Book - A Laboratory Text-Book of Embryology 5 (1903)

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Minot CS. A Laboratory Text-Book Of Embryology. (1903) Philadelphia:P. Blakiston's Son & Co.

A Laboratory Text-Book of Embryology: 1. General Conceptions | 2. Early Development of Mammals | 3. Human Embryo | 4. Pig Embryos | 5. Chick Embryos | 6. Blastodermic Vesicle and Ovum Segmentation | 7. Uterus and the Foetal Appendages in Man | 8. Methods | Figures | Second edition | Category:Charles Minot
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This historic 1903 embryology textbook by Minot describes human development.

This textbook was republished in a second edition 1917: Minot CS. A Laboratory Text-Book Of Embryology. (1917) Philadelphia:P. Blakiston's Son & Co.

See also his earlier 1897 textbook; Minot CS. Human Embryology. (1897) London: The Macmillan Company.

Minot Links: Harvard Collection | 1889 Uterus And Embryo - Rabbit | 1905 Harvard Embryological Collection |1897 Human Embryology | 1903 A Laboratory Text-Book of Embryology | 1905 Normal Plates of Rabbit Embryo Development | Category:Charles Minot

See also: Historic Embryology Textbooks

History Links: Historic Embryology Papers | Historic Embryology Textbooks | Embryologists | Historic Vignette | Historic Periods | Historic Terminology | Human Embryo Collections | Carnegie Contributions | 17-18th C Anatomies | Embryology Models | Category:Historic Embryology
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Pages where the terms "Historic" (textbooks, papers, people, recommendations) appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms, interpretations and recommendations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)

Chapter V. Study Of Young Chick Embryos

Method of Obtaining Embryos

Fertile eggs can usually be obtained from dealers, who can supply them in quantities as needed, or hens may be kept with little trouble especially for the purpose. In that case the hen herself will be found the best incubator, for the number of eggs which develop normally under a hen is larger than in an artificial incubator, and abnormalities of development are less frequent. A good setter will remain upon the eggs, even though some are removed and replaced by fresh ones, for about a month. She should be plentifully supplied with water and soft food, which is best kept at a little distance off, so that she will be obliged to leave the eggs to feed. A box that is somewhat secluded, and affords some protection, warmth, and shelter from the light, should be provided. In order to obtain the most accurate results it is desirable to place the eggs as soon as laid immediately under the hen. Only by this means can an approximate correlation between the stage of development and the duration of incubation be secured.

Artificial incubators are now made to work satisfactorily.* The temperature of an incubator should be maintained at about 38 C. (100.4 F.). It should on no account be allowed to rise above 40 C. (104° F.), for that destroys a portion of the eggs and causes the production of many abnormalities in the remainder; and, if possible, a fall to a lower temperature should be avoided, although the results of a lower temperature are less disastrous. No incubator should be used which does not permit a constant supply of fresh air and of moisture. The date should always be marked on each egg when it is placed in the incubator. If a number of eggs from a dealer are artificially incubated the same length of time, they are pretty sure to cover a considerable range of stages, as, of course, eggs so supplied are of varying ages, the exact time of laying not being recorded.

  • The one used at the Harvard Medical School is heated by a kerosene lamp and has a capacity of loo eggs. It is called the Plymouth Incubator, and is sold by Charles I. Nesmith, Reading, Mass. In the market other incubators may be found, doubtless equally. good, among them patterns adapted for the use of gas, where that is preferred.

In this work two stages of the chick are especially studied. The first is normally produced after about forty-six hours' incubation. The embryo should have about twenty-eight segments and three gill clefts showing externally. Embryos a little less or a little more developed are almost equally serviceable. The second stage studied is that of a chick with seven segments, which is normal after about twenty-seven hours' incubation. The student will find it advantageous to begin his studies with older stages, as these can be more easily manipulated.

Removing the Embryo. — Before the egg is opened a basin should be prepared and filled with normal*salt solution warmed to about 40 C. (104 F.). The basin should be large enough to permit the entire egg to be submerged in it.

Take the egg warm from the incubator or the hen ; allow it to rest quietly in one position for two or three minutes before opening it. This is in order to insure that the side of the yolk which contains the embryo is turned uppermost. After an egg is disturbed the yolk will turn and resume its normal position, for which but a short time is necessary. The egg may now be held in one hand, the shell cracked and the pieces of the shell above the yolk be removed with forceps, making a hole about an inch in diameter. The inner egg membrane may be removed with the shell. If any of the white of the egg tends to overflow, it should be immediately snipped off with a pair of scissors, otherwise it will cause the yolk to roll over, thus concealing the embryo.

The embryo and germinal area are now to be examined with the naked eye, or, better, with a hand lens. The student will detect very easily the area pellucida, which lies at right angles to the long axis of the egg, and also see in the middle of the area a long whitish streak, which marks the anlage of the embryo. Around the area pellucida can be seen the mottled vascular area which will vary in appearance according as the development of the blood-vessels and blood-islands is more or less advanced. The area vasculosa is a portion of the larger area opaca which merges at its periphery into the general yolk. In embryos of the second half of the second day, thirty-six to forty-eight hours, the contraction of the heart can be readily seen, and usually the outlines of the head of the embryo may be made out. The germinal area is now to be separated from the rest of the yolk. To accomplish this, plunge one blade of a pair of sharp scissors into the yolk a little beyond the edge of the vascular area, and cut rapidly around until a circular incision has been completed ; then take a flat spatula and plunge it boldly into the yolk at a depth of perhaps an eighth of an inch underneath the embryo. Next lift out the embryo together with the yolk and the overlying white of the egg, steady it a little if necessary on the spatula with a pair of forceps or needle, and transfer it rapidly to the dish of warm salt solution. With a pair of fine forceps the edge of the germinal area may be seized, and by gentle motion it may be separated from the mass of yolk and also from the thin, whitish, overlying membrane of the yolk, and at the same time from so much of the white of the egg as may have been carried along. As one becomes more practised in these operations, it is not difficult to remove the germinal area without taking much yolk along with it.

The operation may be modified as follows : After the shell is opened the egg mav be tilted so as to allow the white to run off, and as it runs over the edge it is snipped through with the scissors, and as much of the white removed as is possible in this way. The whole egg is then submerged in the warm salt solution, an incision around the germinal area made as above, and the embryo floated off from the yolk.

Preservation of the Embryo. — The next step, after the embryo has been removed from the yolk and lies in the salt solution, is to put a glass slide in the salt solution and carefully float the embryo and germinal area upon it, and then remove them together. The slide is now to be laid flat on the table and the germinal area spread out carefully upon it. In this operation good results may often be obtained by allowing a few drops of warm salt solution to fall upon the center of the germinal area. The currents produced by the falling drops will be sufficient to spread out the blastoderm in its natural form,* and at the same time to wash away any superfluous yolk grains that may be adherent to the preparation. At this stage the preparation should be examined by the student with a low power of the microscope, as described below. To preserve the specimen, four or five drops of Zenker's fluid are allowed to fall upon the specimen gently and quietly as it lies upon the glass slide. The specimen is allowed to stand for about ten minutes and is then transferred to a dish containing a larger quantity of Zenker's fluid. The transfer should be made by submerging one end of the slide in the dish and floating the specimen off. In from two to four hours the hardening of the specimens will be completed. They must then be washed fhoroughly by decanting off the Zenker's fluid and replacing it with water, and this water must itself be replaced several times during the next twenty-four hours. Further treatment of the specimen is as described on page 363.

The Making of Serial Sections. — Specimens are best colored with alum cochineal in toto. They are then imbedded in paraffin and cut into series. The most useful sections are those which are transverse to the axis of the spinal cord. They should not exceed 10 p in thickness.

  • The student will observe that the fresh blastoderm is very easily distorted.

Embryo Chick with about Twenty=four Segments and Three Gill Clefts (about Forty-six Hours' Incubation).

The following description will apply almost equally well to embryos with' from twenty-six to twenty-nine segments.

File:Minot1897 fig152.jpg

Fig. 152. Embryo Chick with about Twenty-foi r Segments. Surface View from the Dorsal Side.

/ , Amnio-cardiac vesicle. Am./, Posterior edge of the amniotic fold. A.o, Area opaca. A./<, Area pellucida. A.'.'i, Arteria vitellma (or omphalo-mesnraica)., Blood-island in the area pellucida. ///, Heart. .l/./\ Mid-brain. Md, Medullary canal or spinal cord. 0/, Optic vesicle. 01, Otocyst. Pr.g, Primitive groove. Seg, Primitive segment. Seg.z, Segmental zone. X '5 diams.

Examination in Toto. — The specimen as a whole, as in the fresh state, has a grayish tint when viewed by transmitted light. As soon as it is hardened the opacity of all the tissues is greatly increased. In the center of the germinal area is the very conspicuous area pellucida, which is somewhat pear-shaped. The portion around the anterior end of the embryo (Fig. 152), A.p, is very wide. In the center of the area vaseulosa appears the embryo, the head end of which is twisted over so that the left side of the head lies against the yolk. This twisting of the neck and head so that they become asymmetrical in position is very characteristic of birds. Below the head and somewhat to the right may be seen the tubular heart, Hi, which, in the fresh specimen, pulsates regularly. Around the area pellucida comes the dark area opaca, in which we readily distinguish the outer boundary, or terminal sinus, of the area vaseulosa. In this there is already a well-developed network of blood-vessels through which the blood is circulating, being driven by the heart. The blood moves out from the embryo by two large vessels, A. vi, which lie symmetrically, the vitelline or omphalo-mesaraic arteries. These arteries arise from the dorsal aorta of the embryo and pass out to the area vaseulosa, over which they ramify. The blood returns to the heart by means of the omphalo-mesaraic veins, of which the anterior branches * are alone clearly differentiated at this stage. The general form of the embryo is indicated by figure 152. In the region of the head we notice the very well-marked headbend which is established in the region of the mid-brain, M. b. The medullary tube in the region of the head is very much enlarged and is divided into three well-marked primary cerebral vesicles. The first of these is quite large, and at its side lies the anlage of the eye, Op, in the center of which one readily distinguishes the commencement of the lens. The second cerebral vesicle is much smaller than the first in every dimension. It occupies the region of the headbend and is separated from the first vesicle by a constriction, and from the third vesicle by another constriction. The third vesicle in length more than equals the first and second combined, and at its widest part is nearly equal in diameter to the second vesicle. It tapers out toward the caudal end of the embryo and passes over into the much smaller portion of the medullary canal, which represents, the anlage of the spinal cord. At the side of the third vesicle we can see the beginning of the formation of the ear or otocyst, Ot. On the side of the neck between the third cerebral vesicle and the heart there are three external depressions which bound the first and second branchial arches, 1,2, of the embryo. Behind each arch the depression marks the site of a gill cleft. The first is the longer, the second the shorter. Between the projecting head and the first branchial arch the outline of the embryo makes a depression, which marks the position of the developing oral cavity. The heart is a large tube, Hi. The omphalomesaraic veins join the venous or posterior end of the heart. The heart is very much bent ; its anterior end turns toward the gill clefts and there gives off the primitive aortic branches, which finally join again so as to form the median dorsal aorta which sends off the two vitelline arteries, .4 . vi. On either side of the medullary canal can be seen the primitive segments, Seg. The first of these which is distinct lies close behind the otocyst. At the posterior end of the embryo additional segments are still forming, and the precise number of segments varies from embryo to embryo. The medullary canal, Md, is closed, but beyond its extreme limit traces of the primitive groove, Pr. g, can still be seen. The network cf blood-vessels over the area vasculosa is very distinct and characteristic. The network, however, does not yet extend into the body of the embryo proper. The limit of the body of the embryo is suggested by the darker tissue, Seg. z, surrounding the spinal cord, Md, on either side. About the hinder end of the embryo, both in the pellucida and in the opaca, appear a number of small spots, the bloodislands, bl. is, many of which have in the fresh specimen a reddish color. In hardened specimens the opacity of the blood-islands renders them conspicuous, especially in the area pellucida.

  • The vessels do not appear in the figure.

Embryo Chick with Twenty=eight Segments

The Study of Transverse Sections. — A series of figures from transverse sections of an embryo of this stage is herewith presented. They have been selected so as to show the principal typical structures. The position of the sections can be followed more easily by comparing each transverse section with figure 166, to determine its place and the organs through which it must pass.

Section through the Right Auditory Invagination (Fig. 153). — Owing to a curvature of the neck-bend of the head the section is not symmetrical. It passes through both the hind-brain, /;. b, and the fore-brain, /. b. Underneath the former appears a small structure, nch, the notochord, and on one side can be seen the auditory invagination, Ot, which is formed wholly by the locally thickened ectoderm, which is elsewhere quite thin. The ectoderm, Ec, covering the dorsal side of the hind-brain is very thin, but the portion in front of the auditory invagination is somewhat thicker. The ectoderm of the invagination is very much thickened and contains numerous somewhat crowded nuclei at all levels. These nuclei are rounded in form and have one or two very distinct nucleoli. On the posterior side of the otocyst there is very little mesoderm; on the anterior side, much more. Between the developing otocyst and the notochord there is a bloodvessel, ve, with merely endothelial walls, a branch of the cardinal vein. Between the hind-brain and fore-brain near the notochord, the two aorta?, Ao, are cut. In their interior there can usually be seen a certain number of nucleated cells varying somewhat in size and appearance, but generally having a rounded form with distinct outline and a well-defined nucleolated nucleus.

Section through the Left Auditory Invagination (Fig. 154). — Owing to the irregular form of the embryo the sections through the otocyst are not symmetrical. The present section shows the opening of the left otocyst, Ot.s, and a closed section of the right otocyst, Ot. d. At its lower inner edge the outer boundary of the wall of the otocyst is indistinct, this appearance being due to the union of the cells of the acoustic ganglion with the wall of the otocyst. The section also passes through the first gill cleft, cl. i , of the right side, and shows very distinctly indeed the closing plate, cl. pi, which is formed by a fusion of the ectodermal and entodermal cells. On the opposite side of the section the same cleft is imperfectly shown. On the posterior side of the cleft is the second aortic arch, Ao. 2, and on the anterior side of the cleft, extending toward the fore-brain, /. b, appear the sections of the two descending aorta?, Ao. D. The part shown in this section is that which connects the dorsal ends of the first and second aortic arches. In the region of the fore-brain appears a shaving from the edge of the optic evagination, Op. The anterior cardinal veins, card, appear just inside of the otocyst close to the ventral wall of the hind-brain, h. b.

File:Minot1897 fig153.jpg

Fig. 153. Section of Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 73. Ao, Aorta. Ec, Ectoderm. F.b, Fore-brain. Hb, Hind-brain, mes, Mesoderm. Nch, Notochord.

File:Minot1897 fig154.jpg

Fig. 154. Section of Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 83.

Ao.D, Descending aorta. A0.2, Second aortic arch. card, Anterior cardinal vein. Closing plate. ft./, First gill pouch. Ec, Ectoderm. Epcit, Roof of hind-brain. /./'.Fore-brain. h.b. Hind-brain, mes, Mesoderm, nch, Notochord. Op, Optic vesicle. 01. tl, Right otocyst. Ot.s, Left otocyst. Ph, Pharynx. ;,< 5° diams.

Section through the Invagination of the Optic Lens (Fig. 155). — This section also passes through the hind-brain, h. b, fore-brain, /. b, and through the openings of both invaginations to form the lens, L , which bears a striking resemblance to the invagination which forms the otocyst. The ectoderm, Ec, over the roof of the fore-brain is very thin and passes abruptly into the thickened layer which forms the wall of the invagination. On the ventral side the ectoderm is somewhat thicker. The wall of the lentic vesicle is quite thick, its nuclei are numerous, but are situated chiefly on the mesodermal side of the layer; so that toward its outer surface the layer is comparatively free from nuclei. The invagination of the lens rests against the optic vesicle, the wall of which, Ret. next to the lens is thicker than the posterior or inner wall of the optic vesicle. The thickened outer portion is the anlage of the retina, the thinner inner portion is the anlage of the pigment layer of the retina. The fore-brain, /. b, has an elongated form with quite thick walls crowded with nuclei. Between it and the hind-brain appears the cavity of the pharynx, Ph, which on the left side of the embryo shows a prolongation, cl. I, which extends almost to the surface of the embryo. This prolongation is the first gill pouch. On the dorsal side of the pharynx appear the two large aortic trunks, Ao. D, and on its ventral side the two smaller first aortic arches, Ao. r. These are situated in the mandibular branchial arch, Mdb, which is well marked externally by a rounded protuberance. The second gill pouch is shown on the left-hand side of the section, cl. II.

File:Minot1897 fig153.jpg

Fig. 153. Section of Chick Embryo with about Twenty-eight Segments. Transvekse Series 92, Section 96.

Ao.D, Descending aorta. Ao.I, First aortic arcli. A0.2, Second aortic arch, card, Anterior cardinal vein. cl.I, First gill cleft. cl.I I, Second entodermal gill cleft. Ec, Ectoderm. Ent, Entoderm. Epen, Roof of hind-brain. /./>, Fore-brain, h.b. Hind-brain. /..Invagination of lens. Mdb, Mandibular arch. mes, Mesoderm, ueh, Notochord. Of, Optic vesicle. Ph, Pharynx. Ret, Retina. ■ 50 diains.

Section through the Optic Stalks (Fig. 156). — The head of the embryo now appears quite isolated from the body. It is bounded by a distinct layer of ectoderm, Ec, and containsthe very large fore-brain, f.b, which givesoff on either side an optic evagination, Op, the walls of which are quite thick, about the same as those of the fore-brain proper. Each optic evagination is widest toward the side of the head and is constricted toward the brain, with which, therefore, it is connected by a stalk in which we can already recognize the anlage of the optic nerve. Between the two optic stalks on the side toward the pharynx the floor of the fore-brain bends downward and almost joins the superficial ectoderm. All of the space between the walls of the fore-brain and the optic evagination on the one hand, and of the superficial ectoderm of the head on the other, is filled with undifferentiated mesenchyma. In this tissue blood-vessels, nerves, lymphatics, and muscles will grow, and the tissue itself is to produce the cutis, the subcutaneous tissue, the skull, the dura mater, arachnoid membrane, and pia mater. We have in the present undifferentiated stage of this mesenchyma a most striking contrast with the complicated histological conditions of the adult. The opposite part of the embryo represents the cervical region. At one side we see a small piece of the heart appearing, Ht, and higher up is the wide pharynx, Ph, underneath which is a blood-vessel, A6, the main aorta. . To the left appears another blood-vessel, Ao. 2, a portion of the second aortic arch. The pharynx shows on one side the prolongation of its cavity which constitutes the second gill pouch, cl. II. On the dorsal side of the pharynx appear the descending aorta?, Ao. D, that on the right of the figure being joined by the third aortic arch, near which appears an accumulation of more deeply colored cells, cl. II, part of the entodermal wall of the second gill pouch. Between the pharynx and the hind-brain we have a round section of

File:Minot1897 fig156.jpg

Fig. 156. Section of Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 104.

Ao, Trunk of the aorta. Ao.D, Descending aorta. A0.2, Second aortic arch, card, Anterior cardinal vein. cl.II, Second entodermal gill pouch. Ec, Ectoderm, f.b, Fore-brain. h.b, Hind-brain. Hi, Heart, mes, Mesoderm. My, Muscle plate, nch, Notochord. Op, Optic vesicle. Ph, Pharynx. X 5° diams.

the small notochord which appears quite deeply stained, and therefore stands out conspicuously from the very loose mesenchyma by which it is surrounded. It is not until later stages that the mesenchymal cells begin to crowd around the notochord to constitute the anlage of the future vertebral column. At the present stage the differentiation of the axial skeleton around the notochord has not begun. As regards the hind-brain, h. b, we know that at its sides it is already considerably thickened, but at its dorsal wall it is quite thin and has already expanded considerably, thus initiating the formation of the thin ependymal roof of the fourth ventricle. On either side of the hind-brain appears a blood-vessel, card, the anterior cardinal, which by transformation and migration is to lead to the formation of the jugular veins of the adult.

Section through the Aortic End of the Heart (Fig. 157). — The cervical region of the head and the tip end of the region of the fore-brain are cut separately. On the lower side of the pharynx is attached the double heart-tube, of which the endothelial portion, endo, is in actual contact with the thick entoderm, En, which forms the floor of the pharynx. The heart-tube shows its bend toward the right of the embryo. There is a considerable space between the endothelial heart and the muscular heart, m. lit, and this space is almost wholly free of tissue, except in the immediate neighborhood of the pharynx itself. Close to the connection of the heart-tube with the pharyngeal floor there runs off on either side the membrane of the amnion. Where it starts from the embryo the amnion has considerable thickness and appears somewhat folded in the section ; but as it turns to cover the embryo it becomes very thin. It consists only of two very delicate layers, mesodermic and ectodermic, both one cell thick. The two layers lie close together, but are easily distinguished. On the right-hand side of the embryo the raphe of the amnion may be observed, raph, and in this section it is constituted by only two strands of mesoderm which pass over from the amnion on to the chorion, Cho, or membrana serosa, as it has been called by many embryologists. The arrangement of the envelopes of the head is somewhat more complicated. Underneath the right* of the section of the cervical portion of the head runs the splanchnopleure, Spl, in which one can readily distinguish numerous sections of blood-vessels, which, on the side toward the embryo, are covered by mesoderm, and on the side away from the embryo are covered by entoderm. If we follow along the splanchnopleure to a point near the section of the region of the forebrain, we find that it encounters a circle of ectoderm, Ec, which surrounds that portion of the head. When the splanchnopleure reaches this ectoderm, its two layers divide or split apart. The mesoderm bends off to the right f and forms, together with a portion of the ectoderm, a part of the true amnion, Am' , of the head. The entoderm, Ent, on the contrary, bends to the left and joins the ectoderm on that side of the head to form the pro-amnion, Pro. am. Beyond the head the entoderm and mesoderm asjain unite and we have a continuation of the splanchnopleure, Spl. Owing to the development of the pro-amnion, the relations of the foetal envelopes surrounding the head are complicated. The student may, however, easily satisfy himself that the layer, Ec, in figure 157, is really ectoderm by following it through in the series of sections, for he will then find that it becomes continuous in other regions, on the one hand, with the ectoderm of the true amnion, and, on the other, with the epidermis of the body proper. In the cervical region we have a transverse section of the lower portion of the hindbrain, Md, corresponding to the part of the future medulla oblongata near its junction to the spinal cord. Underneath it is the section of the notochord, nch, and on either side sections of a secondary segment, Seg. Just below the segments is the cardinal vein, Ve, and below the vein, but nearer to the median line, lies the dorsal aorta, Ao. D. The pharynx expands on each side; the prolongation on the left of the embryo is the second gill pouch, cl. II, that on the right is the third gill pouch. The pharynx itself is lined by entoderm, En, which is very thin in the median dorsal line, but immediately below the dorsal aortae it thickens abruptly and continues as a quite thick layer on to the ventral side. In the median ventral line it forms a deep groove, and in the walls of this groove we find that the nuclei are not distributed through the whole thickness of the entoderm, but occupy chiefly its outer or basal portions, so that the portion of the layer next the cavity of the groove is formed almost wholly of protoplasm. At the tip of the gill pouch the entoderm has come into actual contact with the ectoderm, and the cells of the two germ-layers have there united, without distinguishable boundary being kept between the layers. The fused ectoderm and entoderm constitute the closing plate of the gill cleft, and such a plate is formed at the tip of every gill pouch. On either side of the ventral surface of the pharynx appears the section of the second aortic arch, Ao. 2. By following along through a few sections (in the series here studied, from four to six) the junction of these, arches with the endothelial tube of the heart may be observed. The student should verify this connection and satisfy himself that the endothelium of the blood-vessels is a continuation of the endothelium of the heart. This fact is of great morphological and physiological importance. Of the section of the region of the fore-brain little need be said. The ectoderm has begun to thicken somewhat. The walls of the fore-brain, /. b, itself have not begun to show any differentiation into layers. There is a considerable development of mesenchyma between the brain and the superficial ectoderm.

  • This means the left side of the embryo. f The right of the embryo, — the left-hand side of the figure.

Fig. 157. — Section of Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 114.

Am, Am', Amnion. Ao.D, Descending aorta. A0.2, Second aortic arch. Cho, Chorion. cl.II, Second entodermal gill pouch. cl.III, Third entodermal gill pouch. El, Ectoderm. En, Entoderm of pharynx. endo, Endothelial heart. Ent, Entoderm of proamnion. /./>, Fore-brain. Md, Medulla oblongata, mes, Mesoderm of amnion,, Muscular heart, ncli, Notochord. Pro. am, Pro-amnion. raph, Raphe of amnion. Seg, Segment. Spl, Splanchnopleure. Ve, Anterior cardinal vein. X 5° diams.

Section through the 1 'cnous End of the Heart (Fig. 1 58). — We have now passed in our series beyond the level of the head, so that no part of that is included in the section. The general topography of the part is similar to that of the preceding section (Fig. 157), but there are many important differences of detail. We are now in the region of the spinal cord, Sp. c, proper, which here offers to us its characteristic early embryonic form. It is oval in section, its walls are thickened on each side, but are thinned on the dorsal side, where thev constitute the deck plate, and on the ventral side, where they form the floor-plate; the cavity is narrow and slit-like. The notoehord lies close under the ventral side of the medullary tube and below it is the median dorsal aorta, Ao, a single and very large vessel, which is formed by the union of the two dorsal aortae shown in figure 157, Ao. D. Immediately below the aorta follows the pharynx, Ph, which is now more rounded in form and does not extend far laterally. Its entodermal lining is moderately thick, but it is somewhat thinner near the median dorsal line. On either side of the pharynx the mesothelial layer is very thick and stands out conspicuously, owing to its dark staining. Above the pharynx it thins out and passes over on to the somatopleure, Som, and so on to the amnion, A m. On the ventral side of the pharynx the mesothelial layer passes over into the muscular wall of the heart, m. lit. The heart itself is very large; it has two tubes, the endothelial, endo, and the muscular, m. lit, which are very distinct. The endothelial cavity is very large. It is especially expanded immediately underneath the pharynx to form the auricular end of the heart, which receives the veins. Throughout a large part of the auricle the endothelium is closely fitted against the muscular wall. Further ventral wards the double heart-tube bends to the right of the embryo to form the ventricular limb, Veil, in which the endothelial cavity is also enlarged. The heart as a whole occupies about one-half the area of the entire section of the embryo, being of relatively enormous proportions. The cardinal veins, D. C, have moved down as compared with the previous section, and are now found to lie in the somatopleure, in which there also appear several sections of smaller blood spaces above the main cardinal vessel. The path of the cardinal through the somatopleure carries it toward the heart. The vertical part of the vessel, which affects a union with the heart, is known as the duct of Cuvier. The ducts of Cuvier also deliver the blood from the posterior cardinals to the heart. They are at somewhat different levels on the two sides of the embryo, that on the right side being lower and occupying a sort of prominence on the mesothelial side of the somatopleure. If the cardinal veins are traced along through successive sections, it will be found that they open directly into the auricles of the heart, crossing over the coelom, Ccc. The crossing is accomplished by a growth of the somatopleure which unites with the wall of the heart. The openings of these veins are at this stage morphologically symmetrical and are entirely distinct from the openings of the omphalo-mesaraic veins, which enter the heart further tailwards. If sections in the series between the present one and that through the aortic end of the heart (Fig. 157) be examined, it will be found that the heart in the middle part of its course is entirely detached from the pharynx, so that the heart-tube is suspended by its two ends from the ventral side of the pharynx. By the crossing of the cardinal veins the portion of the coelom, Coe, on either side of the pharynx is shut off from the portion of the coelom around the heart. At the raphe, rapli, of the amnion the ectoderm of the amnion joins that of the chorion, Clio. In the portion of the somatopleure, Am' , which runs from the raphe to the embryo there are a number of spaces of rounded form which appear like so many vesicles. The nature of these vesicles is uncertain.*

Fig. 158. — Section of Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 144.

Am, Am', Amnion. Ao, Aorta. An, Cardiac auricle. Cho, Chorion. Cce, coelom. D.C, Duct of Cuvier. Ec, Ectoderm. EnJo, Endothelial heart,, Muscular heart, mslh, Mesothelium. My, Primitive segment. Ph, Pharynx. Knph, Raphe of Amnion. Som, Somatopleure. Sp.c, Spinal cord. Ven, Ventricle of heart. )< 5° diams.

  • They seem to be bounded on one side by ectoderm, on the other by mesoderm ; but as to this, and as to the significance of these vesicles, I cannot express any opinion. The separate opening of the ducts of Cuvier in lront of, and independently of, the omphalo-mesaraic veins, so far as I am aware, has not been hitherto recorded. It is a condition of morphological importance.

The secondary segments, My, are very characteristic, and should be studied with a higher power. The segment consists of an outer layer and an inner layer of about equal thickness, and these two layers pass over into one another at the dorsal and ventral edges of the segment. They are closely pressed against one another, so that there is no space between them. The outer layer is more deeply stained than the inner; its nuclei are somewhat less distinct and are rounded in form. Those of the inner layer are elongated in form, as may be easily observed by raising and lowering the focus. The outer layer is quite close to the ectoderm, and the inner layer rests against the large mass of mesenchymal tissue which surrounds the spinal cord, notochord, and aorta.

Section through the Anlage of the Liver (Fig. 159). — In this section the general topography is similar to that of the last, so that we need describe only the new structures and relations which appear. A little piece of the ventricular limb of the heart with its double walls, m. ht. endo, still appears. The section is, strictly speaking, beyond the venous end of the heart and passes through the sinus venosus, Si. V, which is formed by the union of the omphalo-mesaraic veins entering the body of the embryo from the splanchnopleure of the yolk-sac, or, in other words, from the area vasculosa. In the splanchnopleure, Spl, there is a thickening, x, of the mesoderm which marks the crossing of the veins from the yolk-sac to the venous end of the heart. The entoderm of the embryo forms a tube, Ent, which is greatly elongated in its dorso-ventral diameter. The entoderm itself is quite thick, except in its median dorsal portion. From the ventral side of the entodermal canal spring two small pouches or diverticula, the anlages of the liver. The left diverticulum is well shown in the figure; the right diverticulum appears a few sections further on. It is especially important to note that the entodermal epithelium of the hepatic diverticulum comes into immediate contact with the endothelium of the blood spaces. During the later development this relation is preserved, and there is a complicated intercrescence of the entodermal cells constituting the liver and of the vascular endothelium.* The intercrescence leads to the formation of the sinusoids, which are highly characteristic of the liver and which give rise to the so-called capillaries of the hepatic lobules of the adult liver. These "capillaries" are, however, always true sinusoids, and morphologically not capillaries at all. Owing to the junction of the veins and liver, a portion of the body cavity, Cce' ', at the side of the pharynx is shut off from direct connection with the pericardial cavity. The ridge of tissue dividing the two cavities from one another is the septum transversum. If the series of sections be followed through tailward, it will be found that at this stage further back the septum transversum is formed also upon the right side of the body of the embryo. The mesothelium between the upper division of the eoelom, Cce, and the sides of the entodermal canal is very much thickened and deeply stained. On either side of the very large median aorta, Ao, and just above the coelom, appear

  • A few sections anterior to this the beginning of the intercrescence is observable.

Fig. 159. — Section of Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 165.

Am, Amnion. Ao, Aorta, card, Cardinal vein. Cho, Chorion. Ca\ Ca', Crclom. Ec, Ectoderm, en . Endothelial heart. Ent, Entoderm. Li, Liver, rites. Mesoderm, m.hl, Muscular heart, mslh, Mesothelium Afv, Primitive segment, nc/i, Notochord. raph, Raphe of amnion. St. l\ Sinus venosus of heart. Som, Somatopleure. Sp c. Spinal cord. Spl, Splanchnopleure. I'e, Vein. )", Accumulation of mesodermic tissue about the omphalo-mesaraic vein. • 50 diams.

the right and left posterior cardinal veins, card. Concerning the foetal envelopes little need be said, except to call attention to the large raphe, raph, of the amnion, which is now a rather conspicuous ectodermal thickening and seems to be formed rather at the expense of the ectoderm of the amnion than at that of the chorion. Such an ectodermal raphe is very characteristic of birds; it has in the chick a considerable extent and therefore appears in many successive sections of the series.

Section through the Omphalo-mesaraic Veins (Fig. 160). — This section is intermediate in structure between figure 159 and figure 161, here described.

FIG. 160.— Section of Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 179.

Am, Amnion. .Jo, Aorta, card, Cardinal vein. Cho, Chorion. Ca, Coelom. Ent, Entoderm. /;/, Intestine. tnsth, Mesothelium. ,1/V, Muscle plate, nth, Notochord. Om.D, Right omphalo-mesaraic vein. Vm.S, Left omphalo-mesaraic vein. Som, Somatopleure. Sp.c, Spinal cord. Spl, Splanchnopleure. . 50 clijms.

We are now beyond the region of the heart and liver. The cavity of the intestine is open on the ventral side, so that the walls of the intestine pass over directly into the extra-embryonic splanchnopleure, Spl, in which are lodged the very wide omphalo-mesaraic veins, Om. D and Om. S, which are entering the body of the embryo to run forward past the liver anlage (Fig. 159) to join the posterior or venous end of the heart. It will also be noticed that the amniotic fold does not join its fellow, and therefore has no raphe. In this condition the amnion is said to be "open." Section through the Anterior Portion of the Open Intestine (Fig. 161). — In this section the intestinal cavity, In, being without a ventral wall, opens directly into the general entodermal cavity under the germinal area and above the yolk-mass. The median plane of the embryo is still inclined to the left. The extra-embryonic somatopleure, Som, rises in two high folds, one on each side of the embryo ; the inner portion of each fold, Am, belongs to the amnion, the outer portion, Clio, to the chorion. The splanchnopleure, Spl, passes without demarcation into the wall of the intestinal cavity, In. The entoderm, Ent, of the extra-embryonic

Fig. 161. — Section of a Chicken Embryo with about Twenty-eight Segments. Transverse Series 92, Section 220. Am, Amnion. Ao, Aorta, b.-w, Body wall, card, Right posterior cardinal vein, card.s, Left cardinal vein. C/w, Chorion. Ec, Ectoderm. Ent, Entoderm. In, Intestine, mes, Splanchnic mesoderm, mst/i, Mesothelium. My, Myotome, nch, Notochord. Som, Somatopleure. Sp.c, Spinal cord. Sp/, Splanchnopleure. Vt, Vein. V 50 diams.

splanchnopleure is very thin, but where it passes into the embryonic region toward the median line, it thickens a little. The splanchnic mesoderm is a thin layer of mesothelium which, of course, bounds the coelom everywhere and can be followed continuously over on to the somatopleure. The splanchnic mesenehyma is loose in texture and surrounds the large blood-vessels. The splanchnic mesoderm on either side of the intestinal groove appears quite dark, owing to the condensation of the tissue. Whether this condensation is developed from the mesothelium or from the mesenchyma it is very difficult to say. The somatopleure, Som, where it becomes embryonic, increases greatly in thickness and forms an arch, b. w, which is the beginning of the formation of the ventral body

wall of the chick. The form of the arcli indicates the commencing closure of the embryonic somatopleure on the ventral side, by which the body of the embryo will ultimately become shut off from the underlying layers of the blastoderm. In the median plane of the embryo we find the spinal cord, cut somewhat obliquely, the notochord, nch, and the very large section of the aorta, Ao. The great transverse width of the aorta is due to its approaching division toward the caudal end of the body to form the two branches which run out to the area vasculosa and are known as the omphalo-mesaraic or vitelline arteries. Before they leave the body of the embryo each of these arteries gives off a branch which continues in the body of the embryo not far from the notochord and close to the entoderm. These branches subsequently become the allantoic arteries. On either side of the spinal cord lie the secondary segments, My. A short distance from the

Fig. 162.— Section of a Chicken Embryo with about Twenty-eight Segments. Transverse Series 92, Section 356. Cce, coelom. Ec, Ectoderm. Ent, Entoderm. Mes, Somatic mesoderm, mes', Splanchnic mesoderm. X. Xephrotome. nch, Notochord. Seg, Segment. Som, Somatopleure. Sf. c, Spinal cord. Spl, Splanchnopleure. Ve, Blood-vessel. VV. D, Wolffian duct. X 5° diams.

aorta on either side appear sections of two rather small blood-vessels, the cardinal veins, card. Between the vein on each side and the aorta there is a little accumulation of denser tissue. If a series of sections is followed through, the Wolffian duct may be traced into this condensed tissue, and when the duct is differentiated, it will take the place of this tissue between the aorta and the vein.

Section through the Middle Portion of the Open Intestine (Fig. 162) . — Comparison of this section with the preceding is instructive as an illustration of the fact that the differentiation of structures is found less advanced as we proceed toward the caudal end of the embryo. In the present section the amniotic folds can hardly be said to have appeared at all, although the coelom, Cce, is very wide indeed, and there is little differentiation in either the somatopleure, Som , or splanchnopleure, Spl, between the embryonic and extra-embryonic regions. The entoderm is a little thicker in the embryo than in the extra-embryonic territory. A similar difference may be observed in the ectoderm. The embryonic mesoderm in both somatopleure and splanchnopleure is considerably more developed and much denser than in the extra-embryonic parts. The axial structures of the embryo — namely, the spinal cord, Sp. c, and notochord, nch — are about the same as further forward, but the mesoderm is much less advanced than further headwards, as is evidenced by the small amount of mesenchyma above the axial structures and by the slight differentiation of the mesothelium. The condition of the segments and their relations to the somatic and splanchnic mesoderm are closely similar to those represented in figure 32. Each segment consists of a larger part, Scg, of rounded outline, close to the medullary tube, and of a narrower part, the nephrotome, N, which connects the inner portion of the segment with the lateral mesoderm. The secondary segment consists of a distinctly marked wall which extends around underneath the ectoderm and against the side of the medullary tube, and of a thick inferior wall which fills up also the center of the segment. Between the nephrotome and the entoderm are small blood-vessels, Ve.

Fig. 163.— Suction of a Chicken Embryo with Twenty-eight Segments. Transverse Series 92, Section 419. C<e, coelom. (V, Diverticulum of the coelora. Ent, Entoderm. In, Intestinal cavity. ««, Mesoderm, nch, Notochord. Som, Somatopleure. Sp.c, Spinal cord. Spl, Splanchnopleure. S.z, Segmental zone. X 5° diams.

Section through the Posterior Portion of the Open Intestine (Fig. 163).— This section is similar to the last, but we may note especially the following differences: The spinal cord, Sp. c, shows a comparatively large cavity, which is widest on the dorsal side, so as to be somewhat triangular in section. In place of the segments we have only the mass of cells, 5. 2, which constitutes the segmental zone, out of which later segments will be differentiated. The segmental zone is of a rather loose texture and merges without boundary into the somewhat denser mesenchyma of the somatopleure and splanchnopleure of the embryo. The dense tissue of the somatopleure extends much further laterally than the corresponding tissue in the splanchnopleure. The notochord is very large and fills out the entire space between the ventral boundary of the spinal cord and the entoderm , and though the mesoderm comes in contact with the notochord, it does not surround it, the relations here representing an earlier stage of development than any which we find further headwards. The entoderm, Ent, of the embryonic region is considerably thickened and forms an intestinal channel, In , of very characteristic form ; for the top of this channel is nearly horizontal, while the sides are vertical and form a distinct angle with the top. In the midst of the mesoderm, on either side of the intestine, there is a small cavity, Coe' , which in two or three sections further forward is found to unite with the general cavity of the coelom. The morphological meaning of this special pocket of the body-cavity is unknown.

Fig. 164. — Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 424.

All, Allantois. cau.i, Caudal intestine. Cm, coelom. Ec, Ectoderm. Ent, Entoderm. Mes, Mesoderm. mes', Splanchnic leaf of mesoderm, nek, Notochord. Sow, Somatopleure. Sp.c, Spinal cord. S/>/, Splanchnopleure. S.z, Segmental zone of mesoderm. />, Blood-vessel, y 50 diams.

From this point onward in the series changes in the appearance of the sections take place very rapidly. The two sections next to be described are quite close in the series to the present one.

Section through the Caudal Intestine (Fig. 164). — In this section we encounter the singular fusion of the germ-layers which is characteristic of the caudal extremity of all vertebrate embryos during early stages. In the median line we see three distinct cavities. The dorsal of these may be readily identified as the continuation of the cavity of the spinal cord. The middle and ventral cavities are entodermal; the upper of the two entodermal cavities, can. i, represents a prolongation of the entodermal cavity into the developing tail of the embryo (compare Fig. 43J- The lower cavity is the anlage of the allantois, All, which is destined to grow out during the next few days into a relatively large round vesicle. The tissue on the ventral side of the spinal cord, Sp. c, is connected by a band of cells with the wall of the caudal intestine, cau. i. If the sections just in front are studied carefully, it can be easily observed that the notochord also passes over without boundary into the same band of cells, which is a mass representing the fusion of the walls of the medullary canal of the intestine and of the tissue of the notochord. In this fused tissue we can, with our present means, detect no signs of the coming differentiation. Just as the walls of the caudal intestine are fused with the tissues on the dorsal side, so also are they fused on the ventral side with the tissue of the allantois. If we follow the tissues laterally, we see that they merge into the mesoderm proper. From the mesoderm there has been a distinct upgrowth of tissue of rather loose texture on either side of the medullary canal to form the segmental zone, 5. z.

Section through the Allantois behind the Intestine (Fig. 165). — This section is only three in the series beyond that last described, yet it is posterior to the caudal is well shown, and it can be readily seen that the hind-brain is nearly equal in length to the mid- and fore-brains combined. In the floor of the hind-brain appears a series of curved notches corresponding to the neuromeres. Only a shaving from the side of the mid-brain, M. b, and two similar shavings from the two parts of the fore-brain, the diencephalon, Dien, and the cerebral hemispheres, H, appear in the section. The optic nerve is cut across and appears as a hollow tube. Underneath the hind-brain a piece of the pharynx, Pli, is cut, and below the pharynx is the large projecting heart, which is very clearly shown to consist of an inner or endothelial tube, Endo, and an outer mesothelial tube,, the anlage of the muscular portion of the heart. The endothelial tube is cut twice, the upper portion, Ao, is the aortic trunk, the lower portion, Ven, corresponding to the ventricle. The heart is, as it were, suspended from the lower wall of the pharynx. The entoderm of the pharynx is very thin on the dorsal side, and thicker on the ventral side. Between the head and the pharynx one can see the projecting mandibular process, Mdb. The small space to the right of this process in the figure, between it and the head, corresponds to the cavity of the mouth. Close to the mandibular process, on the side toward the heart, springs the amnion of the embryo, Am, which passes close around the head of the embryo lying very near it, and can be followed down to where it rejoins the posterior end of the embryo, on the left-hand side of the figure. Underneath the posterior part of the hind-brain can be seen a small piece of the notochord, rich. The notochord appears twice more in the section, rich' and rich", in the dorsal region of the embryo. From the end of the hind-brain the cervical region curves to the right. In it there is a large cavity, D, Ao, the dorsal aorta. To the left of the dorsal aorta we begin to get the primitive segments, which are very distinctly marked. They become gradually wider and wider as we proceed toward the caudal end of the embryo. There also they are less advanced in their development. A small bit of the spinal cord appears in section, Md. From the extreme inferior end of the section a prolongation of the splanchnopleure can be seen which also leads off into the formation of the amnion, Am". There appears again a piece, Sp.c, of the spinal cord and a fragment of the notochord, and on either side of this a segmental zone, Seg. z, of the mesoderm. On the right there shows a small portion of the body-cavity, Coz, distinctly bounded on both sides. Its exterior boundary is a piece of the true body-wall, Som, of the embryo, and close by it is another portion of the amnion, .4 m'. How this is possible may be readily understood by comparison of this figure with figure 161, which represents a transverse section of a similar embryo in this region.

Fig. 165. — Chick Embryo with about Twenty-eight Segments. Transverse Series 92, Section 427. All, Allantois. Ca-, C'celom. Ec, Ectoderm. Ent, Entoderm, lifts, Mesoderm, mes', Splanchnic mesoderm.

msth, Mesothelium. nek, Notochord. Som, Somatopleure. Sp.c, Spinal cord. Spl, Splanchnopleure. S.s, Segmental /one of mesoderm. / >, Blood-vessel. )< 50 diams.

intestine and shows, therefore, more completely the fusion of the structures in the axial region. Except for the absence of the caudal intestine, the description of the last section might apply also to this. The shape of the spinal cord, Sp. c, is somewhat different, and its merging on the ventral side with the underlying tissues is more marked. The cavity of the allantois is smaller and almost slitlike. The other differences do not call for special description.

Horizontal Section. — The student will find it profitable to make a series of sections in the horizontal plane, trying to cut them as nearly as possible parallel with the median plane of the fore-brain and mid -brain.

The accompanying figure 166 is from a section of such a series. It shows very clearly the general form of the embryo, the curvature of the neck, the sharp angle of the head-bend, and the almost straight body’. In the section represented the long stretch of the cavity of the fourth ventricle or hind-brain, Ven. IV,

Fig. 166,-Hoeizontal Section of a Chick Embryo with about Twenty-eight Segments. Am, Am', Am", Amnion. Ao, Aorta., Cardiac aorta. Cm, Coelom. Z)^ Dorsal aorta. £««, Diencephalon. £«<&, Endothelial heart. //, Cerebral hemisphere. AT. *, Mid-brain. Mdb, Mandible 1/,/ Medullary tube. «.*/, Muscular heart. «*5, «*4', »*i", Notochord. 0j>, Optic vesicle. Ph, Pharynx.' 5y, Segment.^ Stg.z, Segmental zone of mesoderm. Som, Somatopleure. Sp.c, Spinal cord. Ven, Ventricle. Ven. IV, Fourth ventricle or cavity of the hind-brain. X 3° diams.

Histological Differentiation of the Chick Embryo with Three Gill Clefts

--Mark Hill (talk) 06:55, 5 April 2014 (EST) "Gill clefts" term refers to the pharyngeal arches.

It is important that the student make a thorough examination and study with a high power of all the cells and tissues of the embryo at this stage so as to familiarize himself with the embryonic characteristics of the germ-layers. The cellular homogeneity of the embryo is strikingly evidenced by the nuclei, which in all parts of the embryo are very similar in size, shape, and structure. They are all rounded in form, varying between spherical and slightly oval outlines, which are seldom quite regular. The outline of the nucleus is always well marked, there being a superficial layer of nuclear substance, which gives a darker appearance to the edge of the nucleus. In the interior there is a single or sometimes two, very rarely three, nucleoli, which are quite large and stain deeply. The strands of substance between the nucleolus and the outer part of the nucleus are very slight, and the space around the nucleolus, therefore, appears light. The protoplasm of the cells is never large in amount, so that the cell-body about each nucleus is not conspicuous, except in the case of the blood-corpuscles, which are, in this respect, somewhat more advanced than the other cells of the embryo.

The ectoderm offers chiefly variations in its thickness, being very much attenuated in some parts, as, for instance, in the posterior portion of the head, where the outer ectoderm overlies the hind-brain. Most of the epidermal parts have begun to increase in thickness, and contain nuclei in two or even three layers. There are several special thickenings of the epidermal layer, for which the name of plakodes has been proposed. At the present stage three pairs of plakodes are seen. The first is the pair of areas which are to be invaginated to form the olfactory pits; the second is the pair which are already invaginated to form the anlages of the lenses of the eyes, and the third pair are also invaginated to form the otocysts. The portion of the ectoderm which forms the medullary tube is also very much thickened, except, of course, so far as the floor-plate and deckplate have been differentiated. In both the plakodes and in the thickened portions of the medullary wall the nuclei occupy nearly the whole thickness of the layer, being themselves several layers deep. They are, however, partially absent from that portion of the ectoderm which is near the original external or free surface. Close to this surface there are, however, a certain number of nuclei, the large majority of which are in various phases of division, as shown by the numerous mitotic figures. No mitoses appear, except in the superficial portion of the layer. Over the greater part of the amnion the ectoderm is so very thin as to resemble almost an adult endothelium, but over the chorion or serous membrane it is a little thicker.

The entoderm appears in three distinct forms: first, the large, long, columnar cells of the area opaca ; second, the very thin cells of the area pellucida ; and, third, the somewhat thicker cell-layer in the embryo proper. For an account of the cells of the area opaca and area pellucida see page 86. The entoderm in the embryo presents considerable variations in thickness which have been pointed out in the descriptions of the sections. Where it is thick enough to permit it, the nuclei are disposed in several layers, and in such places we find that the nuclear divisions take place only in the superficial portion of the entoderm, the phenomenon here being similar to that which we have already noted in the ectoderm. The notochord has a sharply defined outline, as if bounded lay a distinct membrane. It contains nuclei which are quite closely placed, but it does not show, at least in ordinary preparations, any recognizable division into separate cells.

The mesoderm offers several varieties, not so much in the character of the single cells as in their methods of grouping. We notice, first, that there are parts of the mesoderm which are quite thick, and in which we cannot perceive any division into mesothelium and mesenchyma. Such a thick layer of mesoderm may be observed at either side of the pharynx (Figs. 158, Ph, and 159), or, again, toward the caudal end of the embryo in both the somatopleure and splanchnopleure, occupying a larger territory in the former than in the latter (Fig. 163). But for the most part the mesoderm has progressed beyond this stage and shows clearly the differentiation of a thin mesothelial layer lining the coelom and the scattered mesenchymal cells. The mesothelium is quite thin in some parts, almost or quite as thin as adult endothelium. The mesenchyma consists of cells with small protoplasmic bodies connected together by fine threads of protoplasm and with a transparent homogeneous matrix between the cells. It varies greatly in appearance according as the cells are more or less closely crowded together, or widely separated from one another. These differences we designate as varying degrees of condensation in the mesenchyma. The variations occur in a perfectly definite and constant manner, though we are far from understanding yet either the cause or the morphological significance of these variations. The secondary segments vary greatly in structure, because they are in unlike stages of differentiation, those toward the tail being least, and those in the cervical region most advanced. We can, therefore, in a single embryo observe several phases of the breaking-up of the inner wall of the segment to form mesenchyma about the medullary tube and notochord. The transformation is accomplished by a spreading out and moving asunder of the cells, and we can also trace a gradual differentiation of the muscle plate, out of the inner portion of the segment. The external layer, or so-called cutis plate, offers an apparently more or less epithelioid structure in all of the segments. The Wolffian duct is differentiated only through a part of the embryo. It is a small cord of cells that has as yet no central cavity. The blood-vessels are formed solely by the endothelium (angioblast). There is nowhere any condensation of the mesenchyma about the bloodvessels as yet. There are no capillaries whatever in the embryo. One of the most important vascular modifications has, however, been initiated in the anlage of the liver, where we find the vascular endothelium coming into close contact with the entodermal cells of the liver, preparatory to the later complete differentiation of the hepatic sinusoids. The blood-corpuscles are round in form with fairly distinct outlines. Their protoplasmic bodies are much larger than those of any other cells of the embryo at this stage, but their nuclei resemble in size and structure those of other tissues.

Embryo Chick with Seven Segments

(About twenty-seven hours' incubation.) The following description is almost equally applicable to embryos with five or nine segments.

Examination in the Fresh State. — The embryo when first removed from the yolk should be placed in a staining-dish with a small quantity of normal salt solution and examined with a low power of the microscope as a transparent object. The specimen as a whole has a grayish or brownish-gray tint. Most of the germinal area is dark, the transmission of light being stopped by the numerous yolk-grains contained in the entodermal cells (compare page 87). In the center of the germinal area the translucent area pellucida is very conspicuous, and has an edge which is quite sharply defined, more so than after the specimen has been preserved. It is shaped somewhat like an elongated pear. In the axial portion of the area the embryonic structures are partially differentiated (Fig. 167). It should be noted that this figure is taken from a hardened, not a fresh specimen. The head of the embryo is protuberant and is of a bluntly rounded form. It projects freely above the surface of the germinal area. Underneath the projecting head is a very clear area in which there is no mesoderm whatever. This is the pro-amnion, pro. am. On either side of the head are two characteristic spaces, a. c. v, the amnio-cardiac vesicles. The surface of the germinal area rises somewhat dome-like over each vesicle. This is due to the fact that in this region the coelom is already very large and the splanchnopleure or upper leaf of the germinal area is arched on either side of the embryo. The relations may be more clearly understood from cross-sections (Fig. 170). The posterior limit of the head is marked by an arching line, the concavity of which faces the caudal end of the embryo. This line, jov, marks the position of the fovea cardiaca. On the sides of the fovea, running forward toward the median line of the embryo, one can distinguish two darker bands which represent the beginning of the formation of blood-vessels growing in from the extra-embryonic region to join in the median line of the embryo and participate in the formation of the heart on the under side of the head. In order to see the fovea clearly, the focus of the microscope must be lowered. The medullary groove is partly converted into the medullary canal, for at this stage it is closed from the anterior limit of the head to a variable point in the segmented region of the embryo. Specimens, however, vary extremely as to the extent of the closure. The line of closure can be readily seen. It is somewhat wavy and irregular in its course, and the closure itself is somewhat irregular, so that we may find one or several points where the closure is not yet completed, although it is complete behind and in front of these points. At the anterior extremity of the head closure is always incomplete, there being an opening there which persists for some time and is known as the anterior neuropore. Above the primitive segments, where it is not closed, the medullary groove has its edges close together. But a short distance behind the last segment the groove widens abruptly and fades out gradually. This widening is termed the r ho mboidal sinus. By proper adjustment of the focus the notochord may be distinguished underneath this sinus. Just at the posterior limit of the sinus the primitive groove, pr, begins and extends backward, often bending to one side or the other, usually to the left. The groove is shallow in front, deeper behind, and ends quite abruptly. The primitive segments appear as square darkish blocks of tissue symmetrically placed on cither side of the medullary canal. The first pair of blocks lies a short distance behind the fovea and the last pair a short distance in front of the rhomboidal sinus. When new segments are added, they are about the same size as those previously formed. The growth of the embryo in length during these stages depends rather upon the multiplication of the segments than upon the growth of the single segments. The region about the primitive streak is quite dark, owing to the accumulation of cells, which belong chiefly to the mesoderm. This dark appearance extends forward and merges into the so-called segmental zone, out of which the segments are differentiated. More careful examination of the area opaca shows that it already possesses a well-defined area vasculosa, Ao, in which (with some difficulty in the fresh specimen) traces of the formation of blood-vessels and blood-islands can be made out. They can be somewhat better distinguished if the fresh specimen be examined not by transmitted, but by reflected light. In the fresh specimen it is very difficult to make out the shape of the medullary canal in the region of the head.

FIG. 167. — Chicken Emuryo, after Twentyseven Hours' Incubation, with Eight Primitive Segments.

/'r<r\ Fovea cardiaca. pro. am, Proamnion, a.c.v, Ammo-cardiac vesicle, st, Sinus terminalis. /»■, Primitive groove. Ao, Area opaca. A.p, Area pellucida. — {After Duval.)

Examination of the Specimen after Hardening. — The specimen, after it has been hardened , should be examined under the microscope in water or alcohol ; and, again, after it has been stained it should be cleared in oil and further examined. This will enable the student to make out the blood-islands and something of the blood-vessels in the area vasculosa, and also the shape of the brain which (Fig. 167) has expanded widely just behind the neuropore; the lateral expansions are the anlages of the optic vesicles (Fig. 1 56) . The remainder of the brain extends from the optic enlargement to a point a little behind the fovea, fov. It is much wider than the remaining portion of the medullary canal; it tapers from the optic vesicle and extends backward. One cannot yet distinguish in it positively any subdivision into mid-brain and hind-brain. On the contrary, its walls are often somewhat irregularly sinuous and vary considerably from specimen to specimen.

Comparison with a Rabbit Embryo. — In the ovum of the mammalia the ectoderm presents a modification known as the trophoblast. In the rabbit this trophoblast is developed over a limited region which is called the placental area (Fig. 168, a. pi), by which the embryo is attached to the wall of the uterus. When the embryo figured was removed, a portion of the placental area remained attached to the uterus, hence the defect shown in the specimen. The vascular area is nearly circular; its boundary is marked by a well-defined terminal vessel, v.t. The nearly straight embryo lies in the center and exhibits plainly the medullary canal and primitive segments. The optic evaginations are already present. The head is free; on its under side the heart is forming, and beneath it is a relatively large and conspicuous pro-amnion, pr. a. Bloodvessels are present over the area vasculosa, but not yet in the embryo. It will be seen, therefore, that though the proportions differ greatly from those in the chick, the fundamental relations in the rabbit are the same as in the bird.

Longitudinal Section of a Cluck. — In order to facilitate the study of the transverse sections of this stage, figure 169 is inserted, which is a nearly median longitudinal section. In consequence of the head end, H, having grown forward about the pro-amnion, pro. a, it has become free on all sides, and at the same time the entodermal cavity has been carried forward with the head, making the so-called fore-gut of English authors. This fore-gut is the anlage which serves as the connection across the median line with the amnio-cardiac vesicles just described in surface views. We can, therefore, distinguish in the fore-gut the anterior portion from the posterior portion which overlies the eoelom. This coelom is the anlage of the pericardial cavity. The anterior division of the fore-gut forms the pharynx proper. It ends blindly in front. The opening of the fore-gut into the general entodermic cavity, Ach, is termed the fovea cardiaca, fo. At the posterior end of the embryo we have a thickened mass of cells constituting the primitive streak, Pr.s. The line on the under side of the figure represents the entoderm, and the space underneath it is a portion of the primitive entodermic cavity.

Fig. 168. — EMBRYONIC Area OF a Rahbit ok (? Nine) Days, with the Placental Area Partly Torn off.

pr.a, Pro-amnion. a. a, Amniotic area, a.z\ Area vasculosa., Area placentalis. ;■./, Sinus terminalis. — (After van Beneden ami Jul in.)

of the pharynx, the oesophagus, and the stomach. Underneath the posterior portion of the fore-gut there has appeared in the mesoderm a coelomic cavity, />,

FlG. 169. — Longitudinal Section of a Young Chick EMBRYO. //. Head. Vd, Anterior portion of digestive canal (Vorderdarm). met. Mesoderm, fo, Fovea cardiacs. /•, pro. a, Pro-amnion. Ach, Entodermal cavity, in life bounded below by the yolk. Pr.s, I'i imitive streak.

Study of Transverse Sections. — Section through the Anterior Part of the Head (Fig. 170). — The head lies free and is covered by a well-defined layer of ectoderm, Ec, which on the dorsal side is continuous with the walls of the medullary canal, md, which is here quite wide, corresponding to the level of the future hind-brain. Where the outer ectoderm joins the wall of the medullary canal there is an accumulation of cells, Gl, readily distinguishable from the adjacent ectoderm and the medullary wall proper. These cells constitute the so-called neural crest, and represent the beginning of the formation of the true ganglionic cells. On the ventral side we have the very widely distended pharynx, Ph, which has the characteristic crescent shape in transverse sections. It is lined by entoderm which forms a distinct continuous layer, is very thin on the dorsal side and thicker on the ventral side, and on the dorsal side is bent upward so as to form a median longitudinal groove. The base of this groove touches the notochord, rich, which lies closely packed in the median line between the floor of the medullary canal and the roof of the pharynx. The notochord presents a circular outline, and its large size, as compared with that of the mammal, should be noted, for it is characteristic of the Sauropsida. The space between the pharynx and the ectoderm is partially filled by loosely scattered mesodermic cells, mes. Underneath the head of the embryo we have two layers of cells which belong respectively to the ectoderm, Ec, and entoderm, En, and together constitute the so-called pro-amnion. On either side, at a point corresponding roughly to the lateral boundary of the head, the two layers of the pro-amnion separate widely from one another and a third layer of cells appears between them, which belongs to the mesoderm and surrounds the large ceelomic cavity which is characteristic of the amnio

Fig. 170. — Chick Embryo with Seven Segments. Transverse Section of the Head. iii/i, Xotochord. 67, Ganglionic or neural crest, md. Wall of medullary tube, mes, Mesoderm. Ph, Pharynx. Et, Ectoderm. En, Entoderm. Am. /", Amnio-cardiac vesicle.

Fig. 171. — Chicken Embryo with Seven Segments. Transverse Section through the Anlage of the Heart.

J/./, Medullary groove. Ec, Ectoderm, mes, Mesenchyma. Am.ves, Amnio-cardiac vesicle. /'//, Pharynx.

msth, Mesothelium. Emio, Cells to form the anlage of the endothelial heart.

cardiac vesicles, Am.V. The letters in the figure are placed in the coelom of the right-hand vesicle.

Section through the Posterior Division of the Fore-gut (Fig. 171). — In the embryo from which this section was taken the medullary groove, Md, was not yet closed at this point. At the upper edge of the groove traces of the differentiation of the neural crest, so clearly shown in the previous figure, can be distinguished, and the ectoderm of the groove at its edge passes over into the general ectoderm, Ec, covering the head of the embryo. The fore-gut, Ph, the notochord, and the mesoderm, mes, are all very much the same as in the previous section. On the under side of the embryo the structure is quite different. The amniocardiac vesicles, Am.ves, come very close together in the median line. They are lined by a distinct layer of mesothelium, msth, which, for the most part, is very thin, but underneath the wide pharynx the mesothelium has acquired considerable thickness and is somewhat irregular, as shown in the figure. Between the mesothelium and the floor of the fore-gut, Ph, there lie a few cells, Endo, which are the precursors of the endothelium of the heart; the thickened mesothelium forms the muscular wall of the heart. In the figure there is a median partition of mesodermic cells by which the two amnio-cardiac vesicles are separated from one another. During this stage, or very soon after, this partition breaks down and disappears, and thereafter the two vesicles communicate freely across the median line, and the pericardial chamber is said to be formed, though it is not yet delimited from the general coelom of the vesicles.

Fig. 172. — Chicken Embryo, Transverse Section across the Anlage of the Heart in a Stage slightly more advanced than flg. 171. .W, Wall of medullary tube, nek, Notochord. msth, Mesothelium. Ph, fharynx. pro. am., Tip of proamnion. En. lit, Endothelial heart. ///./;/, Muscular heart.

The further development of the heart may be understood by the examination of a somewhat older stage (Fig. 172). As shown in the illustration, the mesothelium has become very protuberant, m. lit, in the median line underneath the fore-gut, Ph. On either side it rapidly thins out, msth. In the protuberant fold we can recognize the future muscular heart, as it is sometimes called. The few cells (Fig. 171, Endo) above described have increased considerably in number and have joined themselves together in such a manner as to indicate clearly the formation of the endothelial heart (Fig. 158, Endo). At first the cells are irregularly disposed and have several irregular cavities between them which soon fuse so as to form two main cavities running longitudinally. As the two cavities enlarge they meet in the median line and remain separated at first by a wall of two layers of endothelium. This wall soon breaks through, and there results a single median tube of endothelium which presently appears to be connected with the mesothelium, in. lit, by long cell processes across the wide, intervening space. The heart is now a double tube connected by the mesothelium with the tissues above. Still later we shall find the endothelial heart enlarged and the muscular heart to have grown across on the dorsal side so as to form a closed tube which separates finally on the dorsal side from the mesothelium with which it is originally connected, and after this separation we have a double free heart tube underlving the fore-gut.

Fig. 173.— Chicken Embryo with Seven Segments. Three transverse sections across the caudal end of the medullary groove : A. Section through one of the segments j B, Section posterior to the segments; C, Section just in front of the primitive streak. Md gr Medullary groove, nch, Notochord. Ec, Ectoderm. »«, Mesoderm. En, Entoderm. ■ 250 dianV

Fig. 174.— Chicken Embryo with Seven Segments. Transverse Section across the Primitive Groove. Ec, Ectoderm, mes, Mesoderm. Ent, Entoderm. Pr.g, Primitive groove. The large black dots represent yolk-grains. >( 230 diams.

Sections through the Medullary Groove. — Figure 1 73 represents three sections at different levels. In the first, A, the groove is quite deep and the young primitive segment is shown. At the edge of the groove its thick walls pass over continuously, but quite abruptly, into the general ectoderm, Ec, covering the embryo. Close under the median line of the medullary groove appears an oval section of the notochord, nch. The entoderm, En, is quite thin and somewhat irregular, as is shown in all of the sections. In B the medullary groove is wide open and quite shallow, the notochord is much larger and extends from the floor of the medullary groove to the entoderm and occupies in part a deep notch in the medullary wall. The notochord prevents the extension of the mesoderm across the median line. In C the medullary groove is fading out and merging into the beginning of the primitive streak, which forms a large mass of cells in the median line in which the boundaries between the germ-layers cannot be determined. Laterally this mass of tissue passes over into perfectly distinct germ-layers, of which the middle or mesoderm, mes, is by far the most voluminous. The walls of the medullary groove are crowded with nuclei which lie at every possible level, some close to the inner, others close to the outer surface, and also in every possible intervening position. The nuclei are much crowded, there being but little protoplasm. No distinct cell boundaries can be made out. The nuclei are further remarkable on account of their very conspicuous nucleoli.

Section through the Primitive Groove (Fig. 174). — The section passes through the anterior region of the groove. Underneath the groove, which appears in the section as a shallow notch, Pr.g, the germ-layers are fused with one another and show no recognizable boundary; laterally, however, they become entirely distinct. The ectoderm, Ec, is quite thick, representing, presumably, the stage of the embryonic shield (compare page 62). The mesoderm, mes, is of about the same thickness as the ectoderm, but its cells are far less compactly crowded. In the median line it also merges without boundary into the tissue of the primitive streak. The entoderm, Ent, is very thin indeed and makes a striking contrast with the appearance in the same layer in the region of the area opaca (compare Fig. 35).

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