Text-Book of Embryology 2-10 (1919)

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

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Chapter X The Practical Study of the Embryology Of The Common Fowl

FOR gaining practical experience in the study of embryology there is’ no type of material so convenient as that of the early stages in the development of the Common Fowl. Freshly laid eggs can be obtained practically anywhere and to obtain the various stages of development all that is necessary 1 is to keep the eggs at a suitable temperature (about 38° C.) either under a sitting hen, or in one of the incubators which can be purchased, or even in a simple water-jacketed even such as can be made by any tinsmith. If an incubator be purchased it will be provided with a proper heat regulator for use with electricity, gas or oil, while with the most primitive water-bath it is possible to arrange a lamp so as to give a temperature sufliciently constant as to carry the eggs through at least the first few days of incubation—the most important period for purposes of study. Bird embryos—apart from their use in learning practical embryology-— provide admirable material for giving practice in the ordinary methods of section-cutting which are in such constant use in Zoology, Anatomy, Physiology, and Pathology. This chapter will then be devoted to giving an account of the development of the Fowl with directions as to the technique involved in its practical study.

In the description which follows the developmental phenomena will be described in their natural sequence but on account of the practical difficulties involved in the extraction and preservation of blastoderms of the first day of incubation it will be found best, in actual laboratory work, after studying the new-laid egg and its envelopes, to proceed to the stage of about 42 hours’ incubation and gain some practice in the manipulation of it before attempting the earlier stages. In the following technical instructions the sequence is followed which has been found to be in practice most convenient for beginners.

TECHNICAL DIRECTIONS 2

I. NEW-LAID EGG.——Fil1 a glass vessel about 4% inches in diameter and 2 inches in depth With normal salt solution [Water

1 Provided the eggs have been fertilized. 9 The reader is assumed to have an elementary knowledge of the ordinary methods of cutting sections. See, however, the Appendix.’

508 CH.X PRACTICAL EMBRYOLOGY OF THE FOWL 509

100 c.c., common salt '7 5 gramme] heated to a temperature of about 40° C. Submerge the egg upon its side in the salt solution and remove the side of the shell which is uppermost by cutting with a pair of strong scissors and then lifting off the isolated piece of shell with blunt forceps. Take care to keep the point of the scissors or forceps close to the inner surface of the °shell so as to avoid risk of injury to the true egg or “ yolk.”

II. EGG AFTER 42 HOURS’ INCUBATION. —— Open the egg as before. On removing the piece of shell the blastodcrm will be seen as a circular whitish area on the upper side of the yolk. Excise the blastodcrm by making a series of rapid cuts with the large scissors through the vitelline membrane a short distance external to the boundary of the blastodcrm. Should the yolk happen to be tilted round so that the blastodcrm is not uppermost but rather at one side make the first cut below the blastodcrm so that the elasticity of the vitelline membrane will tend to pull it upwards when the cut is made. Otherwise the blastodcrm may be lost by its being pulled downwards.

Having isolated the circle of vitelline membrane, with its adherent blastoderm, slide it off the yolk by pulling gently on one side with the forceps. Remove the remains of the egg from the dish so as to keep the salt solution clean. Take hold of the circle of vitelline membrane at one edge with the forceps and wave it backwards and forwards beneath the surface of the salt solution. The blastodcrm will gradually become detached. Should it not do so at once the separation should be started by freeing it from the vitelline membrane with a scalpel at one edge. Notice the difference in appearance between the vitelline membrane and the blastodcrm which has been detached from it. If the blastodcrm is yellow from adherent yolk this should be washed oil’ either by waving the blastodcrm backwards and forwards in the salt solution or by gently directing jets of salt solution 011 the yolky surface of the submerged blastodcrm by a wide-mouthed pipette.

The blastodcrm should. now be brought near the surface of the salt solution and a watch-glass slipped under it by which it may be lifted from the larger vessel. The blastodcrm is so delicate that it must be kept submerged in the fluid: no attempt must be made to lift it above the surface by forceps.

A microscope coverslip slightly larger than the blastodcrm should now be submerged in the watch-glass and the blastodcrm floated over it dorsal side above. The dorsal or upper side of the blastodcrm can easily be identified from the fact that the edges of the blastodcrm tend to curl upwards. Having floated the blastodcrm over the coverslip the latter should be gently raised to the surface of the fluid with a pair of large forceps. Take care to keep the coverslip absolutely horizontal and lift it out of the fluid very carefully so that the blastodcrm is stranded on its upper surface, the lower surface of the blastodcrm being in contact with the coverslip. The superfluous salt 510 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

solution should be drawn away with blotting-paper so as to bring the blastoderm into close contact with the glass; take great care that the blotting-paper does not actually touch the blastoderm as in that event it will be apt to stick to it. Now take the coverslip between the finger and thumb and with the aid of a pipette place a very small drop of corrosive sublimate solution (or other fixing fluid) upon the centre of the blastoderm. This will cause the blastoderm to adhere to the coverslip. Now invert the eoverslip and drop it on to the surface of some fixing fluid in a watch-glass.

The blastoderm is then passed through the various operations of staining, dehydrating and clearing, preparatory to mounting whole oi‘ conversion into a series of sections as the case may be. The advantage of having the blastoderm adherent to a coverslip is that it makes it easier to handle and above all it keeps it from becoming wrinkled or folded. The blastoderm if fixed in corrosive sublimate can usually easily be detached from the coverslip at the stage of clearing if it has not already become free at some preceding stage. Should it adhere obstinately it should be placed i11 acidulated alcohol for an hobr or more.

The examination of the blastoderm should be carried out as follows:

1. Study the blastoderm and embryo as a whole under a, preferably binocular, dissecting microscope while it is submerged in the fixing fluid. As the fixing fluid penetrates the embryo the various details in its structure come into view. Continue the examination of the surface relief in the alcohol which is used for getting rid of the excess of the fixing agent. After examining from the dorsal side invert the blastoderm and examine from below.

2. Repeat the examination of the embryo as a whole as a transparent object after staining and clearing. If the individual embryo is to be cut into sections a careful drawing should be made at this stage, the outline being preferably drawn by means of the camera lucida.

3. Study serial sections cut transversely to the axis of the embryonic body.

[Sagittal and horizontal sections will also be useful for study after the transverse oncs.]

III. EARLY SECOND-DAY BLAs'ro1nmM.—-—The same method is used as for the 42-hour stage but special care must be taken on account of the more fragile character of the blastoderm. In all probability the blastoderm will remain adherent to the vitelline membrane in spite of repeated shaking and the process of detachment will have to be started by gently easing up the edge of the blastoderm on the side next the forceps in which the edge of the circle of vitelline membrane is held.

To get rid of adherent yolk the circle of vitelline membrane should be laid on the bottom of the dish of salt solution, blastoderm uppermost. A pipette with a wide mouth should be held vertically X TECHN ICAL DIRECTIONS 51]

a few millimetres above the blastoderm and the india-rubber bulb squeezed rhythmically so as to wash away the particles of yolk by very gentle currents of salt solution. When the blastoderm is lifted out of the solution stranded upon the coverslip it is very apt to become folded. When this happens, on account of the fragility of the blastoderm, no attempt should be made to stretch it out by the use of needles or forceps. The folds should rather be straightened out by a current of salt solution allowed to flow out from the orifice of a pipette held vertically just over the centre of the blastoderm.

IV. EARLY BLASTODERMS.-—Open the egg as before. Let the albumen run off until the vitelline membrane over the blastoderm is exposed. Raise the egg until the blastoderm touches the surface of the salt solution and then bring a wide-mouthed pipette of Flem1ning’s solution, held vertically, into such a position that its tip just touches the film of fluid over the blastoderm. Let the solution flow down slowly on to the vitelline membrane covering the blastoderm. If there is any albumen overlying the blastoderm this should be carefully stripped elf as it coagulatcs. A small piece of black bristle should be stuck into the vitelline membrane on each side to mark the line joining the chalazae so as to facilitate the orientation of the blastoderm for section-cutting. The fixing fluid should be allowed to act for several minutes and then a circle of vitelline membrane may be excised with the blastoderm adhering. to it. Floatv out the circle of vitelline membrane on a coverslip with the blastoderm above and submerge in a watch-glass of fixing fluid. If the circle of blastoderm adheres to the coverslip so much the better: it may be separated in the clearing agent.

Instead of a pipette as above indicated being used for the fixing fluid a small rim of cardboard, e.g. the rim of a small pill-box lid, may be placed on the surface of the yolk, raised up slightly out of the salt solution, so as to enclose the blastoderm and then the little tank so formed may be filled with Flemming’s solution which will gradually diffuse downwards. Minchin recommends a triangular instead of a circular rim for facilitating subsequent orientation.

For fine work it is preferable to embed the whole yolk in celloidin and then after the celloidin has been hardened to c11t out the portion in the region of the upper pole for sectioning. This method consumes however much more time than does the paraffin method.

V. THIRD-DAY EGG.——A. Open the egg as before.

B. Study the embryo and blastoderm while still alive and in situ. A large outline drawing should he made. The details of the body of the embryo will be seen better later but the arrangement of the blood-vessels can best be studied now while the circulation is still active. As a rule they can be seen distinctly through the vitelline membrane but if not the latter should be carefully stripped off. A Greenough binocular microscope with its lowest power objectives is a useful accessory for examining the blood-vessels.

C. Excise the embryo with the surrounding portion of blasto512 EMBRYOLOGY OF THE LOWER VERTEBRATES OH.

derm, float it on a slide, cover with coverslip supported by wax feet at the corners and examine as a transparent object, comparing the various features with those shown in Figs. 235 and 236.

D. Excise a second embryo with its surrounding blastoderm. Float it on to a coverslip, embryo above, and submerge it in a watchglass of fixing fluid. Watch it carefully under the lens or preferably low-power binocular as the tissues gradually become opaque. The amnion will be seen particularly clearly during this process. A drawing should be made of the embryo enclosed in its amnion as an opaque object.

E. Carefully strip off the amnion with a pair of needles 1 and study the configuration of the head end of the embryo.

F. Stain and mount the embryo.

G. Prepare series of sections (at) transverse to trunk region, (1)) horizontal through trunk region and therefore approximately sagittal in the region of the head which is lying over on its left side.

VI. THE FOURTH DAY.-On placing the egg in the salt solution the broad end will tilt up more decidedly than before owing to the increase in size of the air space. Care should therefore be taken to make the first perforation of the shell close to the broad end so as to allow the air to escape. Care must also be taken not to injure the vascular area as the whole blastoderm is now much closer to the shell than it was in earlier stages. As soon as the egg has been opened a careful drawing should be made while the embryo is still alive and in situ. The main features of the vascular system in particular should be carefully worked out at this stage. If the circulation becomes sluggish through cooling a little warm salt solution should be added but care must be taken not to bring about a great and sudden rise of temperature as in this case the greatly accelerated heart-beat is apt to cause rupture of a vessel.

The body of the embryo, allantois, ete., are covered over by the thin transparent serous membrane or false amnion as becomes apparent if the attempt is made to push a blunt needle down into the space round the allantois. This membrane should either be cut thrpugh with a pair of fine scissors, just outside the boundary of the allantois, or carefully stripped off with fine forceps. When this has been done it is possible to shift the body of the embryo into such a position that it with its blood-vessels can be observed in side view. Until this has been done it is impossible to get a proper view of the body of a well-developed embryo of this age owing to its dipping down out of sight into the yolk-sac.

The embryo should now be excised by cutting round outside the boundary of the vascular area and floated into a watch-glass of clean warm salt solution. The embryo may now be studied as a transparent object on the stage of the dissecting microscope. It is better

1 Bearing in mind that steel needles must not be allowed to touch corrosive sublimate solutions. Picric acid solutions are convenient fixing agents to use for D

and E. X TECHNICAL DIRECTIONS 513

however in the first attempt to proceed at once to fix the embryo. An essential preliminary is to remove the true amnion which closely ensheaths the body of the embryo. In doing this it is best to commence at the region between the heart and the tip of the head where a couple of fine needles may be used to tear the amnion. Its anterior portion may then be seized with fine forceps and pulled backwards over the embryo’s head. The operation is simplified by carrying it out immediately after submerging the embryo in fixing fluid as the action of the fluid makes the amnion slightly opaque and therefore more easily visible. - If however corrosive sublimate be the fixing fluid fine splinters of coverslip should be used for dissecting off the amnion unless this is done prior to immersing in the fixing fluid. The embryo should again be carefully studied during the process of fixation, many details becoming particularly distinct before the creature becomes completely opaque. Finally the embryo should be studied, preferably with the binocular, as an opaque object, and then prepared either for section cutting or for mounting whole.

VII. SIX l)AYs.——Open freely into the air-space. Carefully tear away part of its inner wall so as to expose part of the vascular area, great care being taken 11ot to injure the latter. Notice the direction in which the vessels of the vascular area converge: this will indicate the direction in which the embryo is to be. found. Work towards the embryo, picking off the shell piece by piece, using blunt forceps. Frequently the escape of the air from the air-space allows the vascular area to sink down and leave a wide space between it and the shell membrane. In other cases however it remains in close contact with the shell membrane and in this event the greatest care must be taken not to injure the vascular area as by doing so the very fluid yolk is allowed to escape and the salt solution rendered so opaque that observation of the embryo in situ is made almost impossible.

Notice that the allantois has increased much in size, that it has become richly vascular and that it is spreading outwards in a mushroom-like manner underneath the serous membrane. It has already spread so far as to cover the embryo nearly completely.

It is best new to remove the shell entirely and to examine its contents as they lie submerged in the warm salt solution (as shown in Fig. 242).

With fine sharp scissors cut through the serous membrane just outside the limit of the allantois, commencing on the dorsal side of the embryo where the allantois is not yet closely applied to the yolksac. It is easy to do this owing to the coelomic cavity having spread outwards well beyond the limits of the allantois. The allantois being new no longer flattened out, by its continuity with the serous membrane all round, its vesicular character becomes apparent, as well as the difference in character of the vascular network on its proximal and distal walls. The relations of the vascular allantoic stalk to the vascular yolk-stalk should be noted: also the fact that the amnion is

VOL. II 2 L 514 EMBRYOLOGY 01*‘ THE LOWER VERTEBRATES on.

now widely separated from the embryonic body by secreted amniotic fluid. If the embryo is a well-advanced one towards the end of the sixth day the amnion, which is now muscular, may exhibit periods of muscular contraction during which the embryo is rocked to and fro in the amniotic fluid. These movements must be distinguished from the occasional contractions of the muscles of the embryonic body which also occur about this time though they are much less conspicuous.

After a careful study of the living embryo with the allantois and yolk-sac hanging from its ventral side it may be excised along with a circle of vascular area, floated into a watch-glass and examined alive with a lens or binocular, and then treated with fixing fluid such as Bouin’s solution. The latter brings out the surface modelling which should be carefully studied especially in the region of the gill clefts.

Dissect off the amnion, add more fixing fluid and after superficial fixation renew the llouin’s solution. It is a good plan to suspend the embryo by the yolk-sac so that the weight of the head causes the neck to become somewhat straightened. After the embryo is sufficiently fixed the neck may be cut through and the lower surface of the head studied for the relations of the olfactory rudiments and mouth.

Sagittal sections through the head are particularly instructive at this stage.

VIII. SEGMEN'l‘A'l‘ION.——T0 obtain segmentation stages hens which are regular layers should be chosen. In such cases the egg is laid at a slightly later time on consecutive days. As a rule egg-laying is confined to the forenoon and early afternoon and when an egg is due after the end of this period it is retained within the oviduct and not laid until next day. The retention of an egg in this way inhibits the process of the ovulation so that a new egg is not shed from the ovary until the preceding one has been laid.

HISTORY or THE Eco UP TD run TIME or LAYING:--The egg arises as a single cell of the left ovary 1 which grows to a relatively enormous size as yolk is deposited in its cytoplasm. The yolk is of a characteristic yellow colour but in particular tracts the disintegration of its granules into finer particles gives it a white colour. Of this white yolk a mass occupying the centre of the egg is continuous through a narrow isthmus with a tract lying immediately beneath the germinal disc (“ Nucleus of Pander ”) and this latter is prolonged as a thin superficial layer over the surface of the egg. Between the superficial layer and the central mass are a number of thin concentric layers of white yolk.

‘ The right ovary and oviduct which are present in early stages undergo atrophy, never becoming functional. This is probably to be regarded as an ada tive arrangement which has been developed in Vertebrates with large eggs to avoi the dangers which would be involved in the synchronous passage of a pair of eggs of great size, more especially if contained in a rigid shell, into the narrow terminal portion of the passage to the exterior. ' x ‘EGG or COMMON FOWL 515

As the egg increases in size it bulges out beyond the surface of the ovary, becoming eventually dependent from the ovary by a thin stalk at the end of which it is enclosed within the distended follicle. The wall of this is richly vascular except on the side away from the stalk where an elongated patch—the “stigma ”--marks the position in which the follicle-wall will rupture to set the egg free.

When this process (ovulation) is about to take place the thin membranous lips of the oviducal funnel become active, apply themselves to the follicle containing the ripe egg and grip it tightly. The follicle then ruptures and the egg is as it were swallowed by the oviducal funnel. Within the funnel fertilization takes place provided that spermatozoa are present.‘

The egg proceeds now to travel slowly down the oviduct, propelled onwards by the peristaltic contraction of the oviducal wall, the entire passage occupying about 22 hours. As it does so the albumen is deposited on its surface by the secretory activity of the oviducal epithelium. The first to be deposited is rather denser than that formed subsequently. It forms a sheath immediately outside the vitelline membrane and extending in tapering spindle-like fashion for some distance up and down F'<=- 323

U nincubated egg of the Fowl. the oviducal cavity; the two a.s, air-space; alb, albumen; ch, clialaza; s.m,

- , 3 _ ‘ shell membrane. In the centre-—at the apical pole-prolongatlons a’r(" flu” chalazae is seen the germinal disc with the white “Nucleus of

  Puiulv-1"‘showingllwough it‘.

The envelope of (‘louse albumen enclosing the egg is not merely propelled onwards; it also undergoes a clockwise rotation about the axis along which it is travelling, caused probably by the cilia present on the oviducal epithelium.’ Owing to the prolongations of the albumen in front and in rear of the egg not undergoing this rotation the chalazae become twisted upon themselves in opposite directions.

Layer after layer of albumen (Fig. 223, alb) is deposited round the egg and chalazae until the full size is reached. The character of the secretion then changes and the shell membrane (Fig. 223, 3.-m) is formed. Finally in the dilatedhinder part of the oviduct (“uterus”) the secretion is in the form of a thick white fluid which, deposited on the surface of the shell membrane, gradually takes the form of the hard and rigid shell perpetuating the characteristically “oval” form impressed upon the egg envelopes during the passage down the oviduct. In composition the egg-shell consists of calcium salts infiltrating a slight organic basis of keratin-like material. Structur 1 The spermatozoa remain alive and active within the oviduct for a period of about three weeks. 516 EMBRYOLOGY OF THE LOWER VERTEBRATES CH. X

ally the greater part of its thickness consists of calcareous trabeculae forming a fine sponge work. The inner surface of the shell is rough, projecting into minute conical papillae, while the outer surface is covered by a smooth apparently structureless layer perforated by numerous fine pores.

SEGMEN'1‘ATION.——If the egg has been fertilized it proceeds with its development as it slowly travels down the oviduct. The process of segmentation is accomplished during this period and consequently the obtaining segmentation stages involves the sacrifice of the parent hens. Owing to the difficulties in the way of obtaining a complete series our knowledge remained for long fragmentary but recently (1910) a number of stages have been described and figured by Patterson which give a fairly complete picture of the process (Fig. 224). From these data we may take it that the early phases of segmentation are based on the normal plan where a meridional furrow appears traversing, or passing close to, the centre of the germinal disc ’i.6. the apical pole of the egg, and is followed by a second meridional furrow perpendicular to the first. In the third phase there is occasionally a regular set of four vertical furrows but more usually the process now becomes irregular (Fig. 224, C). In the next phase also there may be a fairly regular development of latitudinal furrows demarcating a group of about eight cells round the apical pole but typically there is no such regularity. The initial furrows, which make their appearance as above indicated, gradually extend. They eat their way downwards into the thickness of the germinal disc,-never however cutting completely through it. They also extend outwards towards the edge of the disc which however again they never quite reach. The apparent segments into which the germinal disc is mapped out by the early furrows are therefore not really isolated from one another ——there being still continuity between the segments on the one hand peripherally and on the other on the lower side of the disc next the yolk.

Complete blastomeres are first marked off when, about the time the latitudinal furrows appear, division planes make their appearance parallel to the surface, cutting off the small segments in the centre from the underlying deep layer of the germinal disc.

The later stages of segmentation are quite irregular. Division planes make their appearance in all directions by which the germinal disc becomes completely divided up into small segments except on its lower surface and round its edge where there remains a syncytial mass in which the nuclei divide without their division being followed by any protoplasmic segmentation. It is to be noted that the process of segmentation throughout goes on more actively towards the centre of the disc, more slowly towards its margin, so that the blastoderm comes to be composed of smaller cells towards the centre and larger towards the periphery.

The result of the segmentation process is that the original FIG. 2'24.———Views of the blastoderm of the F0\vl'.~: egg during segmentation. (Afher Pa.1..tei-son, 1910.)

A, 3 hours after fertilization ; IL 5}, In s. ; U, 4 In-:~'. ; J), 4-5 hrs. ; E, about .-3 hrs. ; l , .5111-s. ;

(L 7 }n'.~:. : H, 8 hrs. . 518 _EMBRYOLOGY OF THE LOWER VERTEBRATES en.

germinal disc comes to be represented by a lenticular blastoderm lying at the apical pole of the egg and corresponding to the mass of micromeres of such a holoblastic egg as that of Lepidostren. The superficial layer of cells become fitted closely together and form a definite epithelium——which is destined to become the ectoderm. The cells of the lower layers on the other hand are rounded with chinks between them representing the segmentation cavity. The lowest of all have the appearance of being incompletely cut oil from what is ordinarily termed the white yolk lying below them but which is really a syncytial layer" full of fine granules of yolk and with scattered nuclei.

Apparently a few accessory sperm nuclei are usually present in the fertilized eggs and faint traces of abortive segmentation may be visible round them (of. Elasmohranch, Fig. 8, B *, p. 14).

At the time of laying the blastoderm forms a small whitish disc covering the apical pole ot' the egg. Sections show it to consist of an upper layer of ectoderm and of a lower layer consisting of numerous rounded micromeres lying about in the fluid of the segmentation cavity. These micromeres become larger towards the lower face of the blastoderm and they are more crowded together round the periphery. _

It must not be supposed that all newly-laid eggs show exactly the same degree of development. As a matter of fact great variation occurs, one of the chief variable factors probably being the length of

time occupied in the passage down the oviduct. Where this time is,

longer, as e.g. towards the end of the laying season, the stage of development of the egg when laid is more advanced.

THE FIRST DAY or l"NcUeA'_r1oN.——After the egg has been laid the lowering of the temperature leads to such a slowing of its vital processes that development appears to come to a standstill. If kept at a'low temperature it retains its vitality for‘ a considerable period but makes no appreciable advance in development. If the temperature be raised by incubation the developmental processes are at once accelerated and comparatively rapid changes come about. The blastoderm increases in size, its margin spreading outwards, and at the same time there comes about a distinct difference in appearance between its central and marginal parts-the central portion assuming a dark transparent appearance (pellucid area) which contrasts strongly with the whiter “opaque area” surrounding it. - The examination of sections at once explains this difference in appearance: the more opaque appearance peripherally is seen to be due to the lower layer cells being there closely crowded together.

An important change soon comes over the lower layer cells, in as much as those next to the yolk, in the region underlying the pellucid area, lose their rounded shape, become somewhat flattened and adhere together edge to edge to form a continuous membrane-—the (secondary) endoderm. This appears first beneath the posterior poi tion of the pellucid area; it gradually extends x FOWL—--FIRST DAY’ 1 519

FIG. 225.-~111ustrating three stages of the blastoderm of the Fowl during the second half of the first day of incubation.

a..o, opaque area; mp, pellucid area; lap, head process; mes, boundary of sheet of mesoderin; m.f, medulla:-y fold ; p.g, primitive groove ; 12.3, primitive streak.

forwards and outwards, and eventually is continuous all round with the thickened marginal part of the b1astoderm.1

1 This thickening of the posterior edge of the blastoderm presents in sagittal section a. striking resemblance to a. gastrular lip growing back over the yolk and Patterson (1907) believes that an actual process of involution-—a reminiscence of gastrnlation by inva ination--takes place, It must not be forgotten that any explanation of such 0 scure developmental “phenomena. in Birds must, to be reliable,520 EMBRYOLOG-Y ()l<‘ '_l‘Hln‘ LUWEJ-t VEl~‘tTEBRATES an.

A gradual change takes plzme in the shape of the pellucid area which, up till now circular, a.ssuim,-s an oval or pear shape (Fig. 225, B)——the long axis perpendicular to the long axis of the eggshell, and the narrow end being next the observer when the broad end of the egg-shell is to the left. This narrow end may be called posterior from its relations to the rudiment. of the embryo which appears later. Together with the gradual change in the shape of the pellucid area there takes place the development of the primitive. streak. This makes its appearance usually during the first half of the first day of incubation, as a linear opacity stretching forwards along the long axi.s of the pellucid area in its posterior third. As the first day of incubation goes“ on the primitive streak becomes more and more distinct. A longitudinal groove develops along its middle———the primitive groove-—while on each side of this it forms a ridge, the primitive fold.

If a number of eggs be examined du.ring the first day of incubation

 ,_m.g»,    n
  rgzasa.

FIG. 2‘26.—-Transverse section through primitive streak of the Fowl.

eat, vet-oderm ; vi-mi, 1-ndoderm; mes, mesoderm; 12.;/, primitive groove.

it will be seen that the primitive streak, as is commonly the case with vestigial organs, shows extreme variability. More especially its hinder end is commonly bent to one side or the other, or even bifurcates into two branches. At its front end one or both halves of the primitive streak swell up into a slight knob while the primitive groove becomes somewhat deeper and wider.

The primitive streak is shown by transverse section to originate from a linear tract of ectoderm along which the cells are undergoing rapid proliferation, as is indicated by the relatively numerous mitotic nuclei. The cells budded off by the ectoderm are aggregated together in a compact mass along the course of the primitive streak while on each side they become loosened out and wander away into the space between ectoderm and endoderm to take part in forming the sheet of mesoderm.

1-;_.:-: L .——r-—.—:-—

~——-:jw—j— -f

rest on a firm basis of knowledge of Reptilian development. At the present time however our knowledge of the exact relationship of these clevelopniental stages of Birds to the corresponding stages of Reptiles is not in the present writer’s opinion adequate to form a trustworthy basis for their interpretation. FOVVL ———FIRS'l‘ DAY 521

H

For a short distance in- the region of its front end the mass of cells forming the primitive streak is continuous not only with the ectoderm but with the endoderm as well: the primitive streak of this region may be defined as a tract along which there is cellular continuity between the ectoderm and the endoderm.

During the latter half of the first day what is known as the “ Head process ” makes its appearance (Fig. 225, B, lap). In a view of the whole blastoderm this has the appear‘-aiice of being a somewhat less distinct prolongation forwards of the primitive streal<——-in front of the knob which marks its apparent front end. 'l‘he study of transverse sections shows that the so-called head process is exactly similar in structure to the primitive streak immediately behind it, except that it is separated fr cm the overlying ectoderm by a distinct split and that there are no primitive folds or primitive groove over it. On its lower side there is perfect continuity with the endoderm—-— as is the case with the anterior part of the obvious primitive streak into which it is continued.

During the same period of incubation there appears the first sign of the surface relief of the body of the embryo in the form of what is known as the head fold (Fig. 227, A, h. f). This is formed by the blastoderm bulging upwards and forwards, forming a projection bounded in front by a steep face crescentic in shape as seen from above, the two horns of the crescent directed backwards. The projection increases in prominence: its front edge soon comes to overhang, the blastoderm becoming tucked underneath it both in front and at the sides, the two horns of the crescent which the fold formed at its first appearance gradually extending farther and farther backwards. The projection is destined to give rise to the head end of the embryo and there are certain important details to be noticed about its structure which can be made out best by the study of sagittal sections.

The region of the blastoderm where the head fold develops is composed of the two primary layers, ectoderm and endoderm, the mesoderm not yet having spread into it. It follows that the head rudiment has a double wall, its outer sheath of ectoderm enclosing an inner wall, quite similar in shape, composed of endoderm. It will be understood that this inner wall of endoderm is continued at its hind end into the flattened layer of endoderm which lies on the surface of the yolk. In other words the endoderm within the head rudiment may be described as forming a very short wide tube, blind anteriorly but opening behind into the yolk. This endodermal tube is the rudiment of the front part of the endodermal lining of the alimentary canal of the adult and is termed the foregut.

Soon after the commencement of the formation of the head fold the ectoderm of the medullary plate becomes raised up into a longitudinal ridge (Fig. 227, A, m. f) upon each side of the median line. Between the two ridges is a groove——the medullary groove: the ridges themselves are the medullary folds: the two medullary folds F10. 227.—Fow1 embryos at about the end of the first day of incubation seen by reflectcd light.

5-6 segments. cr..p, pellucid area‘ aw, vascular area; f.g, foregut; h.j, head; m.f, medulla:-y fold; m..c, nnesoder-n1

A, 3 mesoderm segments; B, no segments yet demamcated; 0, segments ; p.a, proamniun ; p.g, primitive groove; 11.3, pximitive st.reaLk. CH. X FOWL--FIRST DAY 523

are continuous anteriorly. The two medullary folds gradually extend backwards and at the same time they become more prominent and arch over towards one another until at about the end of the first day they meet. It is to be _noticed (h‘ig.‘227, B) that the first meeting of the medullary folds is some little distance back from their anterior end, in about the position in which the division between mesencephalon and rhomhencephalon will develop later. Towards their anterior end the folds remain less prominent than they are farther back with the result that the meeting of the two folds is here con— siderably delayed.

During these later hours of the first day important advances are taking place in the development of the mesoderm. In the first place it is to be noted that the anterior limit of this layer is gradually extending forwards, encroaching more and more upon the proanmion ——the part of the blastoderm in front of the head fold which is still two layered. In the second place the mesoderm becomes considerably thickened and more compact in the region near the median 1ine——— adjacent to the head process or notochord. This thickened portion of the mesoderm becomes divided by transverse splits into a series of blocks——thc mesoderm segments——lying one behind the other (Fig. 227, A and C, m.s). The first pair of splits to make their appearance are placed obliquely, sloping outwards and backwards: they mark the hind boundary of the first or most anterior segment. A little later a pair of similar splits develop a little farther back forming the hinder limit of the second segment, and so on, segment after segment becoming separated oil’ from the still continuous mesoderm lying farther back.

While this portion of the mesoderm is becoming segmented it is at the same time becoming sharply marked off by its greater thickness from the lateral mesoderm lying farther out from the axis. Towards the end of the first day at further important development takes place in the mesoderm in as much as isolated splits appear in it parallel to its surface and these gradually spread and finally become continuous so as to divide the mesoderm into the outer somatic layer next the ectoderm and the inner splanchnic next the endoderm. The cavity which has made its appearance between somatic and splanchnic layers of mesoderm-is the coelome. The portion lying within the myotome, which soon becomes filled up by immigrant cells derived from its wall, is the myocoele (Fig. 228, me). The portion lying farther out, in the lateral mesoderm, is the splanchnocoele (splc). The two layers lying external to this cavity——the somatic mesoderm and the ectoderm —constitute the somatopleure or body-wall: the corresponding layers lying internal to the cavity-—the splanchnic mesoderm and the endoderm——-constitute the splanchnopleure or gut-wall.

While the changes above described have been taking place the blastoderm has constantly been increasing in area and by the end of the first day it forms a cap covering an extent of about 90° at the upper pole of the egg. In the opaque area—--the part of the blaste524 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

derm lying outside the boundary of the pellucid area---there are present the same layers of cells as in the pellucid area-—-the ectoderm, which extends. farthest peripherally, the endoderm which passes into «W thick ¥°1kSYI1cWe1layer vermherallv <2ermina1 wall. and the

 s A v            

flu “L * n’

L1 1

Flu. 2‘28.—-'I‘1‘ansVerse section through the bocl y of a Fowl embryo about the end of the tiist day of incubation. c. act, cctoderm; end, endodurm; me, myocoele; my, myotomu (lll6S0(lCI‘ll1 segment); N, notochord;

'n.'r, neural rudiment; som, somatopleure; spl, splanclnioplenre; splc, splzmclmocoele.

mesoderm the outer part of which is still unpenetrated by the coelomic split. The part of the opaque area where inesoderm is present assumes a very characteristic mottled appearance (Fig. 227, 0, (M2) caused by the rudiments of blood-vessels and blood: hence the name vascular area which is given to this part of the blaste derm. When the embryo has reached the stage with about seven

inesoderni segments the secretion of fluid (plasma) commences within the blood islands.

13313 Saconn DAY or INCUBATIQN:

opened during the second day of incubation is seen in Fig. 229. The blastoderm has increased considerably in size and now covers about 110". The pellucid area has assumed a somewhat fiddle-like shape.

On examining the excised blastederm about the commencement of this day it is seen that the formation of the head fold has progressed considerably and the head rudiment projects more conspicuously above the general level of the blastoderm. Within the head rudiment the foregut can be seen and it is noticeable that it stretches farther back than does the outer wall of the head rudiment. . In other words the head fold of the endoderm has spread farther back

FIG. ‘2‘29.~—-Egg of the Fowl about the middle of the second day of incubation.

a..o, circular opaque area. the dark pellucid area, with the rudiment of the embryonic body lying along its axis.

In the centre is

-——The cg;-,ne1~a1 appearance of an seoegggi X F OWL-—SECON D DAY 525

than that of the ectoderm. This is brought out clearly by a sagittal section such as that shown in Fig. 230. Such a section a.lso brings out the fact that while the greater part of the portion of blastoderm tucked in beneath the head of the embryo is two-layered (proamnion), there being no mesoderm present, this does not apply to the farthest back part of the fold. Here, in the wide space between cctoderm and endoderm, mesoderm has penetrated which will give rise to the pericardiac wall and the heart. The medullary folds have met over a ' considerable extent but still remain separate at their extreme front ends as well as over the whole extent which will later form the spinal cord. Here they bound a deep neural groove. Towards their posterior ends the two medullary folds diverge to pass on either side of a lance—shaped area (rhomboidal sinus) which they enclose by converging towards one another behind it. Along the centre of the

Flu. 230.»-Diagrairiiiiatic sagittal section through anterior end of Fowl embryo with 15 segiiieiits.

am, rudiment of amnion; hr, brain ; er!-, ectmle-rm ; um.-, endocai-dinm; end, endoderm of yolk-sac; _/15;, l'm'v_~,r11t2 h._/‘, p0n'l'-i'I‘l')1' limit or lH.':ul rum of uctoderm; mo, myocardinm; N, notochord; pa, ect.-o«‘lm-In of pm.-nnninn ; -vile, split!|(7lI1I()(1()Ble.

floor of the rhomboidal sinus the primitive streak is still_ visible separated by a knob—li.l<e elevation from the part of the primitive streak which lies farther back.

The mottled appearance characteristic of the vascular area is now seen to be continued inwards, though much more faint, across the pellucid area to the body of the embryo.

An embryo with about ten segments is shown in Fig. 231. The pellucid area is still somewhat fiddle-shaped with the body of the embryo lying along its axis. Apart from the increase in number of the mesoderm segments the most conspicuous advances in development are in the central nervous system. The medullary folds have met and fused together to enclose the neural tube except towards their hind ends where they still bound the rhoinboidal sinus on each side. The forebrain region is greatly dilated, its projection on each side being the optic rudiment (am). It will be noticed that a slight notch in its wall in the mesial plane anteriorly

indicates that at this point the two neural folds have even yet not 526 EM]-3[{.'Y(ie)l'.O(‘:i-Y 01*‘ THE LOWER VERTEBRATES (:11.

completely fused. Posteriorly the neural folds seem to be continuous with the lips of the primitive groove. A faint continuation forwards of the primitive groove may be seen in the floor of the rhomboidal s1nus.

Flu. 231.~-Bla.stml¢_-1'In with l"m\'l .wJl"1J]..‘.‘| will: :n.bm|t l() or ll 1m__e.~'(_)«lt.'-.1'n1 .«-;_;'111u11l-:4.

am, Vtl.s‘(:lll1ll':lI‘o-.1 ; _/'._:/, fur-e;;I11.: la, Imiul : m.‘/1, ma-«lnll:n'_\' rule! ; m..~:. rm-:-.mlerIn .\'I'_4_1_'lll6l|t-2 u./', upliv l'lllllllH_'lll.I /W. ]rmunminn.

Important mesoderrn features are to he noticed. The mottled appearance of the vascular area produced by the rudiments of bloodvessels developing in the splauchnic mesoderin is conspicuous. The formerly isolated vascular rudiments (white in the figure) are now becoming joined up to form a network and the network can be traced —less distinct and on a smaller scale—-across the pellucid area. At x . 'i_«‘oWI._.sil«:(":(_)N1) DAY 527

its anterior and inner corner the network is continuous with a short and wide vessel which slopes obliquely forwards and inwards and disappears beneath the hind end of the foregut (shown more clearly in Fig. 232, 72.22). This vessel is the rudiment of the vitelline vein, which drains the blood from the vascular area towards the heart. Another conspicuous vessel rudiment is the terminal sinus-a marginal vessel which bounds the vascular area externally. In front of the head of the embryo is a somewhat rectangular area of the blastoderm distinguished by its being very transparent (Fig. 232, pa). This is the proamnion—- -its transparency being due to the fact that

FIG. 232.~-—--llmul oi" l“u\vl «-mhr_\'0 of smm-. Hiilgif as that s'hu\\'n in Fig. 231, more highly inugnilieil and Ht-‘vll by tnuus-initlwl liglit.. f.g, foregut; 1!, heart: h.._/',himlerlimil ofhcml fold ()fvC.l'(_)¢ll'l‘lll; inf, infumlibnlnm; m..s, mesoderm

segments; N, notochord; 0.7-, optic rmliuu-nt: ,.u, ]nn:unniuIl ; splc, patent portion of splanchnocoele containing coelomic fluid; -mi. vitellim-. \ win.

the mesoderm has not yet spread into this region of the blastoderm. On each side of the head of the embryo the surface of the blastoderm bulges upwards into a dome-like swelling (Fig. 232, splc). This is due to a precocious splitting of the mesoderm in this region to form a large coelomic space. The bulging appearance is produced by the coelomic space being tensely filled with fluid. The raising up of this region of somatopleure is preliminary to the formation of the head fold of the amnion.

By turni.ng over the excised blastoderm and examining it from below or by staining and then examining it in dorsal view by transmitted light (Fig. 232) it will be seen that between the two coelomic spaces there lines a A-sha_pe<.l structure. The two diverging limbs of 528 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

the A posteriorly are the vitelline veins already alluded to (cw), While the median portion (H)——a straight tube passing forwards beneath the foregut-—is the rudiment of the heart and ventral aorta. It will be noticed that the two vitelline veins when traced backwards from the heart are seen to fit round the tunnel-like opening of the foregut. In the forcbrain region is seen the downwardly projecting pocket of its floor—the infundibulum (Fig. 232, v}nf)———and extending -back from this in the middle line the notochord (IV). On each side of this posteriorly are seen the mesoderm segments (m.s).

In a slightly more advanced embryo with about fifteen mesoderm segments the tucking in of the blastoderm under the head has proceeded considerably further. The neural tube has become closed in entirely except for the slit-like remnant of the rhomboidal sinus posteriorly. The optic rudiments projecting prominently from the forebrain on each side and beginning to be narrowed slightly at their base give the brain a conspicuous T-shape. The wall of the brain in its posterior region shows a series of puckerings one behind the other marking it off into a series of what used to be called brain “ vesicles.” Of these the anterior one, the largest and most distinct, is destined to become the niesencephalon while those behind it enter into the formation of the rhombencephalon. The latter are often interpreted as vestiges of a once present segmentation of the brain, but are regarded by the author of this volume as being adequately accounted for by the active growth of the l.rain within its confined space, aided possibly by the varying consistency of the mesenchyme outside it (see p. 101).

On each side of the head region posteriorly, just in front of the first.obvious mesoderm segment, the rudiment of the otocyst has made its appearance as a cup-like depression of the ectoderm.

The heart, growing in length more rapidly than the neighbouring parts of the body, has been forced into its characteristic bulging outward on the right side. The first traces of haemoglobin are making their appearance in the posterior portion of the vitelline network.

An important new feature becomes visible about this stage in the form of a whitish line on the bulging roof of the splanchnocoele on each side. The lines in front curve in towards one another, meeting in front of the proamnion and sweeping back in a wide curve on each side. This line is the first rudiment of the amniotic fold. As the fold becomes more and more prominent it bends backwards and inwards, arching over the head region, and towards the end of the second day (Fig. 233) forming the anmiotic hood which ensheaths the head portion of the embryo.

Many of the important details in the structure of the second day blastoderm can only be made out by the study of series of transverse sections. In studying the stage new under consideration it is advisable to begin with a section taken from about the middle of x ‘ FOWL—~——SECOND DAY 529

the total length of the embryo such as that represented in Fig. 234A. The blastoderm some little distance away from the median line of the embryo is seen to consist of the usual two double 1ayers——-the somatop1eure(som) composed of ectoderm and somatic mesoderm and the splanchnopleure (spl) composed of splanchnic mesoderm and endo FIG. 233.-—Blastoderm and embryo Fowl with 18 mesoderm segments.

u,.e, hackgrowing edge of amniotic hood; asp, pellucid area; um, vascular area; 1!, heart; of, 0t0(‘,_yst,; sa, sero-amniotic connexion.

derm. In immediate contact with the lower surface of the endoderm in the complete egg there would be the yolk. I'n the splanchnic mesoderm overlying the endoderm are seen the blood-vessels of the vascular area. When traced inwards towards the mesial plane the two layers of mesoderm are seen to come together to formthe narrow protovertebral stalk or nephrotome which joins up the lateral mesoderm to

VOL. 11 . A 2M 530 ']+3MBRY()L()GY or THE Lowm: \’l~}1t5lTfil+}J.:itATES en.

the mesodei-in segment. Immediately above the nephrotome, between it and the cctoderm, is seen the rudiment of the arehinephric du.ct-——a rod of cells which is gradually extending tailwards. i In the centre of the section is the neural tube (s.c) with its thick walls and the solid notochordal rudiment (N) -lying immediately

FIG. 234A.——Transverse section through the middle of El .*i('(‘.(')nIl~(l:l}-‘ Fowl embryo ' (15 segments).

4, p.-iiretl dorsal aorta; a..n.d, fll‘Clllllf‘])lll‘l('f duct; eat, t-(‘totlvrni : end, u-n«l0tl(‘,1‘ln ; my, myotome; N, notoeliord; s.c, spinal cord ; .~'u‘m, somatnpleure; spl, splam-hnopla.-m'e; .s-pin, splanclmoeoele.

below it. The blood-vessel (A) on each side between nephrotome

and endoderm is the dorsal aorta which is at this stage double. Working back towards the tail end of the embryo it is seen that

subsequent sections show less and less advancedstages of development

FIG. 2343.-Transverse section through a second-day Fowl embryo jllst lwliiml the binder limit of the l'(')1‘e;,5l1t.

A, dorsal aorta; and, endoderm; -‘my, myotome; N, not.oeho1.-d; .-.r_.-, spinal em-cl ; min, somatoploure; Sp], splanchnopleure; .~.-plc, splanehum-oele; r, \"('_5.‘€."~‘.(!l.\' ()f\'asc||I:1[‘;1rea,

in concordance with the fact that development proceeds from the head end tailwards. Thus the neural tube opens out by the slit—like rhomboidal sinus; the archinephric duct disappears; the notechord passes back into the undilferentiated tissue of the primitive streak. On the other hand the examination of sections farther forward towards the head region brings into view various important further developments. Such a section as that shown in Fig. 23413 illustrates x Fo_wL.....sEooNn DAY 531

clearly an early stage in the folding off of the foregut from the cavity of the yolk-sac-——a fold of splanchnopleure growing inwards on each side below what will become the foregut. The large vessels seen in the splanchnopleure external to the fold just mentioned are tributaries of the vitelline veins, and a few sections farther forwards they would be found to be united together to form the main vitelline vein on each side.

As the series of sections is traced forwards the two folds of the splanchnopleure are seen to approach one another and finally to meet and undergo fusion, so that there now exists a foregut cavity shut off (as seen in transverse ._section) from the yolk-sac, the walls of the two structures being still connected by a median vertical partition formed by the fusion of the endoderm from

FIG. 2340.~—Transverse S(‘.(‘.t-iml of a .~u-cowl-day Fowl embryo pa-1..<siiig through the I'udiment of the ll(‘iU.'l.

A, dorsal aorta : «l.nu°, 1l()l'H:ll Im-snmmliuiii 1 rm". l‘lIIl()l'L'l.l'|lllllll : crud, cnclorlc-rm : jig/, foregut; ma, myocardium; s.c, spimil cord; so.m, smiiatic nic.-‘ode’-rin; sp.m. spluncliiiiu ino.<o(lo'1'1ii; split, splanclb nocoele; v.m«_-, \'t'-ntl‘£ll mcsocardium.

the two sides. A little farther forward this partition disappears from ‘the section and the foregut as seen in section (l*‘.ig'. 2340) is quite isolated from the endoderm of the yolk-sac wall. The vitelline veins have also fused to form the tubular heart. It is seen that the splanchnic inesoderni ensheaths the endothelial wall of the heart (em) on each side and that where it -does so it is somewhat thickened (me) as compared with the same layer in the region overlying the yolk“-sac. This localized thickening of the splanchnic inesoderm is destined to give rise to the entire thickness of the heart wall except the lining endothelium. It is seen to be continuous with the extracardiac portions of the splanchnic mesoderm by the dorsal (d.mc) and ventral Inesocardium (mnc).

Traced forwards through the series of sections the heart is seen to narrow in calibre as-it tapers off into the ventral aorta. Towards its front end the latter gives off a large branch on each side which 532 EMBRYOL()(_}YOF THE LOWER VER’1‘EBPA'l.‘ES (:11.

passes outwards and upwards round the foregut to become continuous with the dorsal aorta. These two hoop-like vessels which connect up ventral and dorsal aortae are the first pair of aortic arches.

Still further forward the region of the forebrain and optic rudiments is reached (Fig. 2341.)).

Owing to the folding oil‘ of the head rudiment the section of the head itself a.pp1*a.1‘s cmnpletely detached from the blastoderm and the latter is begiiining to form a depression which will later become more marked and in which the head will lie. In the blastoderm it

s will be noticed how away on each side it shows the normal four layers

of cells——-ectoderm, somatic mesoderm, splanchnic inesoderm, endoderm—While on the other hand in the region 1i11_de1-lying‘ the head of the embryo it is only two layered the mesoderm being here absent.

the Optic rudiments.

cot, ec.-tmlm-m : rm/, mulmlu-I-in ; /:H'S, nw.~:mu-liyiiie; n.r, optic. rudiment ; ,:u, pi-oanmion ; splr, n‘];l.'u|('l|ll0C0!..'lI'; Hull, roof ofthalamencephalon.

This two-layered region of blastoderm is the proamnion before alluded to.

The head itself is occupied almost entirely by the brain rudiinent ———the thalanieneephalon in the centre (tltal) continued outwards on each side as the optic rudiment (oxr). For the most part the external ectoderm is closely apposed to thesurface of the brain but dorsally the former is commencing to recede from the latter, the space between the two being occupied by mesenehy me (mes).

THE THIRD DAY or .lNCUI£A'rION.—During the later hours of the second and earlier hours of the third day of incubation there take place a number of important changes which render this period perhaps the most i.nterest'ing of all to the morphologist. For the student who is training llllll.S(..‘.ll" practically in the technique of embryological observation tliem is no finer material than that afi"orded by liinl embryos of about this :.1.g<: for l_o:1.1‘ning one of the

"most important parts of that technique namely the interpretation

of serial sections. OWL--——SEC()N 1) ANI.) '.l.‘H’[R1) DAYb 533

It is mlvisable to make {L (;:.L1‘(3l"111 sbmly of the ana.t;omy of an embryo of about; the Htztgia shnxvn 111 I919‘. 235 01‘ Ii-.’.f_’.(-3,1

Flu. 255.3. 'l'hir«l—«l:ty l*'u\\'l t‘|lliH'_\'u with thv \'u..~"u:1l1:n|':u"u.

u..r, o.-«L-"v ul' :1nminII‘. P5. I-_\'I'*: .‘.’, ln~:H'l; wt, u1uc_\'.w-t; .-.u. >'I'l‘0-flllllliutiv t'Ullll\‘_\iUl|; .~'.I, sinus ternninali. ; I.‘/', Iziil-fnlulg ran. \'il».*llim- .-n'tc_*1-y; . ;u.n'l.iun u!’ splzuurhlmlalelu-v im-'0lnt.eil to form :1

rm-ass Inmul Llw he-:ul of the vInln'yn.

‘ It is custonmr_y In mmml. lI':1)1.w'\':)1'. 0 sections with the posterior or tai1\':rd surface of the section next lilv sli.-In : nulls:-ql1t_‘lltiy the figures represvnt the sectinns as seen frmn in front and Lhv sidu nl' '..:u'.h Iig1u'v tmv.-n-«is H11‘ ri;;l11-lnnici side ml‘ the pzige c<2z't'e.~‘pn|u«ls tn the M't."hu.n«l side at" the crnlrryu. 534 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

On opening the egg it is at once seen that the blastoderm has increased considerably in size, the outer limit of the opaque area having spread downwards as far as about the equator of the egg. The vascular area has also increased considerably and is still bounded by the conspicuous terminal sinus which anteriorly turns inwards and passes back parallel to the corresponding part of the sinus of the other side to open into the vitelhne vein close to its inner end. Of these two veins which run parallel to the long axis of the embryo the right is reduced in size and eventually disappears.

The yolk has assumed a more fluid consistency; the proportion of white yolk has increased; the albumen has shrunk considerably in volume, and the air space has increased correspondingly.

The free edge of the amniotic hood (Fig. 235, a.e) has grown back so as to ensheath all the head and anterior trunk region of the embryo. It follows that when examined in sritw. the front part of the body is seen through two layers of somatopleure. Of these the outer—the serous n1en1brane———forms a kind of roof which passes outwards all round into the general blastoderm. The inner—the true aInnion——c1osely invests the head end of the embryo and is visible in profile as a sharp line immediately outside the outline of the head itself. Anteriorly the amnion very often seems to be prolonged into a sharp peak (Fig. 235, s.a): this is the sero-amniotic connexion.

The free edge of the amniotic fold, somewhat arch-like in outline, may die away posteriorly (Fig. 235) or it may be already continued into the lateral and caudal parts of the fold (Fig. 236)——but even if present these are still low and inconspicuous as compared with the headward part of the fold.

As regards the body of the embryo it is seen that the folding off of this from the yolk is proceeding rapidly. The head and anterior part of the trunk project freely and, correlated with this and with the ventral flexure of the head region, the latter has come to lie_ over on one side, usually the left, so that it is seen in profile when the blastoderm is looked down upon from above. At the extreme hind end the tail region is also seen to be in process of becoming marked off from the blastoderm by a tail fold (Fig. 235, tj) of the same nature as the head fold. Similarly the trunk region between the regions of head and tail fold is becoming demarcated from the blastoderm outside it by a lateral fold (Fig. 236).

The body of the embryo has increased considerably in length and this growth in length is particularly active towards the dorsal. side of the embryo where there is greater freedom from the clogging effect of the yolk. The result of this difference in rate of growth between dorsal and ventral sides is that those parts of the embryo which are detached from the general blastoderm assume a strong flexure towards the ventral side. This is particularly pronounced in the head region, the head being completely bent upon itself so that x I FOWL--THIRD DAY 535

the front end of the brain is reversed in position, what was its ventral side having come to be dorsal.

The mesoderm segments have inc1'ea*‘ed in number there being

FIG. 236.—~Third-day "Fowl embryo (N o. 47) viewed as a transparent object.

cw, edge of amnion; am, amnion; E, eye; H, in-._a.rt;; m.s, mesoderm segments; ut, otocyst; s, indications of preotic mesoderm segments ('9); u,a,, vltellino artery; me, visceral cleft II; *, portion of splanchnopleure bulging downwards into the yolk, forming a recess in which lies the head of the

embryo.

'now about 25-30 metotic segments and those towards the anterior end are showing a considerable amount of dorsiventral growth. , In some embryos (Fig. 236) the ‘series of definitive mesoderm segments is continued far into the head region by what appear to D36 EMBRYOLOGY OF THE LOWER VERTEBRA'.l‘.ES CH.

be the ghostly vestiges of formerly existing segments (see pp. 210,211)

The central nervous system has made important advances in development. The brain shows a relatively large increase in size

‘as compared with the spinal cord : thalamencephalon, mesencephalon

and rhombencephalon are marked off by definite constrictions-—tl1e mesencephalon being particularly prominent at the bend of the head. The greater part of the roof of the rhombencephalon is assuming its definitive thin membranous character. The three great organs of

special sense have made their appearance. The eye (E) forms a large conspicuous cup-like structure lying at the side of the forebrain. Its rim is cleft ventrally by the choroid fissure (Fig. 236). Its mouth is partially blocked by the round lens rudiment. The otocyst (at) is also conspicuous—-—a pea_r-shaped sac, its narrow end dorsal, lying at the side of the hind brain. The olfactory organ is represented by a slight dimple of thickened eetoderm near the tip of the head.

The side walls of the foregut are perforated by visceral clefts. The series of these develop from before backwards and by this stage three have commonly appeared—c1efts I, II, and III of the series.

It is perhaps the vascular system which shows the most interesting features during the third day. The heart is still in the form of a simple tube, but its active growth in length has caused a great increase in the curvature which was already pronounced about the middle of the second day. Its y-like curvature is shown in Fig. 236. At its morphologically front end the heart is continued into the .ventral aorta and this at its end gives off a series of vessels, the aortic arches, which pass up round the sides of the foregut between adjacent gill-clefts and open dorsally into the aortic root which lies just dorsal to the clefts. Like the clefts themselves the aortic arches develop in sequence from before backwards and by this stage arches I, II, and III have made their appearance (Fig. 241, A).

At its front end the aortic root can be traced for some distance into the head as the dorsal carotid artery (Fig. 241, A, d.c). Posteriorly the two aortic roots become hidden from view by the myotomes but the study of sections shows that they have here united to form the unpaired dorsal aorta. Still farther back this vessel again becomes paired and a little behind the point of bifurcation each of the branches gives off a large vitclline artery (ea) which passes outwards at right angles to the axis of the body to supply the vascular area.

Of the venous system the most conspicuous components are the great vitelline veins (Fig. 241, A, 72.7)) which, receiving numerous branches from the vascular area, pass forwards converging towards one another to form by their fusion the hind end of the heart. Examination of the vascular area shows that the branches of the vitelline arteries and of the veins accompany one another in their ramifications. In the living condition, in which all these arrangements .\' .I*‘OWL-.-----THIRD DAY 537

of the vascular system should be studied, the arteries are seen to be more deeply coloured and more conspicuous than the veins. The

. two vitelline veins by their -fusion form the hind end of the tubular

heart and on tracing this forwards a. somewhat Y-shaped vessel is seen opening into it laterally. The stalk of the Y which is very short, though showing considerable variability within its limits, is the right duct of Cuvicr (Fig. 2-11, A, d.C). The branches of the Y are the cardinal veins. Of these the posterior (p.c.'v), coming from the region of the kidneys, is only visible for a short distance, being soon hidden as it is traced backwards beneath the myotomes. The anterior cardinal vein (a.c.v_) on the other hand can be traced forwards for a long distance into the head from which it drains the blood back towards the heart. It will be noted that here in the embryonic Bird we find exactly the same arrangement of main veins—-—duct of Cuvier, anterior cardinal and posterior cardina1—as

F In. 237A.——'l‘ransverse sections through thircl-«lay Fowl (:lnln'_\'t_). (Partly based on figures by Duval.) A, '|‘hrough the hinder part. of the trunk region.

A, dorsal aortne; um, annniotic folds ; «mi, «-ndoderm ; 'm._c/_. xnyotume ; s.«', spinal cord; sum, sonmtoplenre ; spl, s]alams-lannplenre; splc, splanchnocoelt-r.

is characteristic of-the adult condition of lowly organized fish-like Vertebrates.

For the study of such details of structure as cannot be made out in the whole embryo the most useful sections are series cut transversely to the long axis of the trunk region. These should be supplemented by series parallel to the sagittal plane in the head region.

It is well to commence the study of the transverse sections with one through the binder trunk region, about the level of the vitelline arteries. Such a section is depicted in Fig. 237A.

In comparing this section with a corresponding section through the second-day chick (Fig. 23-1A) the same general features will be recognized--—the differences being mainly differences in detail. The most conspicuous of these is caused by the development of the amniotic fold of the somatopleure which rises up on each side, arching towards ‘the median plane over the dorsal side of the embryo (am). Traced forwards through the series the amniotic folds of the two sides are seen to meet and undergo fusion so as to give rise 538 EMBRYOLOGY OF THE LOWER VERTEBRATES CH. rte/the inner true amnion and the outer false amnion or serous inembrane: the T6fif1“e"r"'"'contin11o11s at its inner '"ed‘g‘é"'\vitl1 the somatopleure of the embryo’s body, the latter at its outer edge with that of the blastoderm. It will be readily seen that the space between true and false amnion is morphologically part of the splanchnocoele. It will also be realized that both true and false amnion being somatopleural in nature are composed of ectoderm and somatic mesoderm but that the relative position of these two layers is reversed in the amnion compared with the false amnion. Important changes have taken place in the mesoderm. The mesoderrn segment is no longer connected with the lateral mesoderm the nephrotoine having become converted into renal structures———the archinephric duct and mesonephric tubules. The relations of these will be understood by referring back to the general description of renal

FIG. 23713.—--'l‘r:msvcrs:- section just behinzl the point of union of the two vitelline veins.

A, dorsal aorta ; um, amnion ; r~, umllls .'.lrt,m"i0suS ; err, m-1.m.h-run; emf, enteron ; ,/Lu, false amnion or scrolls membrane; my/, inyotome; N, ]I(_)l.-U(:l1UI‘Il; .s-.e, spinal coral: sn, sc-re-amniotic isthmus; sum, somatopletire; cpl, splanchnoplenru; .s-pic, splmuzlinocoole; I’, \'«-nlrivh-; 42.:-, vitelline wins; y, yolk.

organs in Chapter IV. (p. 254). The inner wall of the segment has lost its epithelial character and broken up into a mass of actively proliferating mesenchyme cells. Many of these cells will wander away in amoeboid fashion and settle down round notochord and spinal cord to form the protective sheath in which eventually develops the vertebral column. Collectively these arnoeboid cells constitute the sclerotome which is therefore much more diffuse in its origin than in the lower vertebrates illustrated on p. 285.

Certain blood-vessels are visible in the section. In the splanchnic mesoderm of the yolk-sac numerous vessels of the vitelline network

are visible : over the mesonephros may usually be seen the posterior.

cardinal vein, while on each side of the mesial plane ventral to the notochord are the two dorsal aortae.

e As the series of sections is traced towards the head the most conspicuous change is the incri:-asing asymmetry due to the body of the embryo coming to lie over niore and more upon its left side. Fig. 237]} represents a section just behind the‘ posterior limit of the foregut. X FOWL --—THIRD DAY 539

The body of the embryo lying over on its left side is closely invested by the amnimi (run) while over this lies the thin roof (_/lam) constituting the serous mcnibrane. At set the two membranes are united by the sero-amniotic connexion. «In the mesoderm of the two folds of splanchnopleure which are approaching one another to floor in the alimentary canal (ant) are Hl‘l_'ll the two large vitelline veins (72.22). The ventricle and the cones are seen out longitudinally in the wide coelomic space lying to the right of the body of the embryo.

A section a little farther forward in the series has the appearance shown in Fig. 2370. The definitive gut (ent) is completely separated at this level frorn the yolk-sac, and corresponding with this the two vitelline veins, which in sections farther back lay one on each

FIG. 237C.—-T1‘£tl1S\'el‘.<U .\f(‘(_‘llHll :1. little in front of the hind «_-ml of the lic:n't..

mu, mnnion: .-l,«l0rs.-il aor-tn ; d..r, «incl us \-mu)sii.s' : «uni, ulinu-m.-u'y 4-.-uml : _1'.vun, i'.'ilso~ mnnion ; /-i.l, 8-Ilte!‘i0l' 1i\'t-1‘ 1'luli1IwI1t ; li.;’, }no.~'.l'erioi' «lit't.o ; N. nolnvlionl : I-.r.r, ])().\'lI'l'lU[' (‘:ll'lllll:ll win ; sum, somatoph-nrv; :=_n/, spl.-in<:lmople-uri: ; splc, splanclinocoele ; I-'_, \'l'lIll‘l(‘lt.‘.

side of the yoll<—sta1l<, are now completely fused into a large median vessel, the ductus venosus (dxv), which is simply the backward prolongation of the heart. The posterior liver rudiment, a blindly ending pocket of the ‘gut-wall projecting forwards ventral to the ductus venosus, is seen in the section figured (Z222), although its communication with the gut-wall is no longer visible, lying as it does several sections farther back. At this level however a second pocketlike outgrowth of the gut-wall has made its appearance (l7§.1). This is the anterior liver rudiment. It will be noticed that it lies dorsal to the ductus venosus. In the coelomic space ventral to the ductus venosus and liver rudiments, and quite isolated, is the rounded section through the ventricular region of the heart (V).

In the sections studied so far the body-wall of the embryo is widely Open on its Ventral side—the opciiiiig l)elllf_,"lml.1I.l(l€(l by the recurved edge along which the soniatopleure ol’ the body is continuous 540 EMBRYOLOGY OF THE LOWER VEil;{.'_l‘-EBHATES OH.

with the non-embryonic region of the somatopleure forming the amnion. As however the _foli_iing oil‘ of the embryo progresses the edge alluded to grows inwards and the opening bounded by it becomes reduced in size. It will be gathered readily from Fig. 237D that through the opening in question the splanchnocoele, included within the definitive body of the embryo, is continuous with that part of the coelome which lies outside (extra-embryonic coclome). In the section figured the heart is seen to be cut through in two places. Reference to the figure of the whole embryo (p. 535) will show that the piece of heart which lies towards the leit side of the embryo (at) is the atrium, while that on the e1nbryo’s right (0') is the ventricle or conus. In the section iigiiiwl a large blood-vessel (d.0) is seen out

Flu. 2371),——'l‘r:i.nsvm-so section a short distance behind the front end of the heart.

.1, dorsal aorta ; am, amnion ; at, atrium ; l..', eonus : «(.!.', duct oi‘ (iuvier: f.«.¢.m., false amnion ; N, notm-hord; 1'-h, pharynx; .-om, sonuttopieure; .-pl, spinnohnopleure; .5-pl:-, splanchnocoele.

longitudinally in the sornatopleure. By tracing this vessel through neighbouring sections it will be found to open at its ventral end into the atrial part of the heart while dorsally it splits into the two cardinal veins-—anterior and posterior. These relations show the vessel in question to be the duct of Cuvier. The only other point calling for special mention in the section figured is that the ventral part of the pharyngeal cavity projects outwards upon either side: this dilated ventral part of the pharynx forms the rudiment of the lung.

In the region in front of the heart the dorsiventral depth of the body of the embryo becomes comparatively suddenly reduced and in the Vacant space within the amnion so provided there appears a_ new structure quite detached from the rest of the section. The structure in question is a section through the recurved tip of the head (see figure of whole embryo). In Fig. 237E this shows the thi.c.k-wallmel forobrain ( with its wide ventriE C9

3"

cc 0 -"1? S9 o C?‘ o v-:

x FOWL-——_-THIRD DAY 541

cular cavity while upon each side and ventrally’ there is seen a localized thickening (olf) of the ectoderm: this is the dimple-like 1 udi o 4'

of the sectionmthere is seen in its centre the wide pharyngeal space and on the embryo’s left side the pharyngeal wall projects out to the ectoderm as an endodermal pocket--~the rudiment of the second visceral cleft (12.0.11). Immediately ventral to the pharynx is the ventral aorta (o.A). On the left side of the embryo the aortic root (am) is seen immediately dorsal to the pharynx, while on the right side--—the section not being accurately transverse—~ ya hoop-like aortic arch (a:.a.III) is seen passing dorsalwards round the side of the pharynx from ventral aorta to aortic root. The large

FIG. 237E.-—-—Transverse section passing through the second visceral cleft and the ' olfactory rudiment.

a.a.IIl, third aortic arch: a.r:.2v, anterior cardinal vein; am, amnion; a.r, aortic root; f.am, false amnion; f.b, forebrain; h.h, hind brain; olf, olfactory rudiment; 11/l, pharynx; ‘I'.A, ventral aorta; no.1], second visceral cleft.

vessel lying dorsaland slightly external to the aortic root (a.c.v) is the anterior cardinal vein. Traced tailwards it is found. to open into the dorsal end of the duct of Cuvier. The neural tube (lab) is seen to have a thin roof and widely expanded lumen indicating that it is now passing into the region of the hind brain.

In tracing the series -of sections further forwards it will be

y organ. To return to the am part“ .

realized that the front part of the head region is, owing to its '

reflexed position, actually being traced in a morphologically tailward direction. In the section figured (Fig. 237F) the reflexed portion of the head is cut at the level of the eye rudiments (opt) which are seen to be in the optic cup stage with the inner or retinal layer

‘ It will be realized from an inspection of the figure of the entire embryo that the recnrved part of the head is reversed in position. Its ventral side lies therefore in the figure towards the right. 542 EMBRYOLOGY or THE Lowna VEI-LTEBRATES en.

distinctly thickened as compared with the outer or pigment layer, and with a narrow optic stalk passing to the thalamencephalon near its floor. In the mouth

of the optic cup is the lens

but this is seen better a few sections farther on in the series.

Turning to the other

Pf half of the section it is

seen that it is no longer
connected with the extraembryonic soinatopleure:
 in other words the series
    e     of sections has now passed

FIG. 2371-‘.—-—.Transverse section passing through the ' the binder limit -of the rmlunents of the eye and otocyst. hemlfold of the SOmatO_ I (l.('.I‘:{|.llt8['ll-ll"(‘fll‘(illHll‘\'|.‘lll; l:.h, hind brain; .."\'t|1nl«-t.-.lu.n-«I; pleura The pharynx opt, optic cup. of. nt.u('_\ sl , 1:14, ph.'-u'_\nx, (no.1, lust \'1.~'(-er:c1 cleft; aum, ventralearotid. Passes out as 3' Pocket

on each side towards the

ectoderm-—4the rudiments of the first pair of visceral elefts (11.0.1). The neural tuhe has become greatly increased in size forming the hind brain with its widely expanded cavity——-the -fourth ventricle. On each side is a large thick-walled sac—-—the otocyst. Examination of

Pin. ..'.‘37(:.- -—'|‘rn.nsv(-rse section passing thrnugln the (*._\'v and just in front of the otocyst.

1I_.I'. 4', il.lli;(‘]'i()!‘ (‘:ll‘(llll:ll Vein : I(..:‘, :u_n'ti(_'. run! ; 41,:-«(,«lm'.~4:nl c-:n-utitl :u't«'I'y; _I[tHI(l. g:u|;;:lin|| of I-i_L,-'l1H'1 H-,-mi,-.1 m-rm: /uh, hind hr.-Lin; lm. pit.-niI_.-n-_\' hotly; .\'_, lllrl_.H(:ll()l‘ll; pin hlmr_\'m.'; pin, ]:ine:ul nr:,r:m; Hull, 1h:'1l:uumn--‘plmlnn: I‘.v'.l. i'l]‘.~}i \i.~:---ml rl--ft.

neighbouring sections shows that it is still connected with the outer skin by a narrow neck. ,In the spongy connective tissue which forms packing between the various organs are seen a x FOWL--THIRD DAY 543

number of blood-vessels such as ventral and dorsal carotids and anterior cardinal veins. ‘

As will be gathered by sliding a straight-edge forward over the figure of the whole embryo, its edge parallel to the plane of the sections, there comes a point in the series where the sections through the reflexed part of the head and the rest become continuous. This happens as soon as the deep niche in the bend of the head is passed. Such a section is represented in Fig. 2370.. Comparison of this figure with the preceding one will make clear the fact that the extreme ends of the section are both “of them morphologically dorsal. The brain is cut through twice—on the right of the figure is the hind brain while on the left is the thalamencephalon distinguished by

FIG. 238.—-Diagrammatic sagittal section through third-day Fowl embryo. '.l‘he notochord and dorsal aorta are omitted Ectoderm and endoderm are indicated by cotltinllous lines, mcsoderm (except endocardium) by dots.

a, position of anus, not yet perforate; ull.,al1antois; am, zumiiong at, atrium; a.e, amniotic edge; f.a,, serous mcinbrane ; fl g, foregut; l, lung rudiment; nws, mesencephalon ; pa.g, postnnal gut; pt, pituitary involution; rh, rhombencephaion; .s~pl, splanchnopleure of yolk-sac; t, thalamencephalon; th, thyroid; V, ventricle; v.A, ventral aorta; 'v.m., remains of velar membrane; ,1/.s, ‘cavity of yolk. sac; 3:/..s-t, cavity of yolk-stalk.

the pocket-like rudiment of the pineal organ (pin). The thin optic

stalk lies outside the section, but the structure of the optic cup

otherwise is well seen.‘ The lens is in the form of a closed vesicle which has by this stage become completely nipped off from the external ectoderm. Immediately ventral to the thalamencephalon is the pituitary involution cut transversely- The section passes through the ganglia of the auditory nerve (gang) and on the embryo’s right through the nerve root connecting the ganglion with the medulla oblongata. Various blood-vessels are cut through: their names and relations with one another are most easily determined by sliding a straight-edge along the drawing of the embryo as a whole.

The study of this stage should be completed by examining series of sections parallel to the sagittal plane in the ‘head region and 544 EMBRY'0LdGY OF THE LOWER VERTEBRATES CH.

interpreting them by what has been made out from the whole embryo and the series of transverse sections. The most instructive sections are those in or close to the sagittal plane. Fig. 238 shows diagrammatically a sagittal section through the whole length of the embryo, but it will of course be understood that, owing to the head of the embryo having come to lie over on its left side while the trunk region retains its original position, a section which is sagittal in the head region will, in actual fact, be practically horizontal in the trunk. ‘

The feature that dominates the section is the cerebral flexure-— the strongly marked curvature of the head region towards the ventral side. The brain is of relatively enormous size: a distinct dip in its roof marks the boundary between the thin-roofed rhombencephalon which lies behind it and the region in front of it-—the cerebrum--whichwill give rise to mesencephalon, thalamencephalon and hemispheres. .

The next instructive feature brought out by such a section is the general relation of gut to yolk-sac. The rounded head-fold of the splanchnépleure has extended far back so as to floor in the foregut (jig). The velar membrane (am) has just ruptured so that the foregut communicates in front with what will become the stomodaeum into which also opens the pituitary involution of the ectoderm (pt). The floor of the foregut dips downwards to form the rudiments of the thyroid (th) and lung (1). In a slightly more advanced embryo

the two liver rudiments would be seen also as pocket-like outgrowths of the enteric floor in the neighbourhood of the atrial end of the

cardiac tube.

The posterior end of the definitive alimentary canal is also becoming folded off from the yolk-sac though the cavity of the yolkstalk—-—the communication between the definitive alimentary canal and the cavity of the yolk-sac—-—is still very wide. The position of the future anal opening is indicated by a thick septum (Ct) composed of fused ectoderm and endoderm. Dorsal and posterior to this the enteron extends back as a blindly ending pocket—-the remains of the postanal gut (pay), while anterior to the anus the enteric floor dips downwards as the rudiment of the allantois (all). The latter is covered with a thick layer of mesoderm and bulges into a dilated portion of the splanchnocoele. Towards the front end of the embryo a still more widely dilated portion of the splanchnocoele accommodates

the cardiac tube. At its anterior (o..A) and posterior ends (at) this,

is ensheathed in the thick mesoderm on the ventral side of the foregut, while its middle portion (V) hangs free in the cavity.

Finally the amniotic fold of the soinatopleure is seen to extend almost completely over the body of the embryo, the amniotic edge (cue) bounding a comparatively small opening near the tail end. '

Having studied in some detail the features- characteristic of an individual third-day embryo it will be convenient now to give a X FOWL-—THIRD DAY 545

general sketch of the chief advances in development which take place during this day.

At the commencement of the day the body of the embryo lay flat along the surface of the yolk: only at its head end was it clearly demarcated from the surrounding blastoderm and this head region owing to the commencing ventral curvature was beginning to lean over on to its left side. During the course of the third day the tucking in of the blastoderm under the definitive body proceeds apace so that the body becomes more and more completely demarcated from the part of the blastoderm forming the yolk-sac wall, and the yolk~stalk becomes correspondingly narrowed. The preponderance of growth activity on the dorsal side which leads to the ventral curvature is during the early hours of the day especially marked in the region of the mesencephalon but as the day goes on becomes very pronounced about the level of the heart and still later in the tail region. Thus the axis of the body develops strong ventral curvature especially marked at three different levels——mesencephalic, cardiac and caudal. Along with this increasing curvature the whole body of the embryo comes to lie over on its left side so that the observer looking down upon the egg from above sees the body of the embryo in profile from its right side.

During the day the embryo becomes ensheathed in the amnion in the manner already described. The vitelline network of bloodvessels attains to its highest development, forming as it does the organ for respiration as well as for absorption of the food and its transport into the body of the embryo. Correlated with the lying of the embryonic body over on its left side the paired venous channels which convey the blood from the vitelline network into the heart gradually lose their symmetry, those of the right side dwindling in size while their fellows show a corresponding increase.

In the brain the main regions become established: the roof of the thalamencephalon and medulla oblongata. assume their thin membranous character while the hemispheres bulge out in front of the thalamencephalon. The central canal of the spinal cord becomes reduced to a vertical slit by the thickening of the side walls. The olfactory rudiment makes its appearance: the auditory rudiment becomes converted into the closed pear-shaped otocyst, still however connected with the ectoderm by a solid strand of cells. In the eye the lens thickening has become involuted and converted into a closed vesicle with its inner wall markedly thickened. The optic cup has been completely formed and the retinal layer differentiated from the thin and degenerate pigment layer. In the latter the first deposition of pigment takes place during the later hours of the day.

The definitive alimentary canal is still open towards the yolk-sac over about half its extent but in addition to the foregut there becomes folded off during the course of the third day a considerable extent of hind-gut, the ventral wall of which commences to bulge out to form the rudiment of the allantois towards the close of the

voL. II 2 N 546 EMBRYOLOGY OF THE LOWER VERTEBRATES OH.

day. The hind-gut is still closed posteriorly but the foregut late in the third or during the fourth day becomes thrown into communication with the stomodaeum by rupture of the velar membrane. The pituitary rudiment makes its appearance. The four gill-pouches are formed and reach the ectoderm, the fourth in the closing hours of the day, and the first or it may be the first two become perforate. The thyroid rudiment makes its appearance and during the latter half of the day becomes closed. The pulmonary rudiment develops and becomes constricted off from the pharynx except at its front end. About the beginning of the day the two liver rudiments appear and during its course the process of anastomosis begins between the branches which sprout out from them. During the latter half of the day the pancreatic rudiments make their appearance——first the dorsal, then the left ventral, then the right ventral.

During the course of the day the mesoderm segments increase from about 20 to 25 up to about 40. Early in the day the Wolfiian duct becomes tubular and in the latter half of the day it completes its backward growth and reaches the cloaca. The germinal epithelium becomes recognizable.

The skeleton remains throughout the day purely notochordal.

The heart retains its S—shape and during the latter half of the day the atrial septum begins to develop. The two dorsal acrtae begin about the commencement of the third day to undergo their fusion to form the definitive unpaired aorta. In addition to the first one or two aortic arches which are already present the third makes its appearance (Fig. 241, A, III, p. 550), then the fourth, and during the latter half of the day the sixth, while the first becomes obliterated. As regards the venous system the most important feature is the assumption of the same general plan of the main trunks as is characteristic of Fishes.

Finally it should be noted that during this day the body of the embryo-becomes enclosed within the amnion.

It will be realized even from the bare summary that has been given that the third day of incubation of the Fowl’s egg'is morphologically the most important of all and the student will be well advised to devote a good deal of time to making a detailed study of embryos of this period. _

THE FOURTH DAY or INCUBATION.-——-By the end of the fourth day of incubation the blastoderm has spread about half-way round the yolk. The vessels of the vascular area are conspicuous, though it is to be noticed that the terminal sinus is becoming relatively less so than it was during the third day. The folding off of the body of the embryo has progressed greatly. By the extension backwards of the head fold the region of the heart has become floored in on its ventral side. Posteriorly the tail fold is deepening in a similar fashion. Between head fold and tail fold the somatopleure of the embryonic body is prolonged ventralwards into a very short and wide tube--the somatic stalk-——the wall of which is reflected dorsalwards as the true X FOWL—THIRD AND FOURTH DAYS 547

amnion. The latter is now complete and closely invests the body of the embryo. Lying loosely within the somatic stalk and of much smaller diameter is the splanchnic or yolk stalk--the continuation of the splanchnopleure in a ventral direction as it passes out into the wall of the yolk-sac. The body of the embryo has undergone a great increase in size. The growth of its tissues has been particularly active in its dorsal region and this has led to a continuation of the flexure towards the ventral side which was already well marked in the third day embryo. ,

An important new feature in the fourth day embryo is provided b y the two pairs of limb rudiments each in the form of a dorsiventrally flattened ridge with rounded edge and broad base of attachment to the body. The head of the embryo at once attracts attention by its relatively enormous size. This is due to the relatively immense size of the brain and eyes. We have here to do apparently with a case of the precocious growth in size of organs which in the fully developed condition possess extreme complexity of minute structure. The main regions of the brain can be seen very distinctly: the relatively large mesencephalon with its bulging dome-like roof, the thalamencephalon with the pineal rudiment, the rapidly growing rudiments of the hemispheres, and the hind-brain with its relatively thin and membranous roof. The three main special sense organs are all conspicuous—-the olfactory organ, the eye with its choroid fissure and lens, the pyriform otocyst. Arranged in a row ventral to the otocysts are the pharyngeal clefts—three or four in number. In the case of cleft I the ventral part of the cleft is becoming much narrowed by the approach of its anterior and posterior walls. The dorsal end of the cleft on the other hand remains dilated: it corresponds to the spiracle of fish-like forms.

The heart, which forms a large structure lying between the tip of the head and the region of the fore limbs, is still in the form of a coiled tube but the appearance of localized bulgings of its wall foreshadows its division into the various chambers characteristic of the

adult. Thus the curve of the tube lying posteriorly and on the right

is becoming dilated to form ‘the ventricle: the part morphologically in front of this leading towards the ventral aorta is slightly dilated to form the conus arteriosus, while the curve lying anteriorly and on the left side shows a slight bulging on each side foreshadowing the two auricles. Slight constrictions separate these various bulgings-—an atrioventricular constriction narrowing the cavity to form the auricular canal, and a less conspicuous one between ventricle and conus. '

The general arrangement of the peripheral vessels is intermediate between that of the third day (Fig. 241, A) and that of the fifth day (Fig. 241, B) and need not be described in detail. Aortic arches I and II undergo in turn a gradual process of obliteration while arches IV and VI make their appearance farther back if they have not already done so. It is also during this day that arch V makes its brief appearance. 548 EMBRYOLOGY OF THE LOWEPt TVERTEBRATES CH.

The allantoic veins, which at first are merely veins of the body-wall, during the fourth day establish their connexion with the allantois, and in the course of the day the right vein disappears.

The allantois itself forms a conspicuous new feature for towards the end of the day it begins to project distinctly from the ventral side of the embryo about the level of the hind limb.

Owing to the increasing size and complexity of the embryo the elementary student will not as a rule prepare complete series of sections later than the third day. He will however find it profitable to have transverse sections through the developingsense organs, sagittal sections through the head, and transverse sections through the posterior trunk region.

From the study of sections the following advances in development during the fourth day may be made out.

In the brain the rudiment of the paraphysis makes its appearance Fm. 239.--Fowl's egg opened at the end and the pineal outgrowth begins to

of the fifth day. The embryo enclosed sprout; out into diverticula, about;

in its amnion is sunk down in the - 1 centre of the vascular area, the allan~ the end of the day‘ rlhe Olfactory

tois projecting upwards towards the» rudiment b3C0m3S Conneclied With serous 1nembrane——a transparent mem- the buccal gavjty by a, slight,

brane through which theembryo and ' allantois are seen. The increasing groove‘ The rudunents of lagena

fluidity of the yolk is shown by the and recess make their appear-.

outward bulging of the yolk-saewall ance as bulgingg of the

?J:2§‘;§1‘;’3l":i1..°2§ii.‘3§ “s::‘°.:.‘..*:.‘.::‘: ototytt watt The cavity of the

Iiowlies completelyunderneath the yolk 13113 b(.3C01T1€3 Obliterated by th9

so as ‘to be invisible in a view from grgwth of its inner wall; pigment,

“b°“"‘ becomes conspicuous in the outer

““‘°;‘éi‘£::§2.‘:‘..2é’s:‘.l':3.§2‘:é wall of the optic cup: the layer of

nerve fibres in the retina becomes

recognizable: mesenchyme begins to invade the cavity of the optic

cup and about the end of the day also intrudes between the lens and the ectoderm.

The post-anal gut becomes reduced to a solid strand of cells and finally disintegrates. The yo1k—stalk becomes narrowed to a fine tubular channel. The gall-bladder begins to dilate towards the'close of the day :. the dorsal pancreas begins to develop outgrowths: and the rudiments of the caeca make their appearance.

The mesoderm segments increase in number to about 50. Early in the day, if it has not done so already, the Wolffian duct opens into the cloaca. The mesonephric glomeruli begin to appear and the tubules become elongated and coiled. In the posterior region of the X FOWL-—-FOURTH AND FIFTH DAYS 549

mesonephros secondary tubules make their appearance while in the anterior region a process of degeneration becomes apparent. During the second half of the day the ureter begins to sprout out from the Wolffian duct and about the end of the day the rudiments of Milllerian ducts and of the metanephric units may become recognizable.

In the heart the atrial septum becomes completed about the end of the fourth day and the endothelial cushions begin to develop.

FIG. 240.—-—Chick extracted from the egg at about the middle of the fifth day of incubation.

all, allantois; C.H, cerebral hemisphere; Is‘, eye; Hy, opereulum; M, mandibular arch; pin, pineal rudiment faintly visible as slight elevation on root’ of thalzuuencephalon; Rh, thin roof of rhombencephalon; som, edge of somatopleure cut through where it becomes reflected back over the body of the embryo to form the amnion; Lu, roof of tnesencephalon (optic lobe); V, ventricle; 47.0,

visceral clefts Ill and IV ; y.s, yolk-sac.

FIFTH DAY.—--The progress in development during the course of the fifth day is illustrated by Figs. 239-241. The albumen has so shrunk in volume as to be no longer visible in a view of the opened egg from above: the yolk has become extremely fluid: the vascular area has increased considerably in size. The allantois is now a conspicuous object and the mesoderm covering its surface is beginning to develop blood-vessels. The ‘head of the embryo is, as before, of relatively very large size: the flexure in the region of the mesen550 TEMBRYOLOGY‘ OF THEALOWER VERTEBRATES; CH.

cephalon is still more pronounced. The operculum (Fig. 240, Hg) is conspicuous, growing back from the hyoid arch over the posterior visceral clefts. The limb rudiments now project freely though their form is that of simple flippers without any of the peculiarities of the leg or wing of the Bird. The body of the embryo is floored in onits ventral side completely but for the rounded opening (som) along

- whose lips the somatopleure is continued into the amnion and through

which emerge the narrowing yolk-stalk and the stalk of the allantois. ilThe study of the living embryo in situ shows the general plan of the blood system to be as is shown in Fig. 241, B. The heart still

FIG. 241.—Diagrarn showing the main parts of the vascular system as seen in a Fowl embryo during the third day (A) and the fifth day (B).

a..a, allantoic artery; a.c.w,-, anterior cardinal vein; at, atrium; (1.17, allantoic vein; d.C, duct of

Cuvier; d.c, dorsal carotid; il.a, iliac artery; p.a, pulmonary artery; p.c.v, posterior cardinal vein; p.v.c, posterior vena cava ; v.A, ventral aorta; 1:.a, vitelline artery ; v.c, ventral carotid ; v.z~, vitelline vein ; I-VI, aortic arches. '

betrays its tubular origin though the chambers are clearly recognizable as dilatations. Three aortic arches (III, IV and VI) are distinctly visible and occasionally the fleeting vestige of the penultimate arch as in the specimen represented in the diagram. In front of the aortic arches the ventral aorta is seen extending forwards as the ventral carotid (ac) :. the pulmonary artery (p.a) passes back from the sixth arch. Dorsally the aortic root extends forwards into the head as the dorsal carotid artery (d.o). A little distance behind the liver the vitelline artery (ua) leaves the dorsal aorta and farther back the allantoic artery (cm) a branch of which, the iliac artery, passes to the hind limb.

In the venous system the duct of Cuvier is seen, continuous at its dorsal ‘end with the anterior and posterior cardinal veins. ' The

‘former (a..o.q2) branches through‘ the head: the latter (19.0.72) can be X FOWL-—FIFTH AND SIXTH DAYS 551

traced dimly back into the region of the kidney. The main blood-'

stream to the heart comes from the vitelline vein (ac) and is joined

within the substance of the liver by the blood from the left allantoic

vein (am) and the posteriorvena cava (p/ac).

Ignoring the vitelline and allantoic vessels which are clearly adaptations to the peculiar conditions of the developing embryo the main plan of the blood system is seen to be clearly the same as is characteristic of Fishes.

By cutting off the head after fixing and viewing it from below (Fig. 245, A) the modelling of the face can be studied. The frontenasal process ( f .72) is bounded on each side by the shallow oro-nasal groove connecting it with the buccal cavity. The ridge forming the outer boundary of the olfactory organ is demarcated from the maxillary process by a faint transverse groove passing outwards towards the eye-—the lachrymal groove. Posteriorly the stomodaeal opening is bounded by the mandibular ridge with a distinct break in the middle line between the two mandibular arches.

Of other developmental features of the fifth day we may note the following. The first indications of turbinals appear on the mesial wall of the olfactory organ, and of semicircular canals in the otocyst. The optic stalk becomes solid: the rudiments of the ocular muscles become recognizable. The pituitary body begins to form outgrowths. The rudiments of thymus and bursa fabricii make their appearance: the bronchi begin to develop branches. The formation of new mesonephric tubule rudiments comes to an end and the mesonephros begins to show signs of functional activity. The atrial septum develops secondary perforations. The fourth aortic arch on the left side, and the portions of aortic root immediately behind the third arch undergo reduction. The horizontal septum of the ventral aorta begins to extend back into the conus and the anterior portions of the posterior cardinal veins begin to undergo atrophy.

SIXTH DAY.-—During the sixth day of incubation the body of the embryo increases rapidly in size and in correlation with this it dips down into the very fluid yolk, pushing the splanchnopleure of the yolk-sac wall in front of it, so that it is almost hidden from view when the egg is first opened. The amnion is, now raised up from the body of the embryo by a marked accumulation of amniotic fluid (Fig. 242). The allantois has increased greatly in size and in the natural condition is flattened rnflushroomwise againetnthfi iIme1‘..Su1:fa0B

\.—__-—-on-n.n-—. -—a of the serous membrane. In the embryo excised as directed on p. 513

‘it will be seen that the somatopleure of the embryonic body is

completely closed in ventrally except for a small circular space round which it is reflected outwards in a funnel-like fashion and continued into the thin membranous amnion. Through the funnel-like opening a slender probe can be passed from the extra-embryonic coelomic space beneath the serous membrane into the portion of coelome enclosed within the body of the embryo which will become the definitive splanchnopleure or body-cavity. Through the opening i552 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

there pass out the stalks of the yolk-sac and the allantois (Fig. 246, B) each conspicuous owing to its large blood-vessels. The peripheral distribution of the vitelline and allantoic vessels shows a characteristic difference (Fig. 242)———the vitelline network (vascular area) terminating, in the now greatly reduced terminal sinus at a considerable distance from the distal pole of the yolk-sac while n the other hand the allan

      : .               ' mic networkis most richly

developed on the distal side of the allantois (p. 474)

The body of the embryo now for the first time’ begins to show indications of bird - like form, and faint traces of digits and of feather-rudiments may become apparent about the end of the day.

In the eye the rudiment of the pecten, which first became recognizable during the fourth day, is now conspicuous as an ingrowth of . mesenchynie through the choroidal fissure, bounded on each face by the inflected lips

, _ of the fissure. FIG. 242.-Coiiinioii Fowl. View of contents of the

, . egg-shell extracted at the end of the sixth day of he tongue beglns 1,30 _ incubation. The serous meinbraiie has been removed Pr0.]eCt and the thyrold

- so as {to gllow the allantois to be f(iltfi1pl&1C(3(l1 slightllly becolneg constrictgd off in or( er 0 give a c carer view 0 e )O(y o t e embryo contained within its ainnion. from the pharynx‘ The oesophagus towards the a. ii, edge or vascular area; all), reimilns of albumin; all’, . outer wall of allaiitols; all”, inner wall of allantois; am, end of the day loses lbs amnion; *, portion of vascular area lying, in the natural cavity; the dilatation of

ppiiaigilcgn. beneath the head of the eiiibryo and free from blood- the gizzard becomes evi_

. dent; the intestine begins to grow actively in length (Fig. 246, B). The three pancreatic rudiments become continuous with one another. ,

The ‘muscles of the body begin to exhibit contractility, the trunk

occasionally showing twitches of ventral flexure. The ureter develops outgrowths to form the primary collecting tubes of the metanephros about the beginning.of the sixth or the end of the fifth day and the terminal part of the duct of the opisthonephros may become incorporated in the cloaca so. as to give the ureter its independent opening. About this time the first indications of sexual differentiation become recognizable, the genital strands beginning to show signs of degeiiera-T tion in the female. X FOW'L-—-—SIX’_l‘l--I T() EIGHTH DAYS p 553

The main portions of the skeleton become laid down in prochondral tissue and, towards the end of the day, in cartilage.

The heart begins to assume its definitive external form; the ventricular septum develops and the conus septum begins to do so. The fourth aortic arch becomes obliterated on the left side.

SEVENTH DAY (Figs. 243 and 244).-——The mushroolm -shaped allantois is spreadin r activel all round beneath t 1e serous membrane. The amnijon is begilining to show waves of contraction passing along its wall. The brain and eyes and consequently the head as a whole are of relatively enormous size. In sections the roof of the fourth ventricle is found to be developing irregular folds in which the vessels of the choroid plexus will appear. All three turbinal rudiments are present in the nose. The crop is beginning to expand. The visceral clefts are all closed. The glands of the stomach are beginning to make their appearance as rudiments. The cavity of the enteron disappears for some distance forwards from the point of Omugln the a'11antmS' The Flu. 243.——Fowl's egg opmuul during the seventh day. Mllllerlan ducts 1113)’ Show The body of the chick is semi dimly through the

incipient asymmetry, The, highly vascular allantois. 'l‘ho \(.'.\‘.\'(.‘lf\‘ of the

. ° ° ' allantois can he tlistitlgllisllc-<l lmm lhn.~'(* of the notochorq 18 beglnnlng to vascular area by their turning back at the edge of be consbrlcted by the Var 179'‘ the allantois while those of the vascular area pass

brace, The first traces of onwards uninterruptedly. The highly fluid charQssification are Inaking their {‘l.Ct(‘.I: of the yolk‘ is .>‘hm\'l1 the _\'w«.»ll<-sac. wall

. . iulgmg outwards oi oi the broken shell at. the appearance, especially in the point m,.,k.,.1 «_ skeleton of the limbs. ,,,,_ .,,,,,,,toi,,_

The septum of the conus arteriosus is complete and the muscular coat extends into it from each side: the pocket-valves are becoming excavated. The fourth aortic arch on the left side has disappeared while the portion of aortic root between arches III and IV on the right side, and behind arch III on the left side, are becoming ulvlitica-1':1.tf.ed.

EIGHTH l)AY.—'_l.‘hc inoveinents of the amnion now reach their highest degree of activity. The fronto-nasal process (Fig. 245, C) is growing out to form the. pointed beak while the lower jaw is taking a siniilar pointed form, the two mandilmlar arches being new con tinua-.<l into um-. :uml<her ventrall,\' \\'il;.hout a l>1'e.ak. 'l‘he rudiments‘

of l'eathers are beginning to inake themselves apparent. In the brain the cerebellum is becoming folded on itself so as to bulge outwards, The oro-nasal grooves are covered in to form the ‘554 EMBRYOLOGY. OF THE LOWER VERTEBRATES CH.

tubular communication between nose’ and mouth. The lachrymal groove is no longer visible: the lachrymal glands are developing as solid ingrowths of ectoderm. The pituitary body now forms a rounded mass of branched glandular‘ tubes lying between the trabeculae and communicating with the buccal cavity by a narrow tubul_ar duct opening immediately over the glottis. The air-sac rudiments make their appearance on the surface of the lung (Fig. 246, C, a.s).

The mesonephric tubules have been growing actively up till now: the metanephric units are making their appearance: theMiillerian duct reaches the cloaca if it has not already done so although no actual communication is established until about six months after hatching.

Ossification becomes conspicuous in the limb-bones and the investing bones of the head. The keel of the sternum forms an ossification distinct from the two lateral rudiments of the body of the sternum.

The terminal sinus of the vascular area has disappeared. The septum of the conus is now completely

traversed by muscle so that both aortic and pulmonary cavi. ties are completely ensheathed by muscle. The splitting apart of the two vessels is inaugurated by the appearance of a longitudinal incision along the line of attachment of the septum. ‘

FIG. 244.—-—Chick extracted from egg during seventh day showing operculum (op).

As regards the further progress of development the following approximate times maybe mentioned.

About the ninth day the oesophagus gradually becomes patent again. On the tenth day the arterial arches have practically assumed the definitive condition and the metapodial skeleton is ossified. x rowL—.;LA'rER ‘DEVELOPMENT 555

Up to about the eleventh day. the contractions of the amnion remain very active, but thereafter they gradually become more gentle until during the closing days of incubation they stop. The mesonephros also attains to its maximum activity and there commences the process of degeneration which will continue till the time of hatching: tubules have developed throughout the length -of the metanephros. ‘

By the twelfth day the duct of the pituitary body has become reduced to a solid cellular strand: the exact time at which this happens is very variable; it may be as early as the sixth or seventh day. The lachrymal duct, which originated as a _solid ingrowth of ectoderm along the line of the lachrymal groove, now-becomes tubular. About the twelfth or thirteenth day the cavity reappears over the greater part of the rectum except just at the hinder limit of the occluded portion immediately in front of the allantois. Here the cavity remains blocked till nearly the time of hatching.

Flo. 2515.-—-View of head of Fowl embryo as seen from below. (After l‘)uval, 1889.)

A, five days; B, six days; C, eight days. fin, fronto-nasal process; mac, maxillary process; olf, olfac-V tory opening; o.'n., oro-nasal groove ; sp,hyomandihu1ar cleft; V, ventricle; I, II, visceral arches.

About_the thirteenth day the cartilaginous skeleton is complete and the rudiments of claws begin to develop.

About \the fifteenth day the Eustachian valve develops in the heart.

By the sixteenth day the albumen has all gone -and the yolk-sac wall becomes completed ventrally.

About the nineteenth day the yolk-sac becomes enclosed within the body-walland the partition between mesenteron and proctodaeum breaks down so that the alimentary canal communicates with the exterior. a .

About the twentieth day the umbilicus closes. The violent struggles of the young bird cause its beak to penetrate the air-space: its lungs are filled with air: its further struggles cause its beak to break the shell and it emerges, leaving behind the broken shell lined with the cast-off allantois and serous membrane.

Correlated with the, process of hatching important changes take place in the circulation? the gap in the atrial septum (foramen 556 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

ovale) becomes closed so thatthe blood arriving in the right auricle can only reach the left auricle by the circuitous route through the

Fro. 246.-—Dissections from the right side showing the general arrangement of the viscera of 9. Fowl embryo at the end of the fifth (A), sixth (B), and eighth (0) days of incuba,tion_ (After Dnval, 1889.)

a.s, abdominal air-sac; all, allantois; c.a; conus arteriosus; wee, caecum; gi, gizzard; li, liver; mp, mesonephros; rr.a, right auricle; r.l, right lung; V, ventricle; y.d, yolk-stalk; y.s, yolk-sac.

right ventricle and pulmonary circulation, and the allantoic vein, duct of Botallus, and ductus venosus in the liver become obliterated. X . EMBRYOLOGY OF COMMON FOWL 557

LITERATURE

Duval. Atlas d'Embryologie. Paris, 1889.

Poster and Balfour. The Elements of Embryology. Second Edition, edited by A. Sedgwick and W. Heape. London, 1883.

Koibol und Abraham. Keibels Normentafeln zur Entwicklungsgeschichte der Wirbeltiere, II. Jena, 1900.

Lillie. The Development of the Chick. New York, 1908.

Marshall. Vertebrate Embryology. London, 1893.

Patterson. Biol. Bulletin, xiii, 1907.

Patterson. Journ. Morpli.. xxi, 1910.

The most complete account of the development of the Fowl is that by Lillie. It, and Duval’s Atlas if a copy can be obtained, for it is unfortunately out of print, should form part of the equipment of every embryological laboratory. CHAPTER XI

HINTS REGARDING THE PRACTICAL STUDY OF THE EMBRYOLOGY OF THE VARIOUS TYPES OF LOWER VERTEBRATES

AMPHIOXUS.-—The interest and importance of Amphioxus to the student of Vertebrate morphology are due to the fact of its position near the base of the Vertebrate phylum. It is true that in its adult structure Amphvlomus is intensely specialized in correlation with its burrowing habit. Further, it is necessary to recognize that a burrowing like a pelagic mode of life, in which the environmental conditions are comparatively uniform, is likely to lead to a kind of fixing of the organization which will be fatal to its adaptability to new sets of conditions and consequently to its capacity for evolving along new lines. We must therefore regard it as improbable that the Vertebrata passed through an ancestral condition of specialization for a burrowing habit and the specialized features of the later stages of the life history of Amphioxus cease on that account to have a phylogenetic interest. The main interest to the Vertebrate morphologist lies therefore in the earlier stages before the specialization of the adult has developed——in such features as segmentation, gastrulation and the origin of the main systems of organs. And the interest of these stages is heightened by the fact that food yolk-— that potent disturbing factor—is present to a far smaller extent in the egg of Ampiaxioxus than in that of any other of the lower Vertebrates.

Unfortunately the known localities in which fresh ernbryological material of Amphioxus can be obtained in abundance are still few, and in most laboratories recourse must be had to preserved material purchased from supply stations such as the Naples aquarium.

The best locality so far known for obtaining developmental stages of Amphiomus is the pantano or shallow lagoon at Faro near Messina. Here the spawning takes place each evening, when conditions are favourable, during the summer months from April to July. The eggs pass to the exterior through the atriopore. If in a dish on board a boat the eggs are liable by its movements. to become distributed through the water and they are then apt to become drawn by the inspiratory current in amongst the buccal cirri. When the

558 CH. xx PRACTICAL HINTS 559

Amphioams becomes inconvenienced by such entangled eggs amongst the cirri it is able suddenly to reverse the respiratory current so as to clear them away, and in this Way there is produced a misleading appearance as if the eggs were being laid through the mouth. The first meiotic division has been completed before oviposition while the second is in the spindle stage at this period. Fertilization probably takes place immediately, spermatozoa being disseminated through the water.

It is best (Cerfontaine, 1906-7) to bring the adults into the laboratory and wait until they spawn which operation may be considerably delayed. To a dish of pure ‘sea-water is added a little sea-water containing sperm then the eggs, collected with a pipette as soon as extruded, are added.

Batches of eggs are fixed periodically, preferably in strong Flemming’s solution or Hermann’s solution. After dehydration they are placed in a mixture of 2 parts clove oil and 1 part collodion in which they may be kept indefinitely. For examination whole the egg or embryo is placed on a slide or|coverslip in a drop of the clove-oil-collodion. After the specimen has been arranged in .the desired position by means of needles a drop of chloroform is applied in order to cause the collodion to solidify. The whole is then cleared with cedar oil and mounted in canada balsam. For the preparation of sections the procedure is similar, only in this case the slide or coverslip should be coated with paraffin as a preliminary to allow the collodion block to become detached, and the latter should be embedded in paraffin. ,

PETROMYZON.-The various species of Lamprey make their way up streams to suitable gravelly spots for spawning in the spring or early summer (April, May, in the northern hemisphere). Material

for emhryological study is best got by “stripping ” the ripe males and females 7}.e. by passing the hand back along the body with gentle

pressure so as to force out the eggs or sperm. The gametes from the male and female are collected separately in two small dishes: they are then mixed together, stirred gently with a feather, and water added. This “ dry ” method gives a smaller proportion of unfertilized eggs than when the eggs are received from the fish directly into water (Herfort, 1901). As fixing agent the ordinary corrosive sublimate and acetic acid is quite satisfactory.

MYxINOIDs.——-The only Myxinoid eggs that have been obtained in any numbers are those of Bdellnstoma which are dredged near Monterey, California, on shelly and gravelly bottom at a mean depth of about 12 fathoms (Bashford Dean, 1899). Much still remains to be done in working out the details of their development but it is clear that this is of a highly peculiar and specialized type.

ELASMOBRANCHI1.-—The eggs are fertilized in the upper part of the oviduct. They may traverse the oviduct comparatively rapidly and be laid as in Birds at an early stage of development [0’h'£mae'ra, Scylliidae, Castration, Rain] or they may remain in the oviduct for a prolonged 560 EMBRYOLOGY or THE LOWER VERTEBRATES

FIG. 247.—-Blastoderm of Torpedo with meulnllary folds x 18). (After Ziegler, 1892.)

A, stage four (b'c.'unmon, 1911); B, stage six; 0, stage ten. The rounded projection near the anterior edge of the blastoder-m is the bulging I‘00f of the so-glm-IlL:1l.inlI (::|\'il.,\-'. In (I the bl()0«‘l-islamls form :1. row of (‘HlI.\‘pl(.'llIJll.*~‘. I-h-\'.'tt-inns of the :«'11rl':-we of the l)l:lh'lA)ll(‘I‘lll p:u':alls-I to its 4*Il;_{4-,

r 14

:

CH.

period and the young born in an

advanced stage [Notidam/us, Mus—'

talus, Galeus, Uarcharias, Zygaena, Lamna, Alopias, Uetorlzxinus, Acumtlmlas, Scymnus, Squatina, Torpedo, Trygonidae,Myliobatidae]. Amongst the viviparous Elasmobranchs preserved developmental stages of Torpedo (Fig. 247) may be obtained from Naples, and of Acanthias from various marine laboratories.

Amongst the oviparous forms certain species of Skate (Rania) are used as food-fishes and their eggs can frequently be obtained in quantity at trawling centres. In such cases arrangements can be made with local fish-dealers to send on by post the “ skate-purses ” taken from the oviducts when the fish are cut up.‘ The eggs of the different species differ in size and in the characters of the shellshape, colour, degree of translucency (Williamson, 1913). Of the European species B. batvls is the most convenient species to use; the normal period of spawning is from December to April but the retarding effect of the low temperature i.s so great that December eggs are practically overtaken in their development by the April eggs. The complete period of development is roughly 20 months, most of the eggs hatching about August.

The eggs should be posted in damp seaweed. On arrival the soft sticky marginal zone of the shell, which separates off except at

one end and serves to anchor the.

egg to the sea-bottom, is removed, and the date is marked in ink with a wooden style upon the flat portion of shell between the two horns.

3 —u-: t ‘

1 Jamieson observed out of many thousa.°nds of eggs only one case of the inclusion

of two eggs within a common shell. XI PRACTICAL HINTS—ELASMOBRANCHII 561

For hatching boxes it is convenient to take ordinary fish boxes freely perforated with anger holes, provided with a cross partition in the centre, and pitched inside and out to discourage the growth of seaweeds. The hatching boxes are moored afloat in pure sea-water within a breakwater or other shelter. About 20 eggs are placed in each compartment.

On alternate days the boxes are drawn a few times backwards and forwards through the water to dislodge any sediment that may have accumulated. Once a week they are hauled’out of the water and each egg-shell tested by rubbing the finger over its surface. If a slippery mucus-like layer has developed on its surface the egg is useless and should be got rid of.

When the egg has reached the desired period of development it is removed from the Water, placed in a horizontal position with the more strongly convex side below and opened by carefully removing the greater part of the less convex side of the shell. The isolated piece of shell must be lifted off very carefully as the albumen is very adhesive and the vitelline membrane extremely delicate.

In the early stages the embryo is almost invisible in the fresh state so the egg, still held carefully in a horizontal position, is gently submerged in fixing fluid. The blastoderm then comes into view and after a short time may be excised and floated into a watch-glass to complete fixation and the subsequent processes.

In later stages (Fig. 248) where the body of the embryo is constricted off from the yolk—sac, it is narcotized by submersion in sea-water containing alcohol and then the yolk-stalk is ligatured with thread and the embryo excised for further treatment.

Embryological material of the Sharks is to be preferred to that of the Skates or Rays on account of their less specialized character but unfortunately it is more diflicult to obtain in quantity. Small sharks of the genus Scyllelum and allied genera occur commonly round the shores of the various continents and their eggs may be found attached to seaweed at extreme low tides.

' On the British coasts a well-known spawning ground for Sag/llium canicula exists at Careg Dion about 2% miles from Beaumaris on the Anglesea side of the Menai Straits in between 3 and 4 fathoms of water and in spots not exposed to strong tidal currents} The eggs are deposited usually in the morning, the shorter stouter pair of filaments which issue first from the cloacal opening being trailed about /amongst tufts of the seaweed Halidrys siliquosa until they become entangled when the fish swims round so as to wind the elastic filaments firmly amongst the seaweed. The eggs can only be obtained at very low and specially favourable spring tides and as White finds at one time embryos of all stages of development it would appear that oviposition is not limited to any definite season. ~ Sag/llwlum not infrequently deposits its eggs in aquaria and at the

1 For the details in regard to this locality I‘ have to thank Professor Philip J. White of Bangor.

\roi.. 11 2 o 562 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

Berlin Aquarium it has been observed that pairs of eggs were deposited at intervals of about ten days. The methods of technique mentioned in connexion with the Skate are also applicable to the eggs of Scyllium.

It should not be forgotten that, as mentioned earlier in this

FIG. 248.—Raia batis, embryos.

at, atrial portion of heart; E, eye; c, conus ; f.g, foregut; H, heart; l, lens; Zi, liver; ot, otocyst; pin, pineal organ ; rh, thin roof of fourth ventricle; v.c.I, etc., visceral clefts; y.s, yolk-stalk; V, VII, VIII, cranial nerves.

volume, one of the greatest desiderata in Vertebrate embryology is an oviparous shark with eggs of small size. ‘

TELEOSTOMI.-—-The most archaic and therefore the morphologically most important surviving member of this group is.Polypterus and strenuous efforts have been made to obtain ‘developmental material. Harrington lost his life on an expedition to the Nile with this object. Budgett made two expeditions to the Gambia, one to. Nigeria and XI PRACTICAL I-IINTS—-FISHES 563

the Nile, and a fourth to the Niger Delta. with the same object in view. The three first expeditions were fruitless but on the fourth he was fortunate enough to obtain ripe males and females and to accomplish fertilization of a number of eggs. Unhappily Budgett did not live to work out this precious material, falling a victim to blackwater fever soon after his return to England. The Budgett material has been investigated (Graham Kerr, 1907) but further material is urgently needed to work out much of the detail.

On the Gambia and on the Upper Nile Budgett found females with eggs in the oviducts during July and August; in the Niger Delta during August and September. During these periods he found that at any one time only a small proportion of males had active motile spermatozoa in their urinogenital sinuses so that it looks as if the actual breeding season of each individual male were very short. The fertilizations which were successful were effected with teasedup testis, the tubules being much distended and the sperm clear instead of opaque as it frequently is. In some cases Budgett found that eggs from the splanchnocoele gave a larger percentage of successes than those from the oviduct. .

The fertilized eggs adhered strongly to the bottom of the dish and this supports the statements made by the natives that in nature the eggs are attached to sticks and stems ol' plants under the water.

Nothing is known regarding the development of the other surviving Orossopterygian-—Oalamichthys.

Of the Actinopterygian ganoids, whose haunts are more accessible

and less unhealthy than those of I’ol_2/pterus, the development has’

been worked out more or less completely in the case of each of the main types———the Sturgeon (Acipenser), the Garpike (Lepidosteus), and the Bowfin or Dogfish (Amia). .

At the large fishery stations such as those on the Elbe or Delaware Rivers ripe Sturgeons are caught during a brief season on their way into the river to spawn. The eggs and spermatozoa may be obtained by “ stripping ” the fish 73.6. by firm pressure passed backwards along the sides of the body, or by opening the fish. The eggs are immediately placed in a dish and a little of the sperm mixed with a small volume of Water is poured over the eggs, the whole being stirred gently for about ten minutes. They are then distributed in a single layer over the bottom of a submerged shallow tray made with coarse mosquito netting to which the eggs adhere firmly within twenty minutes. ‘The trays are then placed in wooden hatching boxes with gauze ends and moored in the river so that they are traversed by a constant current. The dark-coloured somewhat tadpole-like larvae hatch out in from three to six days.

Lepidosteus (Dean, 1895) breeds at Black Lake, N .Y., normally between the middle of May and the middle of June, the eggs being fertilized at the moment of spawning and being distributed over the bottom in shallow water, adhering firmly to stones and other solid 564 EMBRYOLOGY OF THE LOWER VERTEBRATES C1--I.

objects. For laboratory purposes it is best to employ artificial fertilization as in the caseof the Sturgeon. T Amia (Dean, 1896) spawns at Black Lake during the latter half of April or May. The eggs are deposited on a compact site over which the vegetation is pressed aside so as to form a clear space with about a foot of water over it. The eggs, fertilized at the moment of laying, adhere to roots or other portions of the water—plants. The rate of development as in other cases varies greatly with the

F10. 249.—Stages in the development of Symbmnchus. (After Taylor-, 1914.) our, optic rudiment; .I’.F, pectoral nu rudiment.

temperature and from four days to fourteen have been observed to elapse between the deposition of the eggs and their hatching.‘

Of Teleostei (Figs. 249 and 250) by far the most convenient for systematic laboratory work are the Salmon (Salmo salar) and the Trout (S. famlo), eggs of which can be obtained in quantity from the various hatcheries. The eggs obtained by “stripping” are fertilized artificially and may then be sent by post packed in damp moss. Small hatching boxes suitable for laboratory use can also be purchased?

The eggs and larvae of marine Teleosts are often obtained in great

1 Excellent developmental material of Lepidosteus and Anita may be obtained from the Woods Hole Laboratory or from Mr. J. C. Stephenson, Washington University,

St. Louis. 9 E.g. from the Snlway Fisheries Co., Dumfries, Scotland. but these are not so con XI PRACTICAL HINTS——-—I<‘ISHES 565

numbers in the tow-net

venient for investigation on account of their reduced size. As there is little doubt that the 'l‘eleostei have been evolved out of ancestral forms with large eggs investigations are particularly desirable on those teleosts, mostly freshwater forms inhabiting warm climates, in which the large size of the egg has been retained. There is an important field for investigation in the embryology of tropical freshwater fishes. Of individual families the Siluridae, Characinidae and Gymnotidae call especially for investigation.

DIPNOI.-— The Lungfishes form a group of much importance to the Vertebrate morphologist on account of, on the one hand, their great antiquity and the retention of many archaic features in their organization and, on the other hand, of the -fact that they present to us foreshadowings of various features which become prominent characteristics in the tetrapoda or terrestrial animals. A knowledge of their embryology consequently became one of the great desiderata of Vertebrate Embryology. The first

- Fm. 250.——-Bla.stmlei‘1ns anal mubryos of Trout dlscovered of the three (Salmo fa/riu). (After Kopscll, 1898.)

surviving representatives of the gI‘0llp—-L6}9?«d0- y, oxpo.~ae(l su1'fa.ee of yolk.

Iv), eye; at, otocyst; p.f, pect.0r:ll Mn; -rh, rhombeneephalou; 566 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

s7?rem—remained unknown so far as its development was concerned until 1896 when Graham Kerr succeeded in obtaining abundant embryological material in the Gran Chaco of South America.

The developmental stages of Protopterus, the next representative of the group to become known to science, were first obtained on the Gambia River by Budgett who had taken part in the Lepidosiren expedition a few years earlier. Ceratodus, the last of the surviving genera to become known in the adult condition, was the first to be made known embryologically by Caldwell and Semon as already mentioned (p. 435).

The Lung-fishes like other animals living under similar conditions breed at the commencement of the rainy season (Protoptems, Gambia, August; Lepidosiren, Ohaco, November but incidence of rainy season irregular and may be de1ayed—till e.g. J une-—or omitted altogether; Oeratodus, September to December). In the case of Oemtodus the eggs are scattered loosely about amongst the water plants, while in Protoptems and Lepidosiren they are deposited in a special burrow at the bottom of the swamp where they are guarded by the male parent.

¢ Dipnoans live well in captivity and there is little doubt that it will be found easy to induce them to breed by using similar methods to those described under the heading Amphibia. It is particularly desirable that this should be done in the case of Lepidosiren on account of the large size of its histological elements which make it a peculiarly suitable type for the investigation of various problems of histogenesis.

The eggs of Dipnoi, especially of Lepidosiren, are of large size and this makes it especially advisable to use celloidin in addition to paraffin methods of embedding. When paraflin is used it is necessary to remove the egg envelope by slitting it up with fine scissors, care being taken to keep the point of the scissors close to the envelope so as to avoid injury to the surface of the egg.

Corrosive sublimate and acetic acid is a good stock fixing agent. For stages before hatching 107 formalin is convenient.

AMPHIBIA.—-The most easi y obtained embryological material is that of the common Frogs of the genus Roma the masses of spawn of which are familiar objects in pools during the early weeks of spring in temperate climates. The exact time differs with climate and also with species, some species such as R. esculenta in Europe and R. catesbiana in North America lagging several weeks behind the others. The spawn, fertilized as deposited in the early morning, may conveniently be kept during its development in earthenware pans. The water should be left stagnant and unchanged during the period prior to hatching as under these circumstances the spawn is less liable to be attacked by fungus but the hatched larvae should be at once transferred to clean water.

Investigations are greatly needed on the embryology of Anura outside the genus Rana (of. Figs. 251, 252, 253 and 254). The different genera and species differ greatly in the size of the egg XI DIPNOI, AMPHIBIA 567

and its richness in yolk and there is no group of Vertebrates which ofi"ers anything like the same facilities for studying the influence of yolk upon the course of development. Further it will be only after greatly extended studies on different species that we shall be in a position to have a really com- prehensive idea of typical Anuran development.

Many tropical species of Frogs and Toads fire to be Flu. ._')l.—-String of eggs of unknown Frog from the Gambia. Obtalned ahve from Individual variations in the rate of developnu.-nt are indicated animal dealers and hy the varying size of the yolk-plug.

in these it may be taken as a general rule that breeding takes place at-the commencement of the rainy season, or in other words when environmental conditions become favourable after a prolonged period during which they have been unfavourable. By bear ing this principle in mind such tropical T i amphibians may usually be induced to breed in captivity. Bles in his excellent account of the life-history of Xenopus (1905) describes a method which will be found to be of general use. The pair of animals were kept in a Budgett tropical aquarium consisting of a glass bell-jar 20 inches in diameter dipping into a galvanized iron water-tank heated by a small Bunsen burner and oxygenated by plants of Vallisnemla. During summer the temperature of the water in the belljar was kept at about 25° C. The water was not changed. The frogs were fed daily with small earthworms or thin strips of raw calf’s liver until they would eat no more. In December the temperature was allowed to fall to 15°-16° during 1*‘1G-252--EI§ibI_'y0 of 1’hz/llmIwd'u~sja the day and as low as 5"-8° during the "”P"”f;::l”“l’8 "““‘°”°d °“t 1" night. As the temperature rose With the °;lfiI1:ucca'1wity_M bmmpm_ onset of spring the frogs became more N’ mes, n1c.s'odcrnlsnigincnts. 4, actives Wilkmg “P out of the lethargic condition induced by the winter's cold. Breeding was induced by simulating the natural conditions of the rainy season. The temperature was raised to about 22° 0. Each morning and evening about two gallons of the water was drawn off, allowed to cool for twelve hours and then returned to the aquarium in the form of a fountain of spray from the upturned 568 EMBRYOLOGY OF THE LOWER VERTEBRATES CH.

end of a glass siphon drawn out to a fine point so as to produce the effect of a shower of rain. Within a week or two breeding took place.

The chief difiiculty in the way of cutting sections of _Frog’s eggs is due to the presence of the jelly-like envelope. This may be got rid of by prolonged soaking, six months or more, in -5% formalin (Ogushi, 1908), or by fixing in Zenker’s fluid and leaving the efgs 111 this fluid renewing it after 2 to 3 days and continuing the treatment

FIG. 253.-—Stages in the development of ]’l:.y/llunwdusa /1.3/po¢:h¢mal7"£alz's. E, eye; e.g, external gill; op,opercu1um; oz, otocyst.

for 8 to 14 days or longer, shaking gently so as to remove the envelopes (Kallius, 1908). '

For cutting sections paraflin is commonly used but it should be supplemented by celloidin e.g. the clove-oil method mentioned under Ampimloxus.

In the Urodeles the eggs are commonly laid singly in water and attached to water plants (Triton) or other solid objects such as logs or stones (Proteus, Necturus). In Oryptabranchus and Amphiuma they form a beaded string, adjacent envelopes being connected together by a narrow isthmus.

Fertilization is rarely external (0'ryptob'ranchus——Smith, 1912). In the Newts the female takes up a spermatophore into the cloaca. xx ' PRACTICAL HINTS-——AMPHIBIA 569

Such internal fertilization leads up to the condition in the Salamanders where fertilization takes place in the upper part of the oviduct and the developing embryo is retained for a less or more prolonged period within the body of the parent. In Salamcmdm mooculosa larvae about an inch in length are born in May resulting from fertilization during the preceding summer. '

As in the Anura wide differences exist in the richness of yolk and consequent size of the egg—the latter varying from under 2 mm. in the Newts to 6 mm. (Necturus) or 7 mm. in diameter (Org/ptobranchus japom'cus): so that here again though not to the same extent as in

F10. 254.-—Tadpo1e of unknown Frog from Tropical Africa.

A, side view; B, ventral view. inc, huccal cavity; c.o, ('(‘.lllf‘.I|l.r organ ; rz, anus; E, eye; e.g, external gill; u/,/', olfactory organ; up, operculum.

the Anura there is an excellent field for investiga c tion into the influence of yolk upon developmental processes. The eggs of Urodeles are commonly collected under natural conditions and kept in earthenware dishes. Or the adults just about to breed may be brought into the laboratory and allowed to deposit their eggs in a suitable aquarium;

The Urodela form one of the relatively primitive groups of Vertebrates and their embryology‘ deserves much greater attention than it has hitherto received. Most of the older literature deals with special details i.n the development of the Newts but comprehensive monographs, including “normal plates” on the development of such genera as Proteus, Siren and Amphiuma are much wanted. A general account of the development of the American species of Uryptobranohus has been given by Smith (1912), while the Japanese species has been dealt with by Ishikawa (1918), De Bussy (1915) and Dan. de Lange, Jr. (1916). Of Necturus normal plates with accompanying tables have been worked out by Eycleshymer and Wilson (1910).

The Gymnophiona—-—though an aberrant group of Amphibians highly specialized for a burrowing existence—are of much embryological interest and have provided the material for work of great morphological importance, such as that of Brauer upon the excretory organs. A general account of the development of Icltthyophis 570 EMBRYOLOGY or THE LOWER VERTEBRATES on.

will be found in Sarasin (1887-90) and of Hypogeophis in Brauer (1897).

The eggs, fertilized internally, are normally deposited in the soil and the embryologist has, as a rule, to depend upon such scanty material as can be obtained by digging in the damp soil of localities where Grymnophiona are abundant. Ty/phlonectes in South America and _De7~mophz's in West Africa are viviparous.

Of the group in general it may be said that a comprehensive monograph on the development of each genus beyond Ichthyoplmls and Hypogeoph/is is a great desidcratum.

As standard fixing agents for Amphibia corrosive sublimate and acetic acid, and for the later larval stages strong F lemming’s solution, may be used. For the early stages (segmentation and gastrulation) quite good results are obtainable from eggs that have been preserved alive in 10,°/O formalin: in this case it is well to treat the egg before dehydration for an hour or two with corrosive sublimate solution as without this precaution the formalin-preserved eggs are diflicult to stain well. When any other fixing agent than formalin is used it is necessary, as a preliminary, to remove the egg envelopes. In the case of the larger eggs of the Urodela and Gymnophiona this can be accomplished with the aid of fine scissors and forceps.

REPTILIA. — For gaining practical knowledge of Reptilian development the student will find the group Chelonia most convenient as it is possible to obtain 1 excellently preserved series of developmental stages of Terrapins (Ohrysemg/s) and Snapping Turtles (0helg/dm). In particular localities especially in warm climates he may have opportunities of obtaining the eggs of Lizards, Snakes or Crocodilians. In all cases the same technique may be used as in the

case of the Fowl. ' AVEs.—The Birds, although showing conspicuous differences in

external appearance and in minute details of structure, form a very compact evolutionary group and there is little likelihood of important differences in principle existing in their development. Interesting differences in detail however are to be found—such as the presence or absence of neurenteric canals. Groups which there is any reason to suspect of being particularly archaic——such as Divers, Grrebes, Penguins-—-are worthy of careful scrutiny for possible persistence of Reptilian features.

LITERATURE

B103. Trans. Roy. Soc. Edin., xli, 1905.

Brauer. Zool. Jahrbiicher (Anat.), x, 1897. do Bussy (do Lange), L. P. Eerste ontwikkelingsstadién van Megalobatrachcpos

Mamimus, Schlegel. Amsterdam, 1905.

Cerfontaine. Arch. de Biologie, xxii, 1906.

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

1 E.g. from Mr. J. C. Stephenson,‘Washington University, St. Louis,'or The-Marine Biological Laboratory, Wood's Hole. XI - LITERATURE 571

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

Dean, Bashford. Kupifers Festschrift. J ena, 1899.

Eerfort. Arch. mikr. Anat., lvii, 1901.

Iahikawa. Mitt. Deutsch. Gesell. Natur- und Viilkerkunde Ostasiens, xi, 2, 1908. Kalliua. Anat. Anz., xxxiii, 1908.

Kerr, Graham. The Work of John Samuel Budgott. Cambridge, 1907.

Kopsch. Arch. mikr. Ana.t., Ii, 1898.

do Lange, Dan., Jr. Onderzoek. Z061. Lab. Groningon, iv, 1916.

Ogushi. Anat. Anz., xxxiji, 1908.

Sarasin, P. and H. Ergebnisse nnturwissenschaftlicher Forschungen auf Ceylon, ii.

Wiesbaden, 1887-90.

Soammon. Keibels Normentafeln, xii. Jena, 1911.

Smith. Journ. Morph., xxiii, 1912.

Taylor. Quart. Journ. Micr. Sci., lix,\1914.

Williamson. Fisheries, Scotland, Sci. Imwst., 1912, i. 1913.

Ziegler, H. E. and F. Arch. mikr. Ana.t., xxxix, 1892.

APPENDIX

THE GENERAL METHODS OF EMBRYOLOGICAL RESEARCH

EMBRYOLOGY is one of the youngest of the sciences and it offers a wide field for fascinating and important research. Regarded as a branch of morphology its main object is to gain information concerning the lines along which the structure of existing groups of animals has evolved. In the phylum Vertebrata there is an immense amount of work still to be done and it is important that the would-be researcher should be guided by certain general principles as to the technique of the subject, otherwise he is apt to achieve no more than the addition of relatively unimportant details to the vast accumulation of details which during the past few decades has tended to hide away general principles and incidentally to smother interest in the subject.

The incompetent or inexperienced investigator frequently betrays-himself by his choice of subject: he chooses a problem of relatively minor interest when there lie ready at his hand others which are of real importance, or he chooses a subject really important but of such difficulty that the probabilities are heavily against the feasibility of its solution under existing conditions. The beginner then should see that he has the aid of some competent adviser before he decides upon his line of research.

Having chosen his particular problem he has next to decide regarding the particular animals upon which his research is to be carried out. The earlier workers were guided mainly by the accessibility of the material. Fowls and Rabbits -—-of which embryos were easily obtained and easily investigated—--provided the material for the great pioneers of vertebrate embryology and the embryology of to-day suffers much from the difficulty of getting rid of general ideas founded on such narrow bases. N ow that embryology has taken its place as a branch of evolutionary science we recognize the importance of basing our general ideas upon the phenomena of development as displayed by the more primitive existing groups. In attempting any important problem of vertebrate morphology, evidence must be got from Elasmobranchs, Crossopterygians, Lung-fishes, Urodeles, before we can feel completely confident as to general principles: in other words we must go to groups which are admittedly archaic. Apart from directly adaptive features an animal which is archaic in its adult structure may be expected to show primitive features in its development. Naturally we should not look for this in cases where development takes place under peculiar conditions, for these necessarily involve adaptive modification. A pitfall into which investigators frequently stumble is that, starting from

573 574 EMBRYOLOGY- OF THE LOWER VERTEBRATES' APP.

some particular group—-say Ampluloxus, or the Mammalia——with whose structure they'happen to be thoroughly familiar, they assume its general organization to be primitive. As a matter of fact it may be assumed with considerable probability that every existing vertebrate is to a certain extent a mixture of primitive features and specialized. It is only by careful comparative study that it can be decided which features are probably primitive and it is quite certain that these will not be found all within one group. Consequently speculations based upon the intensive study of one particular group are to be distrusted, though there is always less ground for distrust if the group is one which is recognized for reasons other than embryological, as being on the whole archaic. ‘ When minute histological details are concerned another qualification

which should be possessed by the animal chosen for investigation is large size of its cell units.

The material should be abundant. Not only should there be a continuous series of stages but there should be numerous specimens of each stage. There is no such thing as an absolutely normal individual: the conception “normal” is an abstraction based upon the observation of numerous individuals. Only by observing numerous individuals can we therefore arrive at a knowledge of normal development. Work carried out on a? few specimens may of course provide isolated observations of much interest and value but it is inadequate to serve as a basis for general conclusions.

In all descriptive embryology it is necessary to have some method of specifying the stage of development of individual embryos. Unfortunately there has been a great lack of uniformity as to the particular method of doing this. One of the most frequently used is that of specifying the period of time during which development has been going on as for example a “chick embryo of 40 hours’ incubation.” This method is quite unsatisfactory, owing to the fact that the actual stage of development of any individual embryo is a function of other factors in addition to mere time, such as temperature and individual idiosyncrasy. Thus in many tropical freshwater animals a statement of the age of the embryo is practically worthless unless accompanied by a record of the temperature, and even then there remains the unknown element of individual peculiarity such as is for example illustrated by Fig. 251 where a number of sister eggs of a Frog are seen to have “lost step” with one another to a marked extent even at a comparatively early stage of development. In other words eggs or embryos of the same age are liable to vary greatly in their degree of development, and a statement of their age is not adequate as a precise indication of the stage of development. The want of precision varies in different cases: it is less for example in a Eutherian mammal where development takes place at a fairly definite temperature than it is in a Fish or Amphibian inhabiting a tropical pool or swamp where the temperature is liable to great variation. ,

It is necessary then in referring to particular stages of development to define them by structural features. Here however a new difficulty presents itself in the fact that the relative rate of development of different organsystems is not the same in different individuals. It follows that if a. number of individuals be grouped together as being at the same stage of development as judged by a particular organ A it Wlll be found that other APP. METHODS OF EMBRYOLOGICAL RESEARCH 575

organs B, C, etc. are not exactly at the same stage of development—some are less developed some more in the various individuals. Still for practical purposes this is a useful way of indicating roughly the stage of development. For example early stages in the development of Vertebrates may be defined by giving the number of mesoderm segments which have developed——these being fairly conspicuous structures and definable by a number. A much better system, however, is to use numbered stages defined by the general external form——the first structural feature met with in the examination of an embryo. Keibel has published “normal plates” of the development of various Vertebrate types in which standard stages in development are defined by accurate figures. Unfortunately some of the normal plates are incomplete as regards the earlier stages during segmentation and gastrulation, but wherever the plates extend over the whole period of development they should be made use of by the working embryologist as his standard stages. Where no normal plates exist the embryologist should m_ake_it his first business to construct one by carefully working over the external features of development and defining by careful drawing and description a series of stages which he judges to be roughly equidistant. The embryology of any animal is an account of the observable changes which take place in its structure from the zygote stage up to the adult. Logically the investigation of its embryology should proceed similarly from zygote to adult but in actual practice it is better to work in the opposite direction—-—to commence by getting a clear idea of the adult organization and then to work back from the known to the unknown of earlier stages. An embryological investigation should commence with a careful study of the entire embryos or larvae at the various stages. Each stage should be examined first alive by transmitted and reflected light, careful note being taken of any movements due to muscular contraction, ciliary action etc. Particular attention should be paid to the arrangement of the blood-vessels, the time of commencement of heart movements, of circulation of the blood and of the appearance of haemoglobin in the corpuscles. The appearance of chromatophores should be noted: the seat of their first appearance and their reactions-——whether by changes of form, movement of pigment granules in their protoplasm, or by actual migration-—in

response to changes in direction or intensity of light. During this phase_

of the work constant use should be made of the binocular microscope and rough sketches should be made.

Embryos of each stage should be submitted to the action of various fixing agents and it is important to watch the embryo during the process of fixing, for the fluid as it gradually penetrates the tissues often makes special structures stand out distinctly for a short space of time——to disappear again with further penetration. The fully fixed embryo should be subjected to further careful scrutiny by reflected light under the Greenough binocular. To detect small inequalities of the surface it will be found necessary to arrange the lighting carefully. The light from an mcandescent gas-mantle may be concentrated by a large condenser and caused to illuminate the embryonic surface in a tangential direction. It is often well to cover the specimen with a little house of opaque cardboard or metal resting on the stage of the microscope and possessing two apertures one in its roof through which the observation is made and one at the side through which light is admitted. The embryo must of course be 576 EMBRYOLOGY OF THE LOWER VERTEBRATES APP.

completely submerged in fluid and is preferably contained in a round glass dish with a layer of pitch or black wax on the bottom in which, if necessary, small excavations can be made in which the embryo can rest securely in the desired position. The glass vessel should be rotated slowly during the observations so as to allow of the incidence of the light from different directions. It is important to observe a number, preferably a considerable number, of embryos of the same stage, as owing to individual variation particular features may be much more distinct in some than in others.

A number of thoroughly typical specimens of each stage should be picked out for further investigation and these should be carefully drawn under the camera lucida, a piece of millimeter scale being placed by the side of the embryo and drawn at the same time so as to form a reliable record as to dimensions.

' At this stage the normal plates should be constructed if not already in existence and the embryos classified in accordance with them,

For the study of internal structure the great method is that of cutting the embryo into serial sections‘ but a much older method, that of dissection, should by no means be ignored. Careful dissections made under the Greenough binocular are often extraordinarily instructive. It is advisable to experiment with embryos fixed according to various methods as diffcrrent methods give difl‘erent degrees of consistency, opacity etc. Van Beneden and N eyt’s fluid will be found in many cases to give very good results.

In section - cutting a fetish to beware of is excessive thinness of sections. The expert section cutter is liable to become so interested in his feats in accomplishing the preparation of sections of an extraordinary degree of thinness that he is apt to forget that the criterion of good sections is not simply their degree of tenuity but the relation which their thickness bears to the size of the cell-elements of the particular embryo. Thus while in some cases it is of advantage to have sections so thin as 1 [L2 or even '5 ii, in other cases, such as segmentation and gastrulation stages of some of the large heavily-yolked holoblastic eggs, the sections should reach as much as 80 p. or 100 ,u in thickness.

Before an embryo is cut into sections its soft protoplasm has to be supported by infiltration with some suitable embedding mass. For this purpose the two substances used at the present time are parafiin of high melting-point and celloidin. Of these the first is used frequently alone but the student should realize from the beginning that if he is to obtain reliable results, especially yvhere yolk is present in the embryonic tissues, he must use both methods and control and check the results obtained from one by those obtained from the other.

The process of infiltrating the embryo with paraflin is usually carried out in a hot-water oven heated by oil, gas or electricity and kept at a temperature just above the melting-point of the paraffin by a thermostat. The melted paraflin may be contained in small copper pans preferably plated inside with silver or nickel. An essential preliminary is a very thorough dehydration followed by a very thorough soaking in the clearing agent. To get the best results it is well to take the embryo through

1 A useful guide for beginners is.Sect7}on- Cutting by P. Jemieson in preparation. For those who already possess an elementary knowledge of the subject an .xcellent work of reference is Bolles Lee's Miicrotomicfs V ads-nwcum. .

9 1 p.=n1n millimeter. APP. METHODS or EMBRYOLOGICAL RESEARCH‘ 577

three changes each of 90% alcohol, absolute alcohol, and xylol or other clearing fluid. The actual process of infiltration with paraffin should last for the minimum time (which will have to be determined by experiment 1) and be carried out at the minimum temperature.

It may be remembered that the complicated and bulky water-bath with its thermostat is in no way necessary for the embedding process. A very simple apparatus which is perfectly eflicient consists of a small metal trough (copper, or tinplate) resting upon a metal table kept heated at one end by a small flame. By sliding the trough lengthwise along the table a position can be found such that the entire thickness of paraflin is fluid at the end next the flame and solid towards the other end. Between these two points stretches an inclined plane of solid paraffin upon the surface of which the embryo rests without any risk of the temperature rising appreciably above melting-point. A simple embedding trough of the kind indicated is of great use in the field as there is no method of storing and transporting embryos so free from danger of accident or of histological deterioration as having them embedded in solid paraffin.

'I‘o get a block of parafiin in good condition for section~cutting the embryo should be transferred to a bath of fresh paraffiu as soon as it is infiltrated. With certain clearing agents, e.g. cedar oil, it is well to give two or three changes of paraffin. 'l‘he vessel containing the embryo in"a considerable volume of paraflin should now he floated on cold water so as to give a homogeneoustranslucent block of solid paraffin. On no account should the vessel be actually submerged in the cold water for in this event the contraction of the inner paraffin as it cools within the already rigid outer layers will lead to the formation of cavities into which the water penetrates.

For the actual process of section-cutting it is necessary to use a mechanical microtome. The Cambridge Rocking microtome is one of the most convenient for ordinary enibryological work while the ReinholdGiltay ‘microtome is a most excellent instrument both as regards accuracy and rapidity of working. '

The paraflin block containing the embryo is trimmed down so as to be rectangular in section and is then fixed by, the interposition of a hot spatula to the parafiined surface of the microtoine carrier in such a position as may be necessary to give the required direction of sections.

Where the object is a “diflicult” one, e.g. containing much yolk, it is advisable to have it surrounded by a paraffin block of considerable size. A considerable mass of paraffin above the specimen makes it out better, while a considerable mass to the side causes successive sections, with their long edges, to adhere better together and form a continuous ribbon. The embryo should be near one of the lower corners of the block to facilitate exact orientation.

For thorough investigation of the structure of embryos it is advisable to have specimens cut into sections in the three sets of planes-—transverse, psagittal or longitudinal vertical, and coronal or longitudinal horizontal. To obtain these it is tiecessary to have the embryo orientated exactly on the microtome. In most cases this can be accomplished with a sufficieutly close approximation to accuracy when fixing the paraflin block on to the

‘ E.g. for a Chick at about the middle of the second day about 20 minutes will be found to be suflicient. VOL. II 2 1» ' 578 EMBRYOLOGY OF THE LOWER VERTEBRATES APP.

carrier, especially if care has been taken to trim the surfaces of the block parallel to the three chief planes of the embryo.

Where greater accuracy is needed, as in the case of very small embryos, they should be arranged in position in the melted paraffin with warm needles under the prism binocular microscope. This may be done by placing the watch-glass or other vessel on the top of a small flat copper cistern full of water, provided with inlet and outflow tubes, and heated up by contact with the top of the water-bath or hot stage. In the bottom of the embedding vessel is placed a small plate of glass on the upper surface of which are engraved parallel lines intersecting one another at right angles. When the embryos have been accurately orientated with regard to the engraved lines a stream of cold water is allowed to run through the cistern and this causes the paraliin rapidly to solidify. When the block is quite llard the glass plate is picked off and the ridges formed by its engraved lines serve as accurate guides to the position of the embryo.

Still greater accuracy is obtainable by arranging that the melted paraffin in which the embryo is being orientated is already in its definitive position on the holder of the microtome, the paraffin being kept melted as long as necessary by an electric current passing through a loop of high resistance wire.‘

' For the actual cutting care must be taken that the razor (solid ground) or other knife has a very fine edge which does not show irregularities when examined under the low power of the microscope. The blade should be thoroughly cleaned with pure spirit before commencing work. If very thin sections, e.g. of l [L in thickness, are required it is well to commence with sections of 5 pt, then without stopping to change to 4 p., then to 3 p., then to 2 p, then to l p.——cutting a continuous ribbon throughout and going ahead rapidly when the 1 /1. sections are cutting properly.

The celloidin method should be constantly used as a check on the paraffin method. Where yolk.y eggs or embryos are being cut the celloidin method gives the only trustworthy sections as by it the yolk granules are held in position and prevented from sticking on the edge of the knife, ploughing through the tissues and destroying much of the fine

detail, as is always liable to happen if paraffin alone is used under such ‘

circumstances.

In cases where there is no need for specially thin sections (say under 25 pi.) a convenient method is that in which the celloidin block is hardened

by exposure to: chloroform vapour and then cleared by immersion in cedar-wood oil.

The block of celloidin is usually fixed to a block of wood which is

gripped by the holder of the microtome. Care should be taken that such wooden blocks are baked for several days so as to ensure their being

absolutely dry. Otherwise moisture will diffuse out and produce a milky opacity in the celloidin which ought to be absolutely clear and transparent. Sometimes it will be found that the block becomes too hard and will

not cut properly, its edges frilling or breaking. This is sometimes due to the presence of a trace of chloroform in the cedar oil used for clearing.

When this is the case the cut surface of the block should have perfectly pure cedar oil applied to it with a brush just before each section is ‘cut.

‘ A special apparatus for this purpose is made by the Cambridge Scientific Instrument Company. APP. METHODS OF EMBRYOLOGICAL RESEARCH 579

To obtain thinner sections it is necessary to embed the celloidin block containing the object in parafiin. This may be done simply by transferring the block saturated with cedar oil to melted paraffin. A better method is to use a solution of celloidin in clove oil of about the consistency of treacle. The object, thoroughly permeated by this and surrounded by a small quantity of the celloidin, is hardened and cleared in chloroform. The block is then carefully trinnned with one face accurately parallel to the plane of the required sections. It is now immersed in melted paraiiin for a minimum time (ten minutes suilices for a small object). After cutting and mounting the sections the slide is immersed in xylol ,until the parafiin is dissolved out, then in absolute alcohol, then in a mixture of equal parts of absolute alcohol and ether until the celloidin is removed. The slide is now taken down through the series of alcohols and the sections stained and mounted in the ordinary way.

The arriving at a clear idea of the structure of an embryo from the study of a series of sections involves fitting the successive sections together into a continuous whole. To a great extent this reconstruction of the whole from the successive sections can be done mentally but where complicated structures are being investigated, some aid- is either absolutely necessary or at least desirable for the sake of accuracy. The preseht writer finds the most reliable as well as the most convenient of such aids in the method of reconstruction by means of glass plates.‘ Successive sections are drawn with a hard (9 H) lead pencil by means of a camera lucida upon finely ground sheets of glass such as is used for photographic focusing screens and then the successive drawings are fitted together, a fluid of as nearly as possible the refractive index of the glass being interposed between them so that the ground surfaces disappear and the heap of plates appears as a clear block with the structures drawn running through it and appearing as a kind of solid model.

The following details may be noted. Sections are cut to a standard thickness of 10 ,u (z'.e. T55 mm.): the glass plates are 1 mm. thick: the drawings are made at a magnification of 100 diameters. But it will be found in practice that much use can be made of the method even if these three dimensions are not so exactly correlated. The outlines made with pencil of the particular organ that is being studied are filled in with water colour. Vermilion is the most generally useful colour for it retains its opacity and light-reflecting properties to an unusually high degree when submerged in fluid of high refractive index. When the plates are dry N o. 1 is laid, ground side up, on a flat surface——_preferably a glass stage with a_ mirror beneath so that light may be refleeted up through it—a few drops of the fluid used, e.g. clove oil or cedar oil or a mixture of fennel oil (two parts) and cedar oil (one part) as recommended by Budgett 2 are placed by a pipette on the centre of the ground surface and then plate N o. 2 is lowered gently into position and fitted into its place over plate N 0. 1. The outlines of the drawings should be made to coincide exactly, and the two plates should be pressed firmly into contact care being taken to avoid interposed air bubbles which act as elastic cushions and prevent the upper plate from settling down into contact with the other. Successive

1 Quart. Jowm. Micr. Sea, xlv, 1902. 2 Trans. Zool. Soc. Landon, xvi, Pt. 7. 1902. 580 EMBRYOLOGY OF THE LOWER VERTEBRATES APP.

plates are fitted on in a similar manner until the particular organ stands out like a solid model in the mass of plates. _

The same set of drawings may be used for different organs : the clove oil is removed by treating with strong spirit, and the water colour by holding under the tap, and then, after drying, a new organ can be coloured in. By colouring merely the cavity of an organ the relations of the cavity can be displayed as by an injection. When finally done with the drawings are removed by scrubbing with “ Monkey brand” soap.

By this method, after a little practice, reconstructions can he made with great rapidity and accuracy.

Though less accurate and much more tedious the older method of reconstructing with plates of wax is useful for building up a permanent model. Its use is also indicated where only a single specimen is available. Instead of wax plasticine may be used 1 which allows of a kind of dissection being made, in as much as particular parts of the model may be bent out of the way to display structures which would otherwise be hidden.

1n investigating the development of the skeleton the cartilage is often found to pass by imperceptible gradations into unmodified mesenchyme. The absence of sharply defined surfaces in such cases makes the reconstruction method unreliable and it is advisable to supplement it by subjecting the embryo to treatment with a specific stain which picks out the cartilage while leaving the other tissues uncoloured so that the cleared and transparent specimen may be studied as a whole under the binocular microscope.

An excellent stain for this purpose is v. Wijhe’s Methylene Blue.” The embryo is fixed preferably in '5% watery solution of corrosive sublimate, with 10% formalin added just before use, and preserved in alcohol. When about to be stained it should be treated for a day or two with alcohol containing :1-% hydrochloric a.cid——care being taken_ to renew this so long as it develops any yellowness due to traces of iodine. The stain consists of a solution of 1% methylene blue in 70% alcohol to which 1% hydrochloric acid has been added some time before use. The embryo is stained for a week and is then treated with 70% alcohol containing {/0 hydrochloric acid and renewed several times the first day and thereafter once daily until no more colour comes away. The embryo is now dehydrated, cleared gradually in xylol, passed through stronger and stronger olutions of canada balsam in xylol, and preserved eventually in balsam so thick as to be solid at ordinary temperatures though liquid at 60° C.

An excellent method of cleaning small cartilaginous skeletons is to

place. them amongst Frog tadpoles which remove the muscle etc. from the surface of the cartilage by means of their oral combs.

In regard to the general principles of embryological research it need hardly be said that, as in other branches of science, accuracy of observation occupies the first place. And yet, curiously, accuracy may become a fault. In those branches of science which are more effectively under the control of mathematics it is well recognized that in any type of investigation there is a limit of- probable error of observation-—due to instrumental or sensory imperfections or to disturbing factors of one kind or another— ‘ Harmer, Pterobranclyia of Seiboga Expedition, 1905. 9 Proceedings Akad. Wetensch. Amsterdam, J une 1902. APP. METHODS ‘OF EMBRYOLOGICAL RESEARCH 581

beyond which it is mere waste of time to push observation. In all biological observation the limit of probable error is particularly high yet this fact is peculiarly apt to be ignored and it is no unusual thing to find dimensions or other numerical data stated to three or four places of decimals when anything beyond the first place is worthless for the reason indicated. _

To secure accuracy of observation not merely training and experience in the art of observing is needed but also a proper psychological outlook: the observer must be able to take a completely detached point of view and must ever be on the watch to guard against some particular hypothesis or preconceived idea causing actual error instead of fulfilling its proper function of keeping the powers of observation tuned up to the highest pitch of alertness. '

The whole spirit and aim of scientific investigation is directed toward

‘the seriation of facts and the devising of general expressions or formulae _ which unite them together. In this it contrasts with the more primitive

state of mental development which observes isolated phenomena, noting the differences between them but blind to the common features which link them together. In embryology as in other departments of knowledge the able investigator sees the general principles which run through and organize the masses of detail: he interests himself in discovering the likeness which is hidden under superficial difference; he is constructive not destructive.

In this volume embryology is treated as a branch of morphology but it must be borne in mind that morphology and physiology are inseparably intertwined. The living body whether of an embryo or an adult is above all a piece of exquisite mechanism fitted to live and move and have its being, and to ignore this is to make morphology as sterile and as misleading as would he the study of machinery apart from the movements and functions of its various parts. More particularly in attempting to delineate the evolutionary past of .an organ, or set of organs, speculation must always be rigidly controlled by the reflexion that at each phase in evolution it nmst have been able to function.

When at length the stage is reached of putting results into form for publication the first thing to aim at is absolute clearness of expression. It must be remembered that clearness of language and clearness of thought are closely interdependent. Sloppy obscure language means sloppy obscure thought. The greatest care should be taken in the correct and precise use of technical terms. Argumentation in regard to scientific and other matters is, when the disputants are equally well informed, due as a rule to some word or expression being used in slightly different senses. Elegant literary style, however desirable, must always be subordinate to clarity and precision of language. Indeed actual harm is sometimes done to scientific progress by the writer whose literary skill carries away not merely himself but others of uncritical and impressionable mind. Scientific problems are eventually settled not by skill in dialectic but by increase of knowledge.

As a rule the proper presentment of an embryological thesis involves pictorial illustration. In this the elaborate coloured lithographs of former days may conveniently be replaced to a great extent by simple line or half-tone drawings in India ink ‘or process black which can be reproduced photographically and inserted in the text in contiguity with the passage which they illustrate. Their function is to render more clear the statements of the author: they represent as accurately as possible phenomena as observed by the skilled and trained eye with a brain behind it. Actual photographs, which repr'(:sent merely details lying in one particular plane and as seen by the untrained photographic lens, should be avoided. Apart from the imperfections indicated they are so blurred by the ordinary processes of reproduction as to be liable to misinterpretation and in these days of skilful manipulation they are of course useless as guarantees of truth.