it are nuclei, often of enormous size and irregular form. (Joncerning the origin and fate of these nuclei much discussion has raged and the matter cannot yet be regarded as satisfactorily settled. The question is complicated by the fact that, as shown by Ruckert (1890), polyspermy appears to be a normal occurrence in Elasmobranehs. In addition to the ,single microgamete which takes part in the formation of the zygote-nucleus a variable number of extra spermatozoa make their way into the egg and give rise to accessory sperm-nuclei. Where such sperm-nuclei are situated in the coarse yolk they apparently soon degenerate but when, on the other hand, they are within the protoplasm of the germ-disc they remain during the early stages of development in a living and apparently healthy condition, even undergoing mitosis synchronously with the nuclei derived from the zygote-nucleus up to the fourth or even fifth or sixth division in the case of ’1'07'ped0.

The importance of this fact should be noted in connexion with our ideas of the reciprocal physiological relations of nucleus and cytoplasm. It is fully recognized that the nucleus governs and controls cell metabolism: it is not always so fully recognized that conversely the cytoplasm exerts an important influence over the nucleus. Clearly the fact that the accessory sperm-nuclei “keep step" in their mitotic divisions with the embryonic nuclei must be due to some influence exerted on the former nuclei through the cytoplasm. It should, in fact, never be forgotten that cytoplasm and nucleus are merely locally specialized portions of the same common living substance or protoplasm. ,

At first the accessory sperm-nuclei are clearly distinguishable in the germinal disc from the true embryonic nuclei by their smaller size and reduced (haploid) number of chromosomes. After the zygote-nucleus has undergone two mitoses however—or even before the second mitosis——the accessory sperm-nuclei wander—-or become transported by cytoplasmic movements--outside the limits of the germ~disc. They continue their mitotic rhythm for a time so that, for example, at the 8-nuclear stage of the blastoderm they may be seen in groups of eight lying in the yolk-syncytium. During early stages of segmentation numerous such obviously accessory spermnuclei may be seen in the syncytium but as time goes on the nuclei

1 Although Haeckel originally defined the term syncytium (Die 1i'all‘schu.°dmone, Bd. I. p. 161) as a protoplasmic mass formed by the fusion of origwinally separate cells the word has come into such general use for a multinucleate mass of protoplasm which

shows no subdivision into cells, whatever its origin may have been, that there seems no serious objection to the use of the term yolk-syncytium as suggested by Virehow.

VOL. II C 18 EMBRY()L()GrY OF THE LOWER VERTEBRATES on.

of the syncytium are seen to be of a different character. They are now of enormous size and of peculiar lobed appearance. The lobing becomes more complex as time goes on and appears to be due to incomplete and irregular attempts at amitotic division.

The discussions, alluded to above, have centred round the mode of origin of these highly characteristic giant nuclei. Balfour, who first described them (1874), did not express any opinion as to their origin. liickert in his first paper (1885) on Elasmobranch development looked on them simply as specialized embryonic nuclei and gave the masses of protoplasm in which they are embedded the name “ merocytes.” Latterly however Riickert, after his discovery of polyspermy in Elasmobranchs, has taken the view that the yolknuclei are really the accessory sperm-nuclei before alluded to which have altered their character in correlation with the altered environInent in which they find themselves after leaving the germinal disc.

In spite of Riickert’s more recent observations and conclusions, and in spite of their being supported by Samassa, Beard and others,

A B C

Fm. 10. --Views of the segmenting germinal disc of Bdelloslmna stouti. (After Bashford Dean, 1899.)

it must, I think, be admitted that the sperm-origin of the yolk-nuclei is by no means demonstrated. And all general considerations are in favour of Ri'1ckert’s earlier view being the correct one, namely that the nuclei of the yolk-syncytium are genetically of the same order as the ordinary embryonic nuclei. Such general considerations render it extremely improbable that accessory gamete nuclei should really play an important physiological part in the developing embryo: it is far more probable that such nuclei simply persist for a time, undergo mitosis a few times and then degenerate and disappear.

The variations in the process of segmentation are well illustrated by the three cases just described and it will be convenient now to summarize the general characteristics of the process in the various remaining groups.

LAMPREYs.——In the Lamprey the phenomena of segmentation agree closely with those observed in the frog and need not be further described.

MYXINOIDs.—In the Myxinoids the somewhat sausage-shaped egg is heavily yolked and possesses a germinal disc situated close to one pole. A few segmentation stages of Bdellostoma (Fig. 10) I SEGMENTATION , 19

have been described by liashford Dean (1899) and as might be expected the segmentation is meroblastic. Apparently the first two furrows (a and B) -have the normal meridional arrangement the specimen figured by Dean (Fig. 10, A) showing a displacement at the intersection of the two furrows. These latter do not reach the edge of the germinal disc. The third set of furrows (Fig. 10, B) appear to be vertical and in the next stage figured (Fig. 10, C) the furrows have become joined up to form an irregular network which still barely reaches the edge of the blastoderm. CROSSOPTERYGIANs.—-——Our knowledge rests entirely on -the observations of Budgett (Graham Kerr, 1907). These, necessarily fragmentary, observations sufiice to show that the process of segmentation is of great interest. In the earliest stage observed, but not figured, by Budgett the egg was “ segmenting in four equal

FIG. 1'l.—-Segmentation and gastrulation in I’ul;a;p1!erus. (Drawings by Budgett. Graham Kerr, 1907.)

A, represents a view of the apical pole: the remaining figures are side views.

portions, the constrictions being deeper than in the frog.” A second egg (Fig. 11, A and B) is in the eight-blastomere stage. The blastomeres are practically equal in size and it may be inferred with considerable probability that in Pol;/pm-us two meridional furrows are succeeded by a latitudinal one which is very nearly equatorial.

The nearness of the latitudinal furrow to the equator is remarkable

in view of the fact that the egg of Polypterus, as shown by the study of sections (Fig. 1, B, p. 3), is not by any means nearly isolecithal.

ACTINOPTERYGIANS.-—The typical Teleost is characterized by the fact; that its richly yolkecl "eggs show a more complete segregation of protoplasm and yolk than do those of any other Vertebrata. In correiation with this the segmentation is here the most markedly meroblastic in character. These featuresosuggest that in the ancestral Teleost the yolk was large in quantity and that the egg as a whole was of great size. Amongst present-day Teleosts however it is only, comparatively speaking, a few forms mostly inhabiting fresh water, 20 EMBRYOLOGY OF THE LOWER VERTEBRAT IS on.

which produce eggs of very large size 6.{]. (£2,/1/r,ncu'0/ms nilotricus (Budgett, 1901; Assheton, 1907) where they measure about 10 mm. in diameter, or the Salmon or Trout where they measure from 4 to 5 mm. '

'1‘he majority of fishes produce eggs in enormous numbers, amounting in some cases to several millions, and correlated with this the size of the individual egg has become much reduced. The average diameter of a Teleostean egg may be taken as about 1 mm. In an egg of this size segmentation of so markedly meroblastic a character would be puzzling except on the hypothesis that the meroblastic condition had arisen in ancestral forms in which the eggs were much larger.

The larger part of the egg consists of a spherical mass of practically pure yolk. On the surface of this is a thin layer of protoplasm containing droplets of oil, and this layer of protoplasm is more or less distinctly thickened in the region of the apical pole to form a germinal disc in which is contained the nucleus. Irregular prolongations of the superficial protoplasm may sometimes, especially in immature eggs, be traced inwards into the substance of the yolk.

A characteristic feature of many teleosts is the tendency for the yolk to assume a liquid form. This is particularly marked in many pelagic eggs where it is not merely liquefied but runs together at the time of spawning or of fertilization to form a sphere of glassy transparency. There may further be, interspersed amongst the ordinary yolk, droplets of oily looking fluid often with a distinctive colour. These may unite into a few droplets or into a single larger drop forming a conspicuous, often coloured, sphere in the midst of the ordinary_ yolk. The colour and size of such drops frequently afford an easy means of recognizing the species to which a particular egg belongs. They may also have a characteristic position and may be surrounded by a special condensation of protoplasm or, on the other hand, they may simply float freely in the main mass of fluid yolk. Although these droplets may, as already indicated, exhibit peculiarities characteristic of particular species they do not seem to give indications of genetic affinity in regard to genera or larger groups: nor do they show any definite relation to the conditions, pelagic or otherwise, under which the egg develops (Prince, 1886).

The yolk of teleosts is also characterized by a diminution of its specific gravity which causes the egg to assume a reversed position with the apical pole below, and which further, in the case of a vast number of marine fishes, causes the egg as a whole to float freely suspended in the sea water.

Seeing that the Teleostei as a group is above all characterized by specialization for a swimming existence, independent of a solid substratum, we are perhaps justified in assurning that the freely floating pelagic mode of development above mentioned was originally present throughout the group. The demersal type of development, where the eggs are deposited on the "solid substratum, would then I SEGMENTATION 21

be regarded as a secondary reversion to, rather than a persistence of, a pre-teleostean habit. Possibly the reversed position of the egg is to be regarded as a means of protecting its more sensitive apical portion from injury by contact with the surface film of the water in which it floats.

When fertilization takes place the most conspicuous immediate result is the onset of a gradual concentration of the protoplasm in the germinal disc-—-the disc becoming at the same time more heaped up, its vertical diameter increasing and its horizontal diminishing.

The segmentation of the germinal disc in A teleostean fishes is usually of a very regular and characteristic kind. It is illustrated as seen in surface View by Fig. 12. The germinal disc lengthens out into an elliptical shape.

The first furrow to appear (A) is meridional B and occupies the shorter diameter of the ellipse.

The second furrow is also meridional and in a plane perpendicular to that of the first. The third and fourth sets of furrows (B, C, D) are vertical and they become arranged so as to be practically parallel to the first and second, with C the result that the hlastoderni as seen in surface

view assumes a very characteristic arrangement of sixteen segrnen ts arranged in four rows

(Fig. 12, D).

The internal phenomena of segmentation D may he (lescribed from what occurs in the Trout (Kopsch, 1911). In the first place it has to be noted that the early furrows do not extend Fm 1.-_,_ _Segmentati°n right through the substance of the germinal in the blastodermofa disc but leave a continuous basal stratum of teleosteall fish (3% protoplasm next the. yolk. The blastoderm assumes a two-1ayered condition by the 3rd and wnson, 1391,) 4th furrows curving round in their deeper portions so as to intersect the preceding division-planes which were throughoutperpendicular to the surface (Fig. 13, B). Up to the 16cell stage all the segments remain connected by broad protoplasmic bridges apart from the continuous basal layer of protoplasm which connects the deepest cells together.

In the 32-cell stage (Fig. 13, C) the cells of the superficial layer have become completely isolated while the deep cells are still connected together. With the next division the blastoderm becomes three layered, the cells of the intermediate layer being derived some from the superficial, some from the deep layer, as isshown by the evidence of broad bridges of protoplasm which persist here and there between sister cells.

With the next division (128-cell stage, Fig. 13, D) the four-layer 22 EMBRYOLOGY or THE LOWER VERTEBRATES _ on.

condition is reached, the cells of the basal layer being still connected by a thick continuous stratum of protoplasm. By this time it is found that the nuclear divisions of the basal layer are clearly lagging behind those of the other layers. As segmentation proceeds further the continuous basal sheet of protoplasm decreases relatively in thickness. For a time (Fig. 13, E) bulgings of its upper surface indicate that it is giving off cells into the overlying layer but as the thinning process goes on these become less and less numerous. H. Virchow distinguished three zones in the basal sheet of protoplasm-——marginal, intermediate and central, although the latter

Fm. .‘.:’..—-\’m'ii«_-ui sq-.(-t'.ioiis' through the blastoderin of a Tcleost (iS'a..lm.ofa.rz'o) illustrating the prov-ess of Seglnelltntion. (After Kopsch, 191].)

A, and of second division. Section perpendicular to plane of first furrow which is therefore seen cut across. B, commencement of fourth division. Plane of section as in A. The division surfaces

of the third division are seen to curve inwards so as to meet the first division surface. As 9. result the latter has become distorted and no longer forms a plane. 0, middle of sixi.ii division. D, beginning of eighth division. E, beginning of ti-nth division. F, 62-hour l)last;0(,lt-rln. (The dark portion at the top oi‘ Fig. .13 represents the free S1li'f:u-v bounding the second furrow: the dark tone at the lower edge of (.'.ii('ll ligurc represents yolk.)

is not quite central but situated rather towards the posterior or embryonic edge of the blastoderm. The intermediate zone is marked off from the others by the fact that the thinning process has there progressed farther.

Up to about the twelfth division the nuclei all through the blastoderm di.vide practically synchronously except those ‘of the basal layer which as already indicated lag behind. Soon after this however (from about the 41st hour——Kopsch) the divisions become irregular. '

The basal layer becomes the yolk-syncytium: the cell limits visible on its upper side liieeome obliterated and it becomes more and more flattened out. Altlmugli its nuclei l.lli('lGl?g(i repeated mitosis I SEGMENTATION ' 23

there is no longer any budding off of cells, the nuclei simply lying within the substance of the syneytium.

As they increase in number nuclei from the central and marginal regions spread into the intermediate zone, which up to now contained very few nuclei, while others pass outwards into the peripheral protoplasm (Peribla.st;—-Agassiz and Whitman, 1885) lying outside the limits of the blastoderm.

Towards the end of the second day the syncytial nuclei begin to increase markedly in size and they begin to undergo abnormal multipolar mitoses. During the third day they complete the assumption of these peculiarities which are characteristic of the nuclei of a yolk-syncytium——enormous size,curiously lobed appearance, and the tendency for the lobes to become nipped off irregularly so as to give rise to groups of small nuclei.

During these later stages of segmentation the hlastoderm becomes flattened somewhat and instead of bulging out over its attached base all round, its surface passes into the extrablastodermic surface by a slope very much as it did before segmentation began (Fig. 13, I4‘).

ACTINOPTERYGIAN GANOIDS.-—-The ganoid fishes are of special emhryologieal importance because, so far as actinopterygians are concerned, they appear to be the least modified descendants of those ancestral forms from which the Teleostean fishes have been evolved. Study of their developmental phenomena is desirable in order to see to what extent they throw light upon the peculiarities of development which characterize the Teleostean fishes. It will be necessary therefore to review the segmentation processes so far as they are known in each of the three types———the Sturgeon, Amia and Lcpiclosteus.

The only Sturgeons of which anything is known regarding their early development are the common sturgeons of the genus Acipenser. Poly/odon, I’sep/tmws and Scaplmlrhynchus are so far completely unknown, though it is highly desirable that their development should be investigated.

In both Acripenscr Irutltenus (Salensky, 1878) and A. sturio (Bashford Dean, 1895) the segmentation (Fig. 14, A) is of the same general type. The unsegmented egg measures about 2 mm. in diameter in the Sterlet (A. ruthen-Ms), about 2'8 mm. in the Sturgeon (A. sturlio). The lower part of the egg contains coarse yolk granules while in the region of the apical pole it is richer in protoplasm and the yolk is more finely granular. The first furrow (a) is meridional, appearing first at theapical pole and gradually spreading downwards and at the same time cutting more deeply into the yolk. The second furrow (,8) is similar and at right angles to the first. The third set of furrows seem to be typically vertical (A 2) but they show

much variation and may be practically meridional or may show a.

tendency to be latitudinal. The next set of furrows again vary between vertical and latitudinal and from now onwards there is no apparent regularity in the segmenting of the various blastomeres. There 24 EMBRYOLOGY OF THE LOVVER VERTEBRATES CH.

eventually results a blastula (A 5) the upper portion of which, forming rather less than a hemisphere, is composed of micromeres while the lower part is composed of lame, richly yolked, macromeres.

In Amxia the “ oval ”-shaped thgg n’..s=,;..~«.en~.~.a shout 2'5-2‘. mm. hy

about 2-26 mm. In the region of the apical pole which lies at the end of the long axis is s cap-slnapmil portion richer in protop_lasm—an approach in fact to a germinal disc-——while the 1'cS’t of the egg is rich in dark gr:-1.yi_sh brown yolk. ‘

The segmentation (Fig. 14, B) begins, about an hour and a, half I SEGM ENTATION 25

after fertilization, with the successive appearance, at the apical pole, of two meridional furrows (u. and ,8) which gradually sweep downwards to the opposite pole of the egg. Before they reach it, four vertical furrows make their appearance, commencing at a point on furrow (1. not far from the pole and gradually extending downwards over the lower part of the egg (Fig. 14, ll 1).

Before these vertical furrows reach the lower pole a new furrow ~—-latitudinal———deve1ops a short distance from the apical pole, marking oil’ a polar group of eight micromeres (Fig. 14, B 2). At the next division these divide into a superficial and a deep seglnent (the former being separate——-the latter continuous with’ the yolky mass beneath) while the macromeres divide by vertical furrows.

Next an irregular latitudinal furrow develops below the previously existing one, by which a new micromerc is segmented off from the upper end of each macromere.

liegnldostezts.-———Tl1e ellipsoidal or “oval” egg measures about 3'5 mm. by 3'2 mm. and has a cap of protoplasm with fine grained yolk at its apical pole.

Segmentation (Fig. 14, U) in its early stage is like that of A7n'i0r, except that the furrows are more sluggish in spreading downwards over the egg-surface. They never in fact reach much beyond the equator; in other words, in the case of Lepz'zI0steu.s~, the lower hemisphere of the egg does not normally segment at all. The egg therefore has advanced beyond the condition seen in A7nz'a, and has become meroblastic.

In the later stages of segmentation the region of the upper pole is occupied by a lentieular mass of blastomeres which may be termed the blastoderm, and this is bounded at its lower edge and over its lower surface by a set of elements which remain in continuity with the yolk. Later on the divisions between these elements tend to disappear and their place becomes occupied by a “ yolk-syncytium ” containing numerous nuclei.

To summarize then, we have exemplified by the three ganoids Acipenser, A.’ln’t(t and Lepidosteus, three steps in evolutionary change, associated with an increasing degree of telolecithality, from the holoblastic type of egg met with in Lampreys or Amphibians or Orossopterygians to the meroblastic type as it exists in modern Teleosts.

LUNG-FISHES.——Tl1e early stages of segmentation have been observed in two out of the three still existing lung-fishes--C’emtodus (Semon, 1893) and Lepz'dos'i7'en (Graham Kerr, 1900).

In the case of Oeratodus the egg measures about 3 mm. in diameter and is pigmented in the neighbourhood of the apical pole. The first two furrows (Fig. 15, A 2 and 3) are meridional and at right angles to one another. Each appears first at the apical pole and extends downwards with varying rapidity. The third set of furrows are vertical and make their appearance usually before the second meridional furrow (B) has reached the lower pole. The egg 26 EMBRYOLOGY or THE LOWER VERTEBRATES on.

becomes thus divided into eight practically equal blastomeres.

A latitudinal furrow then develops about 715° above the equator, so that the egg now consists of eight micromeres round the apical pole and eight macromeres.

After this stage segmentation usually becomes irregular» although sometimes two additional latitudinal furrows make their appearance in succession so that the egg consists of four tiers each of eight blasteineres. Eventually, as segmentation proceeds, a blastula is formed of the type shown "in Fig. 15, A 7.

The segmentation cavity first appears about the time of the fourth cleavage as a small chink. It rapidly expands and in the blastula figured (Fig. 15, A 7) it is of large size.

In Lepidosiren (Fig.

'15, B) the egg measures usually between 6'5 and 7 mm. in diameter. It is free from pigment in correlation with the fact that it develops in a burrow Sl1mil.ml

'l'1‘OIl1 the action of light. In the region of the apical pole is a whitish cap in which the yolk is in very minute particles while elsewhere i.t is in large coarse« granules.

The first two furrows (Fig. 15-, B 2 and 3) are meridional and at right angles to one another. The third

, set of furrows (Fig. 1.5, B 4) are vertical tlioiigli oceaI SEGMENTATION 27

sionally one or other of them may become latitudinal. The various meridional and vertical furrows gradually extend downwards towards the lower pole of the egg in the order of their appearance, and during the earlier stages the lower hemisphere of the egg possesses only such furrows (Fig. 15, 'B 6).

As the blastomeres go on segmenting there is produced eventually a blastula with an upper hemisphere of small cells which appear white because of the finely subdivided condition of their yolk and a lower hemisphere of larger more yolky elemcn ts (Fig. 15, B 7).

Already at the stage when the egg is divided into four segments a space develops between the blastomeres. As segmentation goes on the micromeres tend to round themselves ofl', leaving wide chinks between containing fluid. By the blastula stage the fluid has collected together into a spacious segmentation cavity which is visible in the whole egg as a dark shadow in its upper hemisphere. At first the cavity is rounded and is roofed in by a single layer of cells but later it spreads out, takes a planoconvex form and its roof comes to be composed of two layers of closely apposed cells.

AMPIIIBIA.——The Amphibia are in the matter of segmentation the most interesting and important group of the vertebrata, for in no other group does there exist so much variety in the proportional amount of yolk present in the egg. Much work still remains to be done in regard to this group in the way of detailed study of the process of segmentation in its relation to the amount and concentration of the yolk.

As already indicated the extent of the influence which the yolk exerts in retarding the living activities of the protoplasm, such as growth and division, bears a rough relation to its proportional amount. As regards the majority of cases this may be said to vary directly with the size of the egg. The largest eggs are as a general rule the most richly yolked. But the rule is by no means an invariable one that the influence on the segmentation is directly proportional to the total amount of yolk in the egg as a whole. For a smaller egg, containing a smaller amount of yolk, may yet have that yolk more concentrated in one region so as to produce there a more intense retarding influence——as is the case naturally in many small Teleostean eggs or as may be demonstrated experimentally by concentrating the yolk artificially through the action of centrifugal force. 0. Hertwig was able by centrifugalizing frogs’ eggs and so causing the yolk to become concentrated in the abapical hemisphere, to bring about a complete cessation of cleavage in that hemisphere so that the egg thus assumed a meroblastic character.

The variety in the size of the egg within the limits of the group Amphibia has already been indicated by the table on page 2. The process of segmentation agrees in the main with what has been described for the frog but there is much variation in detail. The variations have to do both with the position of the furrows and with their appearance in point of time. One gets a good idea of the 28 EMBRYOLOGY OF-THE LOWER VERTEBRATES CII.

general tendency of variation by studying numerous eggs of a single species, for example in the case of Rana pal'ustr75s Jordan and ' Eycleshymer (1894) found amongst other variations in the mode of appearance of the first furrows, those illustrated in Fig. 6 (p. 12). And similar difi“ercnces occur between the eggs of different species.

‘.1

A B C

l"|H. 16. -Variations in topographical relations of early segmentation l'urrows in the egg of [tuna h'))I])(r/'41,!‘/(I. (A. B after Morgan, 1897 ; C after Jenkinson, 1913.)

Tlw llguie in each (-use I'('])['0S(‘lltS a. \ new of the apical pole of the 1-gg,

As regards difference in position of the furrows two of the coninionest variations are the following. At the four-hlastomerc stage two blastomeres may be pressed outwards from the apical pole as in Fig. 16, A. Again meridional furrows may be replaced hy vertical furrows as in Fig. 16, B and 0.

As regards variations in time these are chiefly associated with the retarding of segmentation in the lower yolk-laden segments. This reaches its maximum, so far as Ainphihians are concerned, in the Gy1nnophiona,where segmentation spreads so slowly into the lower

Flo. 17.——Vertical section through apical portion of egg of lclat/z_2/oplus at an advanced stage of segmentation. (After 1’. and F. Sarasiu.)

parts of the egg that during what are ordinarily called the segmentation stages the yolk remains completely uncleaved. It would in fact be concluded from an inspection of these stages alone that the egg is a meroblastic one. Examination of later (gastrulation) stages however shows that the yolk does eventually segment although tardily.

Upon the whole it seems to be the‘ case that the Urodele egg 1 SEGMENTATION 29

segments more slowly-—during at least the first stages—~than does that of the Anura. It may be said also that, on the whole, eggs with a large mass of yolk show a tendency for the first latitudinal furrows to be nearer the apical pole, so that the micromeres which they cut off are relatively smaller. Also it seems to be the case that in the lower, more yolky, parts of the egg the latitudinal furrows are retarded to a particularly great extent, so that in such heavily yolked eggs there is frequently visible a preponderance of vertical and meridional furrows in the lower parts of the egg.

ELASMoBRANUH11.——Of the more typically meroblastic vertebrates the Elasmobranchs call for little in the way of further remarks. The general features of their segmentation have already been described (p. 12).

The eggs of all Elasmobranchs hitherto investigated are of large size and undergo a meroblastic segmentation. Up to the present time no Elasmobranch has been discovered in which the eggs are small and holoblastic, though it is quite possible that such forms exist. It need hardly be said that if they do the study of their embryology will be of extraordinary importance as it Will be of the greatest help in enabling us to disentangle those developmental phenomena of Elasmobranchs which are primitive from those which are merely secondary modifications due to the accumulation of yolk.

SAUROPSlDA.—-The Sauropsida, like the Elasmobranchs, possess large and richly yolked eggs with a meroblastic segmentation, but the process of segregation of yolk and protoplasm has not been carried to such an extreme as in Elasmobranchs, not to mention Teleosts. A germinal disc is present but this still contains a considerable amount of yolk and at its periphery passes by much more gradual transitions into the main mass of yolk. Further in the more primitive Reptiles the blastoderm frequently occupies a much larger proportion of the whole egg than it does in the Elasmobranch.

The general features of segmentation resemble those of Elasmobranchs though the earliest phases depart in many cases less than they do in Elasmobranehs from what is seen in holoblastic eggs. Thus the process may commence with the appearance of a meridional furrow followed by a second at right angles to it and then by two pairs of vertical furrows very much as in an actinopterygian ganoid (Fig. 14, B and C).

This is seen most clearly in the less specialized egg of Reptiles. Even in the Reptile however the process is liable to become irregular at an early stage by the reduction of particular furrows or their irregular orientation. In the Birds (Patterson, 1910) the irregularity is still more marked and even the third set of furrows may no longer be clearly recognizable.

As in the case of other bulky and heavily yolked eggs polyspermy appears to occur normally and an abortive accessory segmentation may make its appearance round the accessory sperm-nuclei. As in the Elasmobraneh (Fig. 8, B”) this is only a transient 30 EMBRYOLOGY or THE LOWER VERTEBRATES on.

phenomenon the accessory furrows flattening out and disappearing as the accessory sperm-nuclei degenerate.

Again as in the Elasmobranch a yolk-syncytium is developed beneath and around the segmented portion of the blastoderm.

A marked difference between the Sauropsidan ‘ and the Elasmobranch type of egg at a fairly advanced stage of segmentation becomes apparent on comparing them with the corresponding stages of eggs of a less markedly telolecithal character (e.g. Fig. 14). It is seen that the blastoderm in an Elasmobranch such as that shown in Fig. 8 I), E corresponds to the mass of micromeres of the holoblastic egg, while in the Sauropsidan it corresponds to the mass of micromeres together with the apical ends of the large macromeres.

This is really an expression of the fact that in the Sauropsidan the germinal disc extends outwards into the main yolk, and shades oil’ gradually into it. The result is that the segmentation process in the outer portion of the blastodcrm is delayed by the presence of yolk precisely in the same way as in the lower portion of the holeblastic egg.

GASTRULATION

The segmentation process is in the more primitive Vertebrates, as in many other groups of the Metazoa, succeeded by a process of gastrulation, in which the blastula becomes converted into a gastrula 'i.e. a type of embryo consisting of the two primary cell-layers, ectoderm and endoderm, enclosing a cavity, the archenteron, which corresponds morphologically with the coelenteron of the Coelenterate and which opens freely to the exterior.

While the process of gastrulation is fairly clear in the most primitive vertebrates it becomes less and less so in the more highly modified members of the group until finally in the Amniota it becomes completely obscured. To facilitate the understanding of the modifications which the process of gastrulation undergoes it will be well to study it first as it occurs in three of the more primitive Vertebrates namely Amp}:/iomus, Poly/pterus, and Lepidosvlren.

(1) AMPHIOXUs.———The blastula of A1n10h'i0ams is composed of a single layer of cells, those towards the apical pole being smaller, those on the opposite side being larger and containing in their cytoplasm larger and more numerous granules of yolk. The process of gastrulation is ushered in by the large-celled portion of the blastulawall becoming flattened as shown in Fig. 18 A. The portion of the flattened area which, as shown by later stages, is anterior in position develops a slight depression (Fig. 18, B) which gradually deepens and at the same time spreads backwards (Fig. 18, C, D, E, F, G) until the large-celled portion of the embryo. is completely invaginated within the small-celled portion and the two-layered gastrula stage is attained. _

‘ See Chapter X., Segmentation of'Fow1’s egg, F, G, H. } ASTRU LAT .10 N 31

Had the process been merely as stated the result would have been a gastrula with a very wide mouth. But along with the

process of involution there takes place an active growth of the lip or rim of the gastrula. This growth is especially active anteriorly as is shown by the fact that mitotic figures are most numerous in this region and become less and less frequent towards the posterior part of the rim. The result is that the original mouth of the gas trula —— the proto-'

stoma.-—becomes gradually encroached upon by the gastrular lip—— the encroachment being most marked anteriorly so that the opening becomes not merely diminished in size but also appears to shift its position towards what will become the posterior end ‘of the embryo. It will be noticed thatwhat really happens is not a process of shifting of the opening as a whole, but rather the persistence of the binder portion of the opening while its anterior portion has disap FIG. 18.—-«Illustrating the process of gastrulation in .-1 mph-z'o:w.s as described by Cerfontaine. The second polar body marks

the neighbourhood of the apical pole. The individual figures are viewed from what is seen later on to be the left side of the Amphioxns, the dorsal side being above and the head end pointing towarils the left side of the page.

peared. Such a remnant of the original protostoma may conveni ently be known by the special term blastopore. Expressed somewhat differently-—the cavity of the gastrula has 32 EMBRYOLOGY 014‘ THE LOWER VERTEBRATES CH.

become roofed in by a process of overgrowth on the part of its lip. The most conspicuous factor in this process consists of backgrowth of the anterior portion of the lip while the growth of the lateral portions inwards towards the mesial plane (so as to narrow the opening from side to side) is less and less active the greater the distance from the anterior end, until finally, in the extreme posterior portion of the lip, such growth as takes place is relatively inconspicuous.

In the process of gastrulation in Awipltiowus there are then two distinct processes at work ( 1) a process of invagination or involution of the large-celled portion of the wall of the blastula and (2) a process of overgrowth, most pronounced in the case of that portion of the gastrular lip which is originally anterior. By the agency of (1) there are established the two primary cell—layers——-ectoderm and endoderm while by the agency of (2) there is formed the dorsal wall of the embryo with its potential later developments such as central nervous system and notoehord.

It will be noticed that the originally anterior portion of the gastrular rim, wl1en it has completed its backgrowtb, lies above, dorsal to, the now greatly diminished gastrular opening or blastepore. Consequently it comes to be spoken of as the dorsal lip of the blastopore.

Although, strictly speaking, the terms endoderm and ectoderm are expressive of topographical relation a11d their use is permissible only after the one layer has become at least partially invaginated within the other, yet it should be carefully borne in mind that these two primary layers have already become distinctly differentiated from one another during the blastula stage, long before the process of invagination begins. One might indeed go farther and say that endodermal characteristics, e._r]. richness in yolk, have already made themselves apparent in the abapical portion of the egg even before segmentation begins.

This fact is of far-reaching importance as we shall find in other Vertebrates that the histological characteristics of ectoderm and endoderm become apparent not merely before the actual process of gastrulation takes place but, it may be, completely independently of that process. '

(2) PoLYP'1‘EnUs.—-ln 1’ol3/pteras an early stage in the process of gastrulation (Fig. 19, B) shows a well-marked groove encircling the egg a short distance on the abapical side of the equator. This groove marks the line along which involution of the egg-surface is taking place and its adapical lip represents the lip of the gastrula. It is to be concluded from a still earlier stage observed and drawn by Budgett (Fig. 19, A) that the involution groove appears first in the region corresponding to the anterior lip of the gastrula of Amjolz/iozzms and gradually becomes extended at its two ends until complete. This is of importance as betraying a tendency for the invaginative activity to be accentuated in this portion of the gastrular lip and diminished elsewhere. - hemisphere. As a

I GA STRU L A'l‘ ION 33

The fact that the yolk portion of the hlastnla consists not of a single layer of cells as in Am,phi0w'u,s but of a solid bulky mass forming a large proportion of the whole hlastula, renders it physically impossible for the yolk hemisphere to he involutcd bodily in to the interior of the apical

consequence we find in Polypterus that the process of involution is replaced to a greater extent than in A71Lp}m'ox~zz.s by overgrowth, the gastrular lip g'I‘0Wing over the mass of yolk-cells as seen in Fig. 19, C, D and E. As this process of overgrowth continues the projecting yo1k—plug-—the mass of yolk-cells not yet enclosed -—gradua1ly ‘diminishes in size and eventually disappears completely in the new narrow blastopore. As yet material isnot available to show definitely‘ whether the overgrowth is more active in what corresponds to the anterior portion of tlie gastrular lip of

Amp}:/iowus but the ,

bili is in FIG. 19. -------lllnsl.)‘:1l.ing prm.-ess of g:ist.1-11l:1tio11 in Poly/ptm--zas. proba 1t-‘y - - (I“i}_r.<. A, U, E, F al'l.er <lra\\'ing.:s by Budgett.) favour 01 this being i

the case and the _r/.1, gastrular lip; ;;.,», yell:-}'»lu;_-'. The egg is in em-h case \'iewml fi ‘ - from the ltffli side and has the dorsal si«.l«.- .-chm-e. The ori;,-in.-Il apirul gures ale Omen‘ pole is directed downwards and towards the left side of the page as in

tated 011 the assump- the preceding figure ol‘ A"-mph.iu.:__u.-.

tion that this is so. The two features to be specially noted in the gastrulation of

l’0l3/ptems as compared with that of Amphz'oa:-us are (1) the

accentuation of the process of overgrowth and the reduction of the

process of involution and (2) the tendency, in early stages -at least, VOL. II D 34 EMBRYOLOGY or THE LOWER VERTEBRATES on.

for the invaginative activity to be diminished along the region corresponding to the posterior part of the gastrular rim of Amphioasus.

(3) LEPIDOSIREN.-——-The first sign of gastrulation is afforded by the appearance, at short distance to the abapical side of the equator, of a latitudinally arranged row of small dimples or depressions of the surface which soon become joined up to form a continuous invaginationgroove. This (Fig. 20, A and B) may extend through about one—third of the circumference of the e g but in contrast with what happens in Polypterus the groove, instead of increasing in length, becomes shorter, flattening out and disappearing at its two ends. ' ‘

The finalstage is seen in Fig. 20, E, where the gastrular lip is

FIG. 20. Illustrating the process of gastrulation in Le}nfdos'zIre'7c.

Fig. A is a side view, the egg being OI'icntab6d so as to correspond with the ligures of .-lm.pIn‘oxu.- and 1’olypte~rus, the largecclled yolky abapicai portion of the egg being above and towards the right hand. Figs. B to E are views looking directly at the gastrular rim (dorsal lip of the blastopore), or in the case of E directly at the completed l)last()p0I‘e. Consequently, as the gastrular rim is during these phases of development not stationary, the views 13 to E are not orientated _morphologica1ly in exactly the same way.

short, and curved into a crescent, forming the dorsal boundary of the blastopore. ' b

At. this stage the large yolk-cells with their conspicuous salmon colour have been completely covered in by small cells-——-a condition that has been brought about through the agency of two distinct factors (1) the process of overgrowth with which we have already become familiar and (2) a new process to which the name delamination is given.

As is shown by the sections drawn in Fig. 21, the yolky or abapical portion of the blastula-wall is in Lepidoszren, as it was in Polypterus, far too bulky to be involuted bodily as was the case in Amplmlozcus. Again the enclosing of the yolky mass by the gastrular lip growing overit as in Poly/pterus is rendered impossible by the fact that the gastrular lip is normally here never completed to form an entire circle. It is, as has been explained, restricted to a comparatively small linear extent.

This small persisting portion of gastrular lip probably does advance over the surface of the yolk by a process of overgrowth, giving rise in this way, just as in Amplmioxus, to what will become the dorsal wall of the embryo with its central nervous system and notochord. That this is the case seems to be indicated by sagittal sections through eggs cut in celloidin while still contained within I GASTRULATION 35

the egg-shell. In these the gastrular lip has a distinct wedge shape this being apparently impressed upon it as it pushes its way between the egg-shell and the surface of the yolk. Corroborative evidence is afforded by the numerous mitotic figures found throughout the

Fm. 2l.——Sagittal sections illustrating gastrulation in Lepidosirm. _f].l, g‘:1sbrul.'u‘ lip.

thickness of the archenteric roof which indicate that it is undergoing rapid - growth.

It will be gathered, however, from a consideration of Fig. 20 that those parts of the margin of the s1nal1—celled region of the egg’s surface which are not involuted to form a groove, must also advance over the surface of the yolk, for the-.sc parts ol' the margin form at first (Fig. 20, A and B) practically a great circle of the egg, while in subsequent stages (0 and D) they form a curve of gradually diminishing radius.

The method by which the small-celled area extends is shown 36 EMBRYOLOGY OF THE LOWER V ERTEBRATES UH.

clearly in Fig. 22, A, where it is seen that portions of large yolk-cells adjacent’ to the small-celled area hecorne split off as small cells which are added to that area. It is to this splitting-ofl’ process that the

1nm1e.delanaina,tion is applied. It is clear, then, that in the gastriilatioii of Lepidosrirevt three

Fm. 22.-——l’orti0ns of sagittal sections of Lepidosilren egg during early stages of gastrulation.

A, showing process of delamination. The small-celled ectoderm is seen on the left: it is becoming extended by the addition, to its lower edge, of cells split on‘ from the yolk-cells. The latter are recognizable by their larger size and by the larger size of the yolk-granules with which their cytoplasm is laden. B and 0 showing involution of the surface along the invagination-groove.

processes are at Work (1) Involution of the surface———this is conspicuous in the first stages (see Fig. 22, B and C), (2) Overgrowth by the gastrular lip—the “dorsal” lip as it is commonly termed‘ from its ultimate position, and (3) Delamination. These same three factors are at work in the gastrulation of the lower Vertebrates in general, and a. clear realizing of their nature is iiecessary to a comprehension I GASTRULATION 37

of the superficially very different gastrulation-phenomena observable in the various groups. .

With regard to the first of these processes, involution of the surface, it must be clearly understood that such appearances as that depicted in Fig. 21, B, point indubitably to the occurrence of a true process of invagination or involution of the surface of the egg. It is necessary to emphasize this as some embryologists are sceptical as to the occurrence of true invagination and believe that a. more important part in the formation of the archenteric cavity is played by a mere cleavage, or splitting apart, of the cells which are to form the roof and the floor respectively. Brachet (1903), indeed, goes the length of stating in regard to Lrpidosireot and Protopterus, with only the data published by myself before him, that “ the first trace of the archenteron is due to a cleavage, the result of which is- the formation of a slit” a statement which is certainly not justified.

On the other hand it should also be borne in mind that there is no difiiculty a priorzl in the way of admitting that portions of enteric cavity may come to arise secondarily by a process of splitting in the midst of a solid mass of endoderm or yolk-cells. This type of modification in the embryonic development of organs which were originally formed by invagination or cvagination is one which occurs quite frequently. Numerous examples of it are mentioned in the course of this Volume. '

Before closing this account of the gastrulation of Lcp'i(losz"re7L attention should be drawn to a remarkable and important phenomenon which has been observed in both L61)z'rI08'i'/'e'It and P7"0t0pte'r71s. During the early stages of gastrulation, while the segmentation cavity is widely patent, the small blastomeres in the neighbourhood of its abapical side are seen, where not distorted by pressure from their neighbours, to be approximately spherical in shape. Elementary physics teaches that this is an indication that they are isolated masscs——that their protoplasmic substance is not continuous from cell to cell. In a later stage (Fig. 21, C) however; the blastomeres in the region of the segmentation cavity do become continuous with their neighbours and form a coarse reticulum traversing the cavity, the fluid contents of which now fill the meshes of the network.

The importance of the phenomenon described lies in the fact that here we have an actual case, clearly demonstrable, of isolated embryonic elements fusing to form a syncytial reticulum-—a type of process which may probably, as will be indicated later, play an important part in the development of the Vertebrate nervous system.

GASTRULATION IN THE VARIOUS GROUPS OF ANAMNIA

LAMPREYS.-—-—The Lamprey (Petromg/zen. fium°atz'l'is) shows in its gastrulation (Fig. 23) an intermediate condition between that of Amphiomus and that of the more heavily yolked holoblastic forms. The abapical portion ‘of the blastula is yolk-laden and thickened, 38 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

though not to the extent seen in Polyptems. The segmentation cavity remains, therefore, relatively capaeious so as to permit of a considerable amount of imagination of the yolk mass into it. ‘

The gastrulation process so far as can be judged seems to conslst mainly if not entirely as in Avie];/mioams of invagination and (2) overgrowth, only in this case the relative importance of the former has been lessened and that of the latter increased,

AMPIIIBIA.--It will be convenient to consider first the gastrulation-phenomena as seen in the common frog (Ra/rm tempowma), this animal having been more exhaustively studied than has any other Amphibian. '

The first sign of the onset of gastrulation is the appearance of a short latitudinal linear involution (Fig. 24, (1,) of the surface of the blas

“\.~.(\\\‘.“\";.llll,‘ , .&‘\‘t l'\'ivil'i’I;i;""i . ’ I . ‘W... "

0 ,9 ‘I

. ‘ - u c O I I ‘ ‘ I "

Flo. 23.——-Gastrulation in J’etrom,yzo7t : based on Goette’s figures (1890).

The individual sections are orientated in the same way as the corresponding sections in Figs. 18 and 21.

tula considerably on the abapical side of the equator(ahout 25 °', Kopsch) and just at the boundary-of the large-celled region. It appears on that side of the egg on which the blastula roof is commonly rather thinner than it is elsewhere. This involution groove, as seen in a surface view of the egg, extends laterally and as it does so assumes a crescentic curvature (Fig. 24, b). The extension of the groove in length continues while its radius of curvature diminishes until finally it forms a closed circle (Fig. 24, b, c, d). i

The groove at its first appearance lies close to the boundary between the small cells of the apical region-—---characterized in the frog by their dense black pigment—and the large pale-yellow yolkcells. During subsequent stages the groove continues to mark the boundary between the two types of cell, so that in the last stage mentioned when the groove forms a complete circle the mass of almost white yolk-cells within it (yolk—plug) stands out in striking contrast with the coal-black cells covering the rest of the egg surface. I GASTRULATION 39

As will be gathered from an inspection of Fig. 24 the gradual covering in of the yolk-cells takes place in an eccentric fashion. On the side opposite to that on which the original involution groove makes its appearance there is comparatively little displacement of the boundary between large cells and small, while on the side where the groove is the displacement is relatively great——l'rom a to (Z in the diagram. Intermediate points of the boundary between large and small cells are displaced more or less according to their greater or less proximity to the point of original involution.

As regards the method by which the yolk-cells become covered

in, it would appear that the “dorsal” lip of the groove advances over the yolk by “ a process of overgrowth, while at those parts of the boundary where there is no invagination-groove the process is one of delamination. The growth of the dorsal lip is clearly indicated .by the outline of the yolk-plug in sagittal sections which indicates distortion by pressure from the dorsal lip.

It will ‘be realized from what has already been said that the outer lip of the circular groove (Fig 247 (1) is simply Fm. 24.--Diagram to illustrate the rim of the gastrula-mouth or prot0— overgrowth by dorsal lip of stoma and that the preceding stages are "“°S‘l°P°"“ i“_”‘° F"°=‘=" i“'‘‘'‘' above all characterized by this rim being M°‘g“"’ 189") i“‘3°mP1"te-. In OW? W013“ the *‘°“i"it3' i,.§r.'.’1°..t1s‘.T§Tsg£i;..i7;Ii,in-i°.333§.‘§3':Z'l.i;.',Z'.3§ concerned in the involution of the gas— of dev(slopme[[t_ trular rim is accentuated at one point (a) while it is suppressed to such an extent elsewhere as only to become apparent at a comparatively late stage when the edge of the small

°celled region has already spread to a great extent over the yolk-cells

by a process of delamination.

It will also be realized that it is not strictly accurate to speak of the circular area bounded by small cells as representing the gastrulamouth until it is completely enclosed by the gastrular rim.

The internal changes which accompany the phenomena just described are illustrated by the sagittal sections shown in Fig. 25. In C the portion of the involution groove which first appeared has become much deepened and runs for some distance parallel to the surface as the archenteric cavity.. It is bounded superficially by a completed portion of gastrular wall showing the two primary cell layers, ectoderm and endoderm.

Some of the yolk-cells round the margin of the segmentation cavity are frequently to be seen, though not in the section figured, to be spreading along the inner surface of its roof, towards the point which was the apical pole of the blastula. ' .

In the later stages the overgrowth bv the gastrular lip, 40 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

accompanied, no doubt, by a eer_tain amount of involution though this is difleieult to determine with certainty, has proceeded much

8.6.

Fm. 25.—-Sagittal sections through the egg of Rana tem_p0-7'u.1'1'a illustrating the process of gastrulation. (After J enkinson, 1913.)

act, ectodenn; ent, an-clu-.ntex-ie (.-.a.vity; g.l, gs.st.rula.r lip; s.«-, segmentation calvity; 31.9), yolk-plug. The arrow indicates the original apical pole.

further so that the archenteron is much deeper. The spreading of the yolk-cells along the roof of L_ the segmentation cavity, already I GASTRULATION 41

alluded to, has in these later stages sometimes proceeded so far that that cavity is nearly completely walled in by yolk-cells.

While the archenteric cavity increases in volume the segmentation cavity becomes gradually rcduced. Normally the latter cavity goes on shrinking until it is finally obliterated but according to O. Schultze (1887) a certain proportion of eggs show a variation from the normal which appears to be of importance for the interpretation of what happens regularly in certain other groups. As seen in Fig. 25, E, the layer of yolk-cells which separates the archenteron from the segmentation cavity is liable to become extremely thin, and Schultze believes that in certain cases this thin partition breaks down and disappears, so that the archenteric and segmentation cavities are thrown into one. What appears at first sight to be the archenteric cavity of subsequent stages would in such cases be really a complex consisting of the true archenteron fused with the remains of the segmentation cavity.

If these observations are to be depended upon, they are of very special interest. For, if the confluence of arehenteric and segmentation cavity really occurs as an occasional variation in such Amphibians as the Frog, this may be taken as a foreshadowing of the similar phenomenon which has become a normal characteristic of the development of many of the higher Vertebrates.

It must however be borne in mind that there exists a dangerous source of possible errors of observation, which it is difficult to guard against, namely that when an egg of the stage of Fig. 25, E, is transferred from one fluid to another, as in the ordinary technical processes which precede section-cutting, violent diffusion currents are set up between the fluid in the segmentation cavity on the one hand and that in the archenteron on the other, and these currents must be very liable to cause rupture in the intervening partition, even when in life this is quite continuous.

As gastrulation nears its end the circle formed by the gastrular lip becomes gradually smaller. Finally its lateral edges come together so that it takes the form of a short longitudinally placed slit, the remains of the yolk-plug at the same time passing out of sight. The gastrula is now complete.

As regards the subsequent fate of the slit-like blastopore it may be mentioned that, for the most part, it becomes obliterated by fusion of its two lips. The portions at its two ends, however, remain open as two pores of which the more anterior becomes the neurenteric canal while the posterior becomes, either directly or after temporary obliteration, the anus.

The process of gastrulation in the majority of Anurous and Urodele Amphibians pursues a course similar in its main features to that of the frog. Detailed studies of the process are, however, urgently needed in those Amphibians which have particularly small

eggs and in which therefore gastrulation is less modified by the presence of yolk. 42 - EMBRYOLOGY OF THE LOWER VERTEBRATES CI-I_.

GYMNOPIIIONA.--—Thcrc are certain Amphibians in which the telolccithal condition of the egg is so pronounced as to lead to a condition nearly approaching the meroblastic. Any such forms occurring either amongst the llipnoi or the Aniphihia must necessarily be of great importance owing to the fact that these groups are less far removed than are any other Vertebrates, from the line of descent of the Ainniota and that, in consequence, the study of their development may be expected to throw light upon features occurring in the meroblastic etr s of Amniotes.

Amongst od;in}iliibians of this type the Gymnophiona alone_have

been subjected to careful study (P. and F. Sarasin,

_. . 1887-1893; Brauer, 1897). The following sum mary of the main features in their g'astrulation

' processes is based on Brauer’s description.

_ _, The egg of Hg/pogeophis shows at the period

(‘} preceding gastrulation a round patch of micro meres, or blastoderm, covering of the ._ surface of . the egg in the neighbourhood of its

O apical pole. Gastrulation commences with the

posterior edge of the blastoderm losing its forward

curvature and becoming curved backwards (Fig.

26), the curved part of the edge becoming . sharply demarcated by the formation of a slight

- cleft-like invagination of the egg-surface-—-which & is deepest in its centre and shallower towards

its extremities. In front of this invagination the Flu. 26. ——Su_ccessive superficial (ectodermal) cells of the blastoderni

““‘8°* °‘7g““"“1.“”“1’ take on a distinctly columnar form. The edges in llypogcophzs as _ . . . . . see“, 9;“ (AM. of the blastoderm apart from the line of 1nvagmaBrauer, 1897.) tion are in the meanwhile gradually spreading outwards over the yolk. As shown in Fig. 27, A, the cells (g.l) forming the anterior wall of the invagination are columnar in form, and the fine-grained character of their yolk makes their general appearance resemble that of the ectoderm cells. This is, however,- to be taken, not as meaning that they really are of ectodermal nature but rather merely as an indication of active metabolism associated with active growth. The invagination-groove gradually, by backgrowth and ingrowth of its lateral portions, assumes a more pronounced backward curvature (Fig. 26) taking first the shape of a crescent, later of a horseshoe and finally of a closed ring.

The central part of the groove almost from the beginning increases rapidly in depth so as to form a narrow cavity——the archenteron-—-which extends forwards. The roof of this cavity is formed of cells agreeing in their fine-grained protoplasm with those of the ectoderm, while its floor on the other hand is composed of cells which"in their coarse-grained character resemble rather the yolk-cells.

In front of the archenteroni are the irregular remains of the segmentation cavity and a. communication becomes established I GASTRULATIO-N 43

between the two cavities so that they form a continuous space-—the broader front part of which is derived from the seginentation cavity, the narrower posterior part from the, true a.rchcnte1-on (big. 27, U

Flo. 27.—-—-Sa.gittnl sections illustrating the pl‘()('('.\'H of _L,":1.\'i.l'lIl:1tl0ll in lI_:/,'2u_:/cu)»//:'.s:. (After Bram-r, 18$-97.)

ecl, e-ctmlu-nn ; cur, :m-lucntmir «-:m't_\' ; _«:.I. 3.-;a.<trnl:Lr lip: .s-.c, se;.rmen1.a1.ion cu\'it_\'.

and D). The two sections of the cavity remain for it time clearly distinguishable by the character of the cells which form the roof —-—those of the archenteric portion being composc;-.«l of fine-grained protoplasm like that of the ectodcrm while those of the portion derived from the segnientation cavity are typical yolk-cells. 44 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

At a stage when the involution groove forms a nearly complete circle a sagittal section presents the appearance shown in Fig. 27, C. The ectoderm is thick and columnar posteriorly, but in front and laterally it thins out into a cubical epithelium which has extended over the whole surfa.ce of the egg with the exception of a small area behind the gastrular rim. 111 the roof of the enteric cavity the boundary between the archenteric portion formed by overgrowth (and probably involution) and that formed from yolk-cells is marked by an abrupt change in the char'acter of the cells which at once become less tall and less columnar. Farther in still the yolk-section of the roof shows marked irregularities of its inner surface and its cells assume a more rounded form. The anterior limit of the blastocoelic portion of the enteric cavity is not, as yet, clearly defined.

The last section figured (Fig. 27, 1)} is taken from an egg in which the gastrulation lip forms a complete ring. Jonsequently the section shows a conspicuous yolk-Iilug (3/.1») within the gastrular lip which, it will be noted, has developed a covering of small iine—grained cells over its surface. The inrolling of the gastrular lip visible in the section indicates that the enteric roof is growing actively in length though Brauer does not make it clear to what extent the formation of the arehenteron is due to this and to what ex tent to actual involution. Naturally it would be very dillicult if not impossible to decide this point definitely without experiments on the living egg. The gastrular opening gradually decreases in diameter (the yolk—plug disappearing from view as it does so) and eventually it closes from before backwards. by its lateral lips coming together (Fig. 26); its posterior part however remains open as the anus.

In the foregoing description is given merely a summary of those features in the gastrulation of H3/pngeopimls which appear to be of importance in relation to the corresponding phenomena of the Amniota: amongst these may be specially mentioned the process of constriction of the gastrular opening, and the double origin of the enteric cavity from archenteron and blastoeoele, only its hinder portion being derived from archenteron.

Another important feature not specifically alluded to in the text but which is indicated clearly by. Fig. 26 is that during the process of gastrulation the boundary of the small-celled area is sweeping onwards over the egg’s surface. It does this probably by a process of delamination as in Leyoridosiren. The important point to notice, however, is that the small-celled boundary is not blocked in its extension onwards by the gastrular lip. The yolk-plug becomes covered with small cells and after the ends of the rim have met so as to form a complete circle the small-celled region still spreads onwards, so that the slit~like blastopore of later stages lies well within the margin of the small-celled area. Thus were development modified by the slurring over of the early stages of the invagination-groove so that this only became apparent at the period_when it had assumed the form of a longitudinal slit, it would at the time of its first appearance I GASTRULATION 45

be situated Well within the small-celled area instead of at its hinder margin. The importance of this consideration will become manifest later on in connexion with the interpretation of the developmental phenomena of the Amniota. ' ELASMOBRANCHII.-——The egg of the Elasmobranch at the time immediately preceding gastrulation difliers from the blastula of the ordinary Amphibian or Lung-fish in its much greater size. The smallcelled or micromeric apical portion of the blastula is represented here by a relatively small mass of cells-——the blastoderm--in the region of

Fro. 28.-«Sagittal sections through Elasmohru.nel: hlastmlerms ('I'm'1}r’¢/u) illu.~'t.rating the process of gastrulation. (A.flcI‘ Ziegler, 19052.)

,,_], ;_.-a_,-tm];u- lip; ,u_c, ,s;e,«_gu1anta.t'.ion 1-.-n'it_\' : lI.7l, yolk nuclei.

the apical pole while the large-celled portion is represented by the yolk. This latter is composed, practically, of a mass of yolk granules, the protoplasmic matrix being reduced almost to van1shing—point. As in the eggs previously described, the nncromeric portion gradually spreads round and encloses the yolk and here again_we find the same three factors at Work-—involution, overgrowth and delamination. The first step in the gastrulation process consists in the involution of the surface along the posterior edge of the blastoderm. This involution groove spreads outwards on each side until it may extend along 5 to 3; the circumference of the blastoderm. The blastederm is meanwhile spreading outwards all round and, as it does so,

the central part of the groove becomes deepened to form a tubular’ 46 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

cavity, the archenteron, which runs forwards from the mid-posterior margin. In the roofing in of this archenteron it is apparently a process of overgrowth which plays the main part-«but along the rest of the blastoderm margin the process of overgrowth appears to die away and its place is taken by delamination very much as was the case in ])ep'£(l()s'zI'rm. This is shown by the fact that the invaginationgroove, which, as already remarked, extends outwards on each side for some distance, never deepens to any considerable extent except in its middle part.

In the region in front of the archenterou the deeper or lower layer cells of the blastoderm increase greatly in number and spread forwards so as gradually to fill up the segmentation cavity. The remains of the latter persist longest near the anterior margin and the ex,-torlerm covering the last remnant of the segmentation cavity commonly projects as a small but conspicuous elevation above the general surface of the blastoderm.

’l‘hese lower cells eventually take on a mesenchymatous character for the most part. These lying next the yolk-syneytium however give rise to a definite epithelium, known as the yolk epithelium. Some of them are said to penetrate actually into the yolk where their nuclei assume the characters of the nuclei of the yolk-syncytium. The floor of the archenteron is formed by the yolk epithelium which is continuous round the inner, or anterior, end of the archenteric cavity with the endoderm of its roof.

ACTINOPTERYGII.-—lt is unfortunate that in the more familiar Actinopterygians belonging to the group Teleostei-—— of which it is so easy to obtain developmental material——-the phenomena of gastrulation .are obscure and their investigation is impeded by technical difficulties in the way of making satisfactory sections. We shall therefore confine ourselves to indicating in a few words the more conspicuous features of the process.

On the whole the features of gastrulation closely resemble those met with in Elasmobranchs—-—a resemblance which however we are not justified in regarding otherwise than as a phenomenon of convergence, seeing that the general evidence of morphology points to the ancestors of the Teleosts being much more closely related to the holoblastic Ganoids than to the existing Elasmobranchs. A characteristic feature to he noted is that here, as will be found to be the case in various mammals, the superficial cells of the blastoderm become much flattened and form a thin protective covering layer which takes no part in the development of the embryo.

When gastrulation is commencing the posterior margin of the blastoderm presents in longitudinal vertical sections the appearance of being turned inwards to form the two primary layers. There is no actual patent archenteric cavity though the inflected portion clearly represents the archenteric roof, the floor being apparently represented by the underlying syncytial layer.

The growth in length of the archenteric roof seems to be brought I GASTRU LATION 47

about mainly by a process of overgrowth similar to that met with in other forms.

A point of special interest is that the posterior portion of the archenteric roof, in the neighbourhood of what will become later the mesial plane, is without the distinct demarcation between ectoderm and endoderm whichis present elsewhere. This continuity of the two primary cell layers apparently represents what is known in the Amniota as the primitive streak~—a structure of great morphological interest which will be discussed later on (Goronowitseh, 1885 ; Jablonowski, 1898).

While these processes are in progress the margin of the blastederm elsewhere is also advancing over the surface of the yolk so as gradually to enclose it. This enclosure of the yolk clearly corresponds to what we have seen in other cases but it is ditiicult to be quite certain as to how far it takes place by actual delamination and how far this has been replaced by a secondary independent growth.

. It is only when the exposed surface of yolk becomes reduced to a small round patch that the cell-margin bounding it shows inflection all round so as to justify us in speaking of a blastopore.

In the surviving Ganoid members of the group Actinopterygii we find that the process of gastrulation, as is the case with other characteristics, repeats conditions which are probably to be looked on as ancestral. The gastrulation clearly belongs to the same general, type as that of Lampreys, Amphibians, and Lung-fishes. That of Aczpenser (Salensky, Bashford Dean, 1895) seems more nearly to resemble that of Poly/pterus, and that of Amia. (Bashford Dean, 1896) and more especially Lepwldosteus (Bashford Dean, 1895) to point towards the mode of gastrulation found in the modern Teleosts.

GASTRULATION IN AMNIOTA

In comparing the process of gastrulation in the Amphibians and Lung-fishes with that in Am.p/m.'oa:us or Pul_2/pterus we have seen that there is a tendency for the greater part of the gastrular rim either to become completely obsolete or to be, at least, greatly delayed in its appearance, for example in the frog the greater part of the gastrular rim makes its appearance only in a comparatively late stage in the process of gastrulation.

In the Amniota we find that this tendency has gone further. It is only in the lowest group-—-the Reptilia —~—that an undoubted gastrular lip is clearly recognizable. In the two remaining groups, the Birds and Mammals, there is no convincing evidence that it has not completely disappeared from development.

REPTILES.-——In a Reptilian egg before the commencement of gastrulation the apical portion is covered by a blastoderm consisting of a superficial layer of flattened ectoderm cells and, underneath this, rounded lower layer cells which are separated by interstices containing fluid.

In the centre of the blastoderm (Fig. 29, A) an area, circular 48 EMBRYOLOGY OF THE LOWER VERTEBRATES OH.

or elliptical or pear-sliapecls with its narrow -end posterior, becomes distiriguisliable from the rest of the hlastoderm by its slightly greater opacity. '.l.‘he area in question is known. as the embryonic shield (as), and its opacity is due to its ectoderm being thickened, the individual cells having taken on a columnar form.

Either enclosed within or projecting beyond the posterior outline of the embryonic shield (Fig. 29, B) is a small area in which there is

3'

FIG. 29.--~—lllustratiu;: gasti-ulul.ion in the Gecko (I’/Ml;/4/m-/3//us). (_Al.'tcr Will, 1892.)

A, showing complete lll.'l\'ttNli'l'lll with the embryoni(.- .~'«l|l1‘l(l in th-- centre. This is l)()lIn(ll.'d behind by the gasti-ul:u- r-im, prev.noia_ms1y developed in this spc-cimen. B, embryonic shield of specimen at the st.:igl- in which the 2tl'L_'ll4.-lllHl'i(' floor is breaking down. C, embryonic shield at, Int-,¢_-r stage where gasl.i-ulzir rim is be-nt hack into it A-shape bounding the yolk-plug: l..lu- outline of the mesoderm sheet is seen on each side. 1), embryonic shield showing stage at which tlw «.,;usl1-i1l:ui- lips h:m- mum-. 1'.o;:«-tlicr so as to bound in longitudinal slit. b.¢t, edge of blastodcrm; o-..-, «:nibryuni«.- .~liiuld; _4/J, ;;-nslrular lip; -mes, limit. of Ina-smlcrni.

no layer of columnar ectoderm sharply marked off from the lower cells. This forms the primitive plate (Fig. 31, pp). The boundary of the embryonic shield gradually spreads outwards and the primitive plate comes to be, if it is not. already, enclosed within it.

Within the limits of the primitive plate the surface of the egg now becomes involuted to form a groove bounded anteriorly by a lip

which from its correspondence with what we have seen 1n lower forms, more especially 111 the Gymnoph1ona,'1s clearly to be recogI GASTRULATION 49

nized as the gastrular lip. This lip gradually shifts backwards and, as it does so, undergoes alterations in shape, which differ somewhat in different species and even in different individuals of the same species but which in their main. features are illustrated by Fig. 30. In its later stages the lip becomes bent or curved backwards so as to have the shape of a 11 or a A (Figs. 29, C, and 30).

Considerable variation occurs between differeiit individuals in the time of the first appearance of the gastrular lip, and in the Gecko 1’lat_7/dactylus Will (1892) observed a correlated variation in shape. Where it appeared relatively early, the involution had the form of an elongated creseentic groove, while in cases where its appearance was delayed the involution formed a shorter and more rounded ‘opening.

As in other cases the central part of the invagiiiatioii groove becomes deepened to form a cavity which is clearly homologous with the

main part of the archenteric cavity in, say, a ‘Z A 5 ‘ o_ ‘I '. ' ‘ o o frog. 'lhis cavity starts by passing directly ,-/‘K

inwards, perpendicular to the egg-surface, but it soon bends forwards and runs parallel to the surface (Fig. 31, C and 1)). The cavity just [3 mentioned (Fig. 31, 1), am.) being an archenteron the layer of cells lining it corresponds to that O which in the lower forms is called endoderm. _ _ It is therefore misleading to replace this by any F“f’t- ,:‘3°'1,”“‘f““i‘_’°“5_’."° other name : to emphasize the fact that they line :15 g::e,:ui:r the true archenteric cavity it may be advisable faceview. A,(,‘h.elom'a, to speak of the cells in question as the wrchentemfi: (fin-““““_"‘v 1396" .3‘

. . _ . _ . __ . .atg/tlactji/lzts, (Will, endoderm in spite ol the clunisiiiess of the ex- 189.4 pression.

In the nieantime the lower -layer cells immediately underlying the ectoderm assume a flattened form and become joined together by their edges to form a definite epithelium which may conveniently be termed the secondary endoderm (Fig. 31, C and D, end’). The more deeply situated cells underlying the secondary endoderm remain spherical and are separated by wide spaces forming a segmentation or subgerininal cavity. 'l‘liese deeper spherical cells have their numbers constantly reinforced by additional cells which are apparently budded off from the underlying yolk-mass.

The floor of the archenteron becomes closely apposed to the secondary endoderm immediately beneath it (Fig. 31, D). The two cell-layers fuse, irregular perforations develop in the membrane formed by their fusion, and the result is that the archenteroii is thrown into communication with the “ subgerininal” cavity (Fig. 31, E). Shreds of the partition persist for some time. but eventually the two spaces form a perfectly continuous cavity just as happened with archenteric and segmentation cavity in the Gymnophiona.

The portion of the primitive plate which is embraced by the 50 EMBRYOLOGY OJ?‘ 'I‘TlE I.()WER VER'l‘EBRA’1‘ES CH.

. horas shoe. l-'»l'll-L}._)U(.l g:lsLru1a.r lip corru.sponds to the yolk-plug of

E ' » '7»

FIG. 31.—-Sngit.t:l..l .s'H'.tio11.~.: t1n'oup;h early .‘~'l:l-;.fL‘..\‘ of I‘./rt(3/aim‘/y/u..<. (At‘tu.1' W ill, 1892.) Ala of the .~.1:n_-.:»- ~'lmwu in |"i_~4_ .1... A ; E is of I luv .~s1,:1;_-,'n- of Fig. '.-'0, B: 1}, C, um! I) :m- ml" int.-rm.-.]i_-,tp

stages.‘ (LS, e.mln-ymnic. shiulll; rut, vc-tuulerwn; uni‘, .~u-urnul:u'_\' --mlm_1:-run‘, no/, :m-lu-.nL«-I-'1: (J:L\'ll}'; 11.1., gash-ular lip; m./:_. Lhickelxed +'.ct;odex‘m which will gi\'e risv later to the central nervous system

(medulla:-_v [n|:(Lu); 12.1), pr-imitzive plate; g/.p, _vulk-plug.

amphihi.'u'1s aml in 801116 cases too (_La._certa~—Wi1l) it becomes completely enclosed, the taps of.‘ the 11o1‘se-shoe Cl11‘V1lIf__I' lnwnrds zmd 1 GASTR ULATION 51

meeting to form a closed ellipse. The yolk-plug di11'ers from that of amphibians merely in its being elliptical in outline instead of circular.

During these changes the anterior or dorsal part of the gastrular lip grows actively backwards over the surface of the yolk-plug, _the portion of yolk-plug which is covered over in this way becoming added to the floor of the archenteron and its superficial cells becoming converted into archenteric endoderm.

The last phase in the closing of the gastrular opening consists in its lateral walls approaching the mesial plane so that the opening assumes the form of a longitudinal slit (Fig. 29, D). l’art of this slit persists for some time as a neurenteric canal-—a communication between the enteric cavity and the cavity of the neural groove or tube (Fig. 32, ())——-while a portion farther back seems to be represented by the anus although in this case the patent opening disappears temporarily so that no absolute continuity can be traced. In the region where the lips have undergone fusion there persists for a time complete continuity between the different cell-layers. The study of sections shows this continuity to be precisely the same as that which occurs in the primitive streak of Birds and Mammals (Fig. 32, B, I), E), and we have thus suggested a clue to the meaning of that otherwise enigmatical structure.

It will have been gathered that the archenteric cavity has become greatly reduced in importance in the Reptile as compared with the more primitive vertebrates. It has become much reduced in relative size,‘ and it soon loses its individuality, becoming merged with the irregular segmentation spaces lying beneath the blastoderm. Correlated with this we can no longer speak of direct conversion of the archenteric cavity into the enteron or alimentary canal, except to a trifling extent. The latter arises, for the most part, as will be shown later, in a quite different manner from the secondary endoderm.

BIRI)S.———In the Reptile, as compared with one of the more pri1nitiv,e anamnia, the main peculiarity of the gastrulation process lies in the fact that the cavity which opens to the exterior by the blastopore is normally of double origin, only its posterior portion being derived from archenteron. Consequently the layer of endoderm which lines it is only to a comparatively small extent derived from the archenteric lining, the much greater anterior part being formed from elements of independent origin.

In the Amniota above Reptiles the replacement of archenteric by secondary endoderm has gone still further, inasmuch as the formation of an archenteron has in them either completely disappeared from development, or at the least is reduced to a faint vestige, and the endoderm is therefore entirely secondary.

1 In some forms, such as Lacerta, the archenteric portion of the enteron appears to be for a time much shorter relatively than in others (e.g. Platydactylus) but this is corrected later on by active overgrowth on the part of the dorsal lip (Will, 1895). 52

EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

As regards the Birds, which of the higherAmniotesalone concern us in this_ volume, there is complete agreement that they are to be looked on as highly-speciaL ized descendants of Reptiliati ancestors. It follows therefore that their develop— mental phenomena should be considered in relation to the corresponding phenomena in Reptiles.

Leaving out of account certain vague phenomena which have been interpreted, in the present writer's opinion unjustifiably, as re1nin~ iscences of gastrulation processes (see Chapter X.), the formation of a gastrular lip seems to have

Fm. 32.——~'l‘rmisverse sections through region of ncurenteric canal of Chelorzia embryo with about 16 segments. (After Mitsukuri, 1896.)

The mid-dorsal ectoderm has become covered in to form the neural tube (s.c.) as will be described in Chap. 11. Fig. C shows the neurenteric canal opening upwards through this, while Figs. 13, D and E, taken from sections anterior and posterior to the neurenteric opening, show the continuity of tissue from eclodcrin to endoderm which is a characteristic feature of a. primitive streak. ect, ectoderm; end, endodcrm; mes, mesoderm; N, notochord; ne.c, neurenteric canal; 3.0, spinal cord. I GASTRULATION 53

been eliminated entirely from ontogenetic development in Birds. What is conspicuous is a well-marked primitive streak which makes its appearance in the posterior half of the blastoderm along what will be the axial line of the body of the embryo (see Chap. X.). A groove develops along the surface of the streak —-- the primitive groove.

Histologically the primitive streak is, in its early stages, a line of proliferation from the inner surface of the ectoderm, the blastoderm being composed only of the two primary layers at the time of its appearance. That the ectoderm alone is responsible for the first appearance of the primitive streak, a point difficult to make absolutely certain by ordinary observation, appears to be demonstrated by the study of an abnormal 36-hour embryo Peawit (Vanellus eristtttus) described by Riithig (1907) in which the endoderm was completely absent while ectoderm and primitive streak were quite normal.

An inspection of blastoderms at successive periods in development shows the primitive streak lying always behind the medullary folds (of. Fig. 227, Chap. X.), and it might therefore be readily assumed that the embryonic body develops entirely in front of the primitive streak. That this is not so is clearly shown by experiments (Kopsch, 1902) in which a scar is made with a hot needle about the front end of the primitive streak during an early stage in its development. If the egg is carefully scaled up again it may go on with its development, and in such a case the scar is found later on to be situated not near the hind end of the embryo but well forward in the head region.

What apparently happens is that the primitive streak grows actively in length with the general growth of the blastoderm but that all the while it is becoming correspondingly shortened at its headward end. As a matter of fact its anterior end becomes gradually converted from before backwards into notochord and'the adjoining parts of the mesoderm. The front part, which is undergoing this change, loses its connexion with the ectoderm while it becomes on the other hand continuous with the endoderm and is reinforced by proliferation from it: it then forms what is known as the Head process.

The point of special morphological importance to notice about the primitive streak is its continuity with the two primary celllayers. Throughout the greater part of its length it is continuous with ectoderm, in its front half with both ectoderm and endoderm, and in its forward prolongation-——-the head process--with endoderm. Correlated with this is the further fact that in some cases (Tern, Goose, Duck, Wagtail, Melopsvlttacus) the tissue of the primitive streak is traversed by a typical neurenteric canal.

Taking these various features into consideration it is impossible to avoid the conclusion that the primitive streak represents the line of coalescence of the gastrular lips just as it actually is in 54 EMBRYOLOGY OF THE LOWER VERTEBRATES cu.

Reptiles, and that the neurcnteric canal represents a persisting portion of a once slit- like gastrula mouth which is otherwise

obliterated.

ORIGIN OF‘ 'l‘lllt' MESODERM

(_ll«‘.NERAl. REl\1Al{KS.--AlT(-Etuly during the process of segmentation the dilltsrentiation of the two primary cell—l-ayers commences--the superlicial cells towards the apical pole dividing more actively, being smaller, and being less laden with food-yolk and thus establishing a character of their own as ectodermal cells. The full establishment of the primary layers however is only consummated during the process of gastrulation when the cctoderm comes by the various processes already described to enclose the remaining cells the (archen teric) endoderm.

The establishment of the two primary layers is followed immediately (indeed the two processes frequently overlap) by the development of the intermediate cell-layer———the mesoderm—-—wln'ch will in the adult form the great mass of the body— all in fact except the epidermis and its derivatives on the one hand and the enteric epithelium and its derivatives on the other.

The problem of the evolutionary history of the mesoderm of Vertebrates is one upon which there is little agreement. Anything of the nature of elaborate and detailed treatment of the subject would be out of place in a textbook of moderate size and a short sketch such asthe following is necessarily coloured by the general

unorpliological views of the wri_ter. While the views set forth in the

following paragraphs seem to the author to fit most satisfactorily the facts so far as these are established beyond reasonable doubt there are other embryologists who would give an account differing considerably from that given here.

To the present writer it seems of importance in endeavouring to arrive at reliable general conclusions from the facts of observation to bear in mind particularly the risk of reaching erroneous conclusions through basing arguments upon phenomena observed in the head region or tail region of the embryo. Intense cephalization, «Le. intense structural modification of the anterior region of the body, to form a head, is admittedly one of the fundamental characters of the phylum Vertebrate. In this modification the mesoderm has been deeply involved so that there is always a considerable weight of probability against conditions observed in the head region being primitive. Again the tail region is also intensely modified, as is indicated e.{}. by the transient appearance within it of a vestigial portion of alimentary canal with surrounding body-cavity. Here again then, though not to the same extent as in the head-region, suspicion rests upon the primitiveness of all phenomena of development peculiar to this region of the body.

It is advisable then, for these reasons, to exercise great caution in I ORIGIN OF THE MESODERM 55

making use of any developmental phenomena except those observed in typical trunk segments as a basis for speculations upon the evolutionary origin of the mesoderm.

It has, further, to be borne in mind that observations upon the development of the mesoderm in its early stages have to be made by the method of serial sections, and that in the interpretation of such sections the liability to error becomes greatly increased if the sections are not exactly in one of the three following sets of planes——(1) transverse to the morphological axis, (2) “ horizontal," and (3) parallel to the sagittal plane. This type of technical difficulty is in many Vertebrate embryos most marked in the head and tail regions.

For these reasons it seems safest, in considering generally the ontogenetic development and the probable evolutionary history of the mesoderm, to ignore all observations except those made on typical trunk segments between the level of the otocyst in front and of the anus behind. This will accordingly be done in what follovs s.

It is agreed by the majority of students of Vertebrate embryology that the most nearly primitive condition of the mesoclerm known to occur in the embryos of Vertebrates is that seen i11 Amp]?/ioxus, where it consists for a time of a series cl‘ endodermal pockets, converted later into closed sacs, upon each side of the body (Fig. 34, B).

It appears fully justifiable to conclude that both of the stages mentioned represent ancestral conditions in the evolution of the Vertebrate mesoderm. The excretory organs of the Vertebrate, in the form of paired segnientally arranged tubes, afford in themselves strong evidence that at one time the Vertebrate coelonie was in the form of isolated segmentally arranged chambers.

In the case of Aznplmfoxus the segmented character of the mesoderm persists only dorsally, the ventral portions of the successive segments becoming fused together so as to give rise to a continuous unsegmented splanchnocoele or peritoneal cavity.

In the Craniata the smallest departure from the condition in Amp/mjowas is seen in such comparatively primitive forms as Lampreys, Crossopterygians and Lung-fishes. In these a solid continuous mesoderm rudiment becomes split off from the endoderm on each side, remaining for some time continuous laterally with the endoderm (Fig. 40, B, C, p. 65). In the outer or lateral part of this mesoderm rudiment the segmentation, which even in Amp}:/ioams was only temporary, never makes its appearance. The dorsal portion does segment but the segment is a solid block of cells in which a cavity only appears later on. It is fairly clear that these mesoderm segments, except for the fact that they are continuous in their ventral portions and that they are at first solid modification of development which is very common in hollow organs), agree closely with the segments of Amplmlocms and that they are homologous structures merely somewhat modified from the primitive condition met with in Amphioxm.

In endeavouring to institute a mor_e precise comparison of the 56, EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

mesoderm segment in its earliest stage, in the typical Vertebrate, with that of Amplzxioxus, the way is found to be blocked by a secondary adhesion (or absence of separation!) having come about between the mesoderm segment and the endoderm from which it has arisen. The young mesoderm pouch of Amplvioxus is attached to the endoderm at its base————7I.e. its ventral end. Its homologue in the more typical Vertebrate, on the other hand, is continuous with the endoderm in two different regions, one ventral and one dorsal. This is illustrated by such a diagrammatic section as that shown in Fig. 33, B, in which the solid mass of mesoderm on each side, indicated by the medium tone, is continuous with the mass of endoderm or yolk-cells at the points a and b. The question is, which of these two points is to be interpreted as representing the root of the meso— derm pocket in Amphiomzs ? Clearly only one of them can represent

I

FIG. 33‘.-—-Diagram illustrating (B) the origin of mesoderm lfroni endoderm in an Amphibian,

and (Aand (3) the two methods of correlating it with the mode of mesoderm formation in A mphicwus. ‘

n, b, see text; cct, ectmlerm; en-t, enteric cavity ; N, notoeliordal rudiment.

this and the other region of continuity must represent a secondary fusion of mesoderm with endoderm.

. The majority of embryologists, following 0. Hertwig (1882), believe that the dorsally situated region of continuity marked 12 is the primary connexion as is illustrated by Fig. 33, C. On this view the mesoderm segment of the Vertebrate springs from the endoderm at a point about the level of the notochord, it grows downwards on each side of the alimentary canal and eventually its tip meets the tip of its fellow of the other side of the body in the mid-ventral line.

-In this view, again, the .continuity which can sometimes be shown

to exist between mesoderm and endoderm at the point a would be regarded as secondary and without evolutionary significance.

If however due weight be accorded to what is observed in the development of the lower holoblastic vertebrates it seems more reasonable to the present writer to conclude that the more ventrally situated connexion, that marked a, is the primitive one and that the more dorsally situated, 12, is the secondary aequirement (Fig. 33, A). .tion of mesoderm.

I ORIGIN OF THE MESODERM 57

Upon tl1e ;i'ormer hypothesis the extension of the mcsoclerm l.ater~ ally by delamination from the endoderrn, which does certainly occur in some forms (see below), would be an inexplicable mystery.’ On the second hypothesis on the other hand this splittilig off of mesodei-m would be comparable with a gradual deepening of the iLIlgl.G which bounds the mesodermal pocket of Amp/uloanm on its mesial side (see F'g. 34, B). The dorsal attachment is, on this view, to he rega1‘Cle(l as a secondary fusion between mesoderm and (m<‘lo(leI‘Ii1. In the higher vertebrates e is o s l this region becomes the ‘seat of active cell proliferation which plays a great part i.n the produc _ After these general remarks we may proceed to consider shortly the details of the early development of the mesoderm in a few examples of the lower Vertehrata. AMPHIOXUS.--— In Amqokioxus the development of mesoderm begins with the formation of a longitudinal fold or outpushing of the endoderm on each side of the

mud " dorsal e Fm. 34.——'1‘ransverse sections of young ..47n.p}i.-('0.-rcus illustrating

(Fig. 34, A, mes). the origin of the mesoderm. (Ai'tci'I-1at.s-click, 188.1.) _In thls W3)’ there col, ectodvrni: 4'-nt, enteric cavity; m.p, im-dull.'u‘_\' [|l.‘|U‘I tiles, 18 formed on each mesodi-.rm; N, notocliordal rudiim-ni.-.

side an upwardly projecting groove or gutter, the narrow cavity of which is a prolongation of the archenteric cavity (Fig. 34, B). Constrictions appear now in the wall of this gutter which divide it up into successive segments --—"—’- the constrictions developing in order from the head-end backwards. 'l‘he groove or fold is in this way converted into a series of pockets‘ the coelomic or enterocoelic pouches. The cavity of these pouches except in the case of the first two usually becomes for a time practically obliterated by the outer and inner walls coming into contact. Finally the communication between pouch and archenteron beeoliies closed and the pouch 58 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

itself hecomes nipped oil’ from the remainder of the are-henteron (Fig. 34, O). _ The‘origina1 a.rchenteron is now replaced by a. main portion, the

Flu. 35.——Transverse sections through embryos of Lepidos1.'7‘en to illustrate the origin of the mesorlerm.

A, stage 12 ; B and C, st.ug-- 14. act, ectodex-in ; et-rul, endorlerm ; «mt, enteric cavity; mes, mesoderm ; N, notochordul rudiment.

enteron, the wall of whieh——the definitive eiidoclerm-—-will eventually become the lining epithelium of the :_Lli.1nenta,ry canal, and, lying dorsal to this on each side, a series of closed sacs, or practically solid I ORIGIN OF THE MESODERM 59

blocks (their outer and inner walls being in contact). These sacs or blocks are the mesoderm segments and their cavities are the segmentally arranged rudiments of the coelome. The subsequent fate of the mesoderm segments will be traced later (Chap. IV.).

Of the lower holoblastic forms amongst the Vertebrata in the stricter sense we will consider lirst Lepidosiren, in which, owing to the large size of the cell-elcmen ts, the details of mcsoderm formation are particularly clear and unmistakable.

The mode of origin of the mesoderm which occurs in Lep'id0s7§rre'n. is illustrated by Fig. 35. The section shown in Fig. 35, A is taken from an egg of the same age as that figured on p. 35 (Fig. 21, Q) in illustration of the disappearance of the segmentation cavity. Immediately below the eetoderm is a mass of rounded blastomeres with intervening chinl<s——rc1nnants of the segmentation cavity: towards the mesial plane the blastomercs are more closely packed together. The small blastomeres in question are clearly distinguished by their finely-grained yolk from the large yolk-cells with their coarselygrained yolk which form the bulk of the egg. The mass of small blastomeres is destined to give rise laterally to the mesoderm and mesially to the notochord. It must clearly be borne in mind that the mass is composed simply of small blastomeres and that it passes at its outer margin without any break into the ordinary yolk-cells.

As development goes on, the mass of small elements becomes compacted together (Fig. 35, B), the chinks between the cells disappearing. At the same time the boundary between them and the yolk-cells becomes more definite, so as to delimit more clearly the mesoderm rudiment (mes) from the definitive endoderm.

Fig. 35, C is taken from an egg of the same age but here the mcsoderm rudiment has become limited also on its mesial side by a split which marks it oil‘ from the notochord (N).

At a somewhat later stage, the mesoderm mass on each side becomes divided into segments by splits, transverse to the axis of the body, which make their appearance at regular intervals from before backwards, but it is to be noted that in Lepidosziren (as in all Vertebrates except Amphz'0am.s) this splitting of the mesoderm is confined to its dorsal portions. There is thus produced along each side of the body a series of incomplete mesoderm segments1 which pass at their lower or ventral ends into an unsegmented sheet of “lateral”

1 Such incomplete mesoderm segments as are described above occur in all the typical vertebrates and are known by various names such as mes-ohlastic somites, protovertebrac, myotomes. These names are in various degrees erroneous or misleading. The word somite means a complete body segment and it is not allowable to apply it to a. single organ. The name protovertebra dates from the days in which these structures were supposed to be the embryonic vertebrae, which they are now known not to be. Of the three terms mentioned myotome is the least objectionable as at least the greater part of the segmented portions of mesoderm become definite myotomes later on. On the whole however it seems most convenient to retain the expression mesoderm segment, the word segment not being necessarily used in the precisely defined way in which such a purely technical morphological term as “somite ” must be used. 60 EMBRYOLOGY OF ’l‘HE LOWER V ERTEBRATES CH.

mesoderm. This latter gradually spreads ventralwards by delamination from the large yolk-cells and eventually the mesoderm sheets on the two sides become continued into one another ventrally.

As will be noticed there are no coelomic spaces within the mesoderm rudiments at these early stages: they arise secondarily later on.

If we review the abovc—dcscribed stages in the early development of the mesoderm segment in Le])'id0s'ire'n., in which, as already indicated, the large size of the cell-elements ensures unusual freedom from the danger of errors of observation, we see that the last described stage is clearly in agreement with the hypothesis that it is a repetition of the stage in Amplzxiowus when the mesoderm existed in the form of a series of enterocoelic pouches on each side. The only conspicuous dill'crence is that, whereas in A7n.};/I/ioasas these were actual pouches, here they are solid blocks of cells in which a cavity only makes its appearance at a later stage of development. That this difference is in no Way a serious one will become apparent to the reader as he realizes that it is one of the commonest modifications of developmental phenomena, when yolk is abundant, that primitivcly hollow organs develop in the embryo from solid rudiments and only form their cavity secondarily.

It may be accepted then with coulidence that the solid mesoderm segments of Lepidosiren at the stage indicated, continuous ventrally with the endoderm, represent the enterocoelie pouches of Amp}mJo:2cu..~: modified in correlation with the abundance of yolk.

The first stages in the development of the mesoderm of Lexp’id0siren are obviously very dilibrent from what are found in Amph'ioa:us and the differences here also we may justifiably attribute to the immense thickening of the cndodermal wall of the archenteron correlated with the storing up of a large amount of yolk in its cells.

In the other groups of holoblastic vertebrates the main features in the early development of the mesoderm agree with those just described for Lep'idosi'ren. In all of them the archenteron is provided with a thick wall of heavily yolked endoderm cells, those forming the roof or dorsal part of the wall being smaller and provided with finer yolk-granules. Out of this smaller-celled mass the mesoderm segments become carved by the development of splits very much in the same way as in Lepidosiren (cf. Fig. 40, B—Petrom3/zon).

Amongst these groups the Amphibia call. for a little further consideration. .

In the frog a split develops on each side which separates the roof of the archenteric cavity into two layers, an inner layer, one cell thick, of definitive endoderm and an outer, two cells thick for the most part, the mesoderm. This split is seen in Fig. 36 which represents a section, transverse to the axis of the archenteron, through an egg with large yolk-plug. The split in this section terminates below at about the level of the floor of the arehenteric cavity while above it stops short of the level of the notochord. I ()Rl(a‘rl.' N 01*‘ 'l‘llE .\Iul*}f_\_‘(Q).l)l4}l*i,M 61

A little later a split at its dorsal end C.lmn:_-u‘ea.tcs the mesoderm rudiment from the notoehord. The mesoderm rudiment, forming now a broad band on each side of the embryo, becomes divided into segments by splits which cut it across and a condition is reached corresponding closely with that already described for Legivlrlostren where the mesoderm consists of a series of solid segments on well side continuous ventrally with the mass of yolk cells forniing the main part of the endoderm.

As in Lepwldosiren the ventral unsegmented part of the Inesodcrm becomes prolonged ventrally by the extension downwards of the split between it and the endo- ' derm. In other words the mesoderm extends ventrally by a process of delamination from the endoderm.

In the anterior part of the body the sheet of mesoderm becomes split off completely from the yolky endoderm before it quite reaches the mid - ventral line so that the sheets belonging to the two sides are discontinuous ventrally but in the binder region the two splits meet ventrally so as to give rise to a sheet of mesoderm continuous across the middle Fm. 36,-—'.l.‘ransverse section tlIl'ou;_~,'h an t‘llll)l'_\'U or hne. - Under Ordlinary c1¥._ ougm ol th lm~.Ul(lllJ. cumstances the Iuesodemn e't ectoderm° and Pll'l0(lPl"ll ‘ m Ill ode ' sheets in the anterior " ‘ i ’ ’ i N: ,‘m0c1;W',j cs’ °s ""‘ region grow ventrally and eventually fuse with one another (as will be described later) while in the posterior region this fusion is anticipated by the two lateral rudirnents being continuous from the beginning.

So far everything seems fairly simple, but it now remains to allude to certain peculiarities which have done much to obscure the clear understanding of the hietliod of 11u'.s'o(‘le1"'In_ formation and which are especially important for the proper emnprehension of the flrst formation of_n1esode1'm in the nneroblastic vertebrates.

The peculiarities in question are to be seen in the hinder part of the trunk region. In this region the split which separates off mesoderm from endoderm remains _for a time incomplete at a point just external to the notocliord. Ear-.11 svgiiieiit therefore remains for a time continuous with the endoderm at this point. The level oi" these junctions of mesoderm and endoderm is marked by a longitudinal 62 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

groove of the inner surface of the wall of the archenteron so that where the junction exists the archenteric cavity may be said to project slightly into it. The cells at this point develop pigment in their protoplasm (Fig. 37, B); they frequently show mitotic figures and appear to be actively proliferating, cells being added at this point to the mesoderm.

'J.‘he peculiarities which have just been described, and which occur in various amphibians, have important hearings in two different directions. In the first place they form an important part of the basis for O. Hertwig’s hypothesis of mesoderm formation in the Vertcbrata, the junctions, which have just been described, between endoderm and mesoderm being interpreted by him as representing the original stalks of the mesoderm segments as they occur in Ampltioxus. As already indicated there do not appear to the writer to

Fm. 37.—-Transverse sections through embryos of (A) T‘I‘l'tu-IL and (B) Rana teonpziramia showing continuity of endodcrm and mesoderm on each side of the notochord. (After 0. Hertwig, 1882 and 1883.) -'

end, emloderni; m.p, medullary plate ; mes, mesoderm; N, notochordal rudiment.

be sufficient reasons for regarding these connexions as primitive rather than those more ventrally situated. The balance of probability appears rather to favour the view that of the two connexions it is the ventral one which is the persistent original one, and that it is the dorsal which is to be interpreted as "due to secondaryfusion. .

The second bearing is at least equally important. It rests on the occurrence of active cell-proliferation on each side of the notochordal rudiment. For in some of the meroblastic vertebrates (Amniota)-—~correlated with the more and more complete segregation of yolk from protop1asm——this zone of proliferation becomes apparently the main source of the mesoderm.

ELASMOBRANCHII.-—-In the Elasmobranch, while there are still traces of formation of mesoderm by a process of delamination from the main mass of endoderm or yolk (Fig. 38, A), a more conspicuous mode of formation is provided by active proliferation of I ORIGIN OF THE MESODERM 63

the endoderni cells along the inner and outer edges of the sheet of

mesoderm.

In early stages and in the anterior part of the enihryo this proliferation process may alone he in evidence, so that in place ofa l)r0ad continuous sheet of inesoderm there are found two narrow strips—~ one (Fig. 38, 0, mos’) arising from the e1l(lOCl.€I‘l11 just external to the notochordal region and the other ('mre-23”) arising from the endoderm

FIG. 38.-—-Three transverse sections through an embryo of [’rz7sti-m'u.s- (Stage B, Balfour), illustrating the origin of the mesoderm. (After C. Rah], 1889.)

Section A, through the posterior half of the mnln-yo; B, through the iniddlc ; (3, through the anterior half. eat, ectoderm ; and, endoderm ; mes, I11(?SOfleI'I1l ; 3/.n, yolk nuclei.

peripherally. The two strips are known respectively as the axial (Riickert; or Gastral, Rabl) and the peripheral mesoderin (Riickert; or Peristomal mesoderm, Rabl).

Much discussion has centred round this double origin of the mesoderm and attempts have been made to distinguish axial and peripheral inesoderm in holoblastic forms including even Amplmloasus, thus infringing one of the chief canons of embryological sciencethat developmental phenomena in the higher forms are to be explained by those of the lower and not vice versa. 64 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

In the formation of axial mesoderm we recognize a zone of fusion of niesoderiii and endoderm accompanied by proliferation of mesoderm entirely analogous with that which occurs in Amphibians but which had not yet made its appearance in lower holoblastic forms.

Whether'it is justifiable to regard the outer zone of mesoderm formation in the Elasinobranch as equivalent to the region of delamination (a process which necessarily involves cell-proliferation) is doubtful. lt is indeed doubtful to what extent there is justificatioii for drawing any morphological distinction between axial and peripheral inesoderin, seeing that the two regions of proliferation are on the protostonia hypothesis morphologically closely related to one another (see Chap. lX.).

If we look at the matter from the point of view of physiology rather than of morphology we may probably recognize in the active formation of axial iiiesoderin an expression of the general tendency in the iiieroblastic egg for all processes of growth and cell prolifera

Fiu. 39.—--'l‘ransverse section through the hlastoderin of a siiake(T7~opi¢i0n.otus) illustrating the origin of the niesodei-in. (After 0. llertwig, 1901).)

act, ectodei-iii ; mil, Bn(l()Il¢*1'lIl : mes, inesoderin.

tion to become concentrated towards the mesial plane dorsally and to slacken off peripherally and ventrally.

l‘tEPTII.ri«:s.——-According to the View taken in this book the inescderni in the holoblastic Uraniates at one period spread outwards by a process of delaiiiination from the yolk-laden endoderm. _ _

In the Amphibians we have seen that a new source of addition to the mesoderm had made its appearance in the form of a zone of proliferation on each side of the Il()t0(3llO1'(l,ll1 which region cells are hudded off into the inesoderiii. _ ' O

In the Reptiles--—adinitted1y descendants from Amphibian-like ancestors-—-—in correlation with the concentration of developmental activity towards the mid-dorsal line brought about by the accumulation of the yolk ventrally, this parachordal source of mesoderm has become predomiiiant while the lateral source has become greatly

reduced. In Fig. 39 is represented the typical mode of mesoderm forma tion as seen in a transverse section through the trunk region of a reptilian embryo. The mesoderm is seen to be spreading o_ut.a.s a wing of cells towards either side from the iiotochordal or primitive streak region between the two primary cel1—1ayers.

BIRDS.---ln the Birds also the method of first mesoderm formation 1 ORIGIN or THE MESODERM 65

appears to be closely comparable with that of Reptiles and Amphibians. Here, at the time when the mesoderm begins to make its appearance, the position of‘ the notochord is occupied by the primitive streak. The mesoderm fornis a loose sheet of irregularly shaped. cells spreading out on each side and added to from two distinct sources: on its inner side by proliferation from the primitive streak and on its outer side by delamination from the endoderm of the germ wall. It will facilitate comprehension of the evolutionary changes which

0 FIG. 40.——Sen1i~¢li:1.gramma1.ic transverse sections through the embryos of various vertebrates to illllstmte the origin cl‘ the inesmlerin.

A, .~lmph.-i.mus; ll, 1'c!rumy:nn. : 1‘, Imp-idosire-n; D, Amphihism; I11, l‘lln.-nmbrum:h. ect, (‘.(_'.tO(l6l‘ll1; end, emlomnnz vent, 1-nt'nrlc x-:u'it_\'; mes, mesoderm; N, inotoclmnl; n.r_, neural rluliments. '|‘l'n-. .~nnall crosses _in«_li«_-nt_«~ regions in which active extension of the nxesoderm is taking place.

the writer beli.eves to have taken place in the mode of development of the mesoderm within the phylum Vertebrata if the main steps are summarized in a diagram. In Fig. 40, A shows the primitive condition where the mesoderm segments are in the form of enterocoelic pockets (Ampholoxus). In B, with increasing amount of yolk, the hollow pocket is represented by a. solid block in which the cavity will develop secondarily (Petromyzon). In C the condition is similar but the dorsal portion of the embryonic body is more flattened out, the bulk of the yolky endoderm .over which it is spread having VOL. II F 66 EMBRYOLOGY OF TIIE LOWER VERTEBRATES CH.

become greater (Lung-fish). In D the secondary continuity of the mesoderm with the endoderm just oiitside the notochord is present and proliferation of mesoderin cells has commenced in this region (Amphibia). Finally, in E, with the very great increase in the bulk of the yolk, the dorsal part of the embryo is still more flattened out, and the addition to the mesoderm by proliferation of‘ endoderm cells into it close to the notoehord has now become conspicuous (Elasmo branch). THE MEH l+JN(“lTYMl«1 °

The fate of the mesoderm whose origin has just been traced is to give rise directly to the peritoneal epithelium which lines the body cavity and covers the organs lying within it, and also to the muscular system. lndireetly it, however, also plays a great part in the formation of what is known as the meseuchymc.

Whereas for a time the Vertebrate body is composed of compact masses or layers of cells, it is a general characteristic that, as development goes on, individual cells detach themselves and wander away through the body, multiplying by fission accompanied by mitosis, and behaving‘ in fact very much as if they were independent organisms. In the course of the many generations of these cells which arise during the process of individual development, they become divided into various strains which show marked differentiation for the performance of difierent functions.

Some retain a relatively primitive amoeboid form and undertake such functions aslthe transport of food material, the absorption of moribund tissues in regions where shrinkage in volume or atrophy is taking place, and the ingestion and destruction of attacking organisms such as. disease germs. Some, their protoplasm laden with insoluble excretory products as a result of their active metabolism, wander towards the light and settle down near the surface of the body as pigment cells or chromatophores which serve on the one hand to protect the underlying tissues from the light and upon the other to give distinctive coloration to the animal. Others again settle down in an abundant jelly-like intercellular matrix to form connective or packing tissue, which in turn shows evolution in various directions in accordance more particularly with different developments of the intercellular matrix. Of special importance are these types in which the matrix becomes hard and rigid so as to form skeletal tissues such as bone and cartilage.

Another important strain of these cells is characterized by the fluidity of the matrix and the independence of the individual cells which float in it. This liquid connective tissue forms the blood which, pumped through an elaborate system of vessels, serves on the one hand for the transport of food and oxygen to the tissues, and on the other for carrying away the waste products of metabolism to the special excretory organs the duty of which is finally to remove these harmful substances. ' I MESEN CH YME 67

The sum of these amoeboid cells, which proceed along the various evolutionary paths above indicated, were, by O. Hertwig, given the name Mesenchyme—to distinguish them from the mesothelium, or mesoderm in the restricted sense, in which the cells remain in the form of continuous layers or epithelia.

The original mesenchyme cells arise by emigration from the pre-existing cell layers. Possibly all three layers give rise to mesenchyme cells. It is the mesoderm however which does so most conspicuously. In an Elasmobranch embryo, for example, active budding off of mesenchyme cells is seen over large areas of the

somatic mesoderm and similarly from the inner surface of the splanchnic mesoderm. Most active of all is the production of mesenehyme cells from the splanchnic mesoderm near the lower end of the mesoderm segment, where the proliferating mesenchyme cells may form a conspicuous mass projecting towards the mesial plane and termed the sclerotome.‘ The special consideration of the sclerotome and of the mesenchyme in general will come most conveniently after the other derivatives of the mesoderm (Chaps. IV., V., VI.).

1 The use of the word sclerotome in this restricted sense has come to be practically universal in embryological literature and is therefore followed in this volume. The word was invented by Goodsir and defined by him, at the British Association meeting in 1856, as meaning a segment of the supportin tissue or framework (whether “ fibrous” or cartilaginous or osseous) in a segmcnte animal.

LITERATURE

Agassiz and Whitman. Mem. Mus. Comp. '/lool., xiv, 1885 and 1889. Assheton. The Work of John Samuel Budgett. Cambridge, 1907. Balfour. (.,!t1a;'lt.1.3Io1%rri. Micr. Sci., N .S. XIV, 1874.

Brachet. Are . io ., xix, 1903.

Brauer. Zool. Jahrbiicher (Anat. Al)t.), x, 1897.

Budgett. Trans. Zool. Soc. London, xv, 1901.

Cerfontaine. Arch. (le Biol., xxii, 1906.

Dean, Bashford. Journ. Morph., xi, 1895.

Dean, Bashford. Quart. Journ. Micr. Sci., xxxviii, 1896.

Dean, Bashford. Kupllcrs Fcstschrift, 1899.

Goette. Ahhamll. zur Entwickelungsgesch. d. Tiere, v, 1890. Goodsir. Edinbiggh }Ne}vlPll1ilosophical Journal, v, 1857. Goronowitsch. or )1. a 11'). x, 1885.

Hatschek. Arb. zool. Inst. Univ. Wien, iv, 1881.

Hortwig, O. Jenaische Zeitschrift, xv and xvi, 1882 and 1883. Hertwig, O. Handbuch d. Entwieklungslehre, Jena, 1906. Jablonowski. Anat. Anzeiger, xiv, 1898.

Jenkinson. Vertebrate Embryology. Oxford, 1913.

Jordan and Bycleshymer. Journ. M01'pl'l., ix, 1894.

Kerr, Graham. Phil. Trans., B, excii, 1900.

Kerr, Graham. The Work of John Samuel Budgett. Cambridge, 1907. Kopsch. Internet. Monatsschrift f. Anat. u. Phys., xix, 1902. Kopsch. Arch. mikr. Anat., lxxviii, 1911.

Kowalevsky. Mém. Acad. Sci. St. Pétersbourg, Ser. 7, xi, 1867. Mitsukuri. Journ. Cell. Sci. Tokyo, x, 1896. . Morgan. The Development of the Frog's Egg. New York, 1897. Patterson. Journ. Morph., xxi, 1910.

Prince. Ann. ltfiag. Fat. Hist., Fifth Series, xviii, 1886.

Rabi, C. Morp . Ja1rb., xv, 1889.

Rothig. Arch. mikr. Anat., lxx, 1907. 68 EMBRYOLOGY OF THE LOWER VERTEBRATES OH. I

Rilokort. SB. Gas. Morph.'Phys4. Miinch., 1885.

Rfickort. SB. Goa. Morph. Phys. Miiuch., 1890.

Rfickort. Kupffers Festschrift, 1899.

Balensky. Memoirs of the Society of Naturalists of Kasan, vii, 1t. 3, 1878. (In Russian. A French translation of the more important parts will e found in Arch. do Biologic, ii, 1881.)

8a.ma.asa.. Arch. Entwickclnngsmecluinik, vii, 1898.

Barasin, P. and 1-‘. Ergcbnisse naturwiss. Forsch. auf Ceylon. Wiesbaden, 1887 1893.

Schultze, O. lidllikcrs Festschrift. Leipzig, 1887.

Bchultze, 0. Arch. mikr. Anat., IV, 1899.

Schwink. Die Entwicklung dos mittleren Keimblattes und dcr Chorda. dorsalis dcr Amphibien. Munich, 1889.

Semon. Zoologische Forschungsreiseu, i. Jena, 1893.

Will. Zoolog. Jalu'bi1cher(Ana.t.), vi, 1892.

Will. SB. Burl. Almd., 1895.

Wilson, E. B. Journ. Morph., viii, 1893.

Wilson, H. V. Bull. U.S. Fish-Commisesiun, ix, 1891.

Ziegler. Entwiukelungsgcsch. (1. niedercn Wirbeltiere, Jena, 1902.

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Textbook Chapters: 1 Formation of the Germ Layers | 2 Skin and Derivatives | 3 Alimentary Canal | 4 Coelomic Organs | 5 Skeleton | 6 Vascular | 7 Internal Body Features | 8 Adaptation to Environmental Conditions | 9 General Considerations | 10 Common Fowl | 11 Lower Vertebrates | Appendix

Reference

Kerr JG. Text-Book of Embryology II (1919) MacMillan and Co., London.

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