Book - Vertebrate Embryology (1949) 2

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

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Part II The Development of the Frog The Frog - From the Production of the Germ Cells through Gastrulation

The embryology of the Frog, Rana sp., will be taken up as the first example of the development of a true Vertebrate, being a valuable object for such study for the following reasons: In the first place its earlier history furnishes an excellent transition between the corre sponding stages in Amphioxus and those in animals which are more

highly evolved. Second, the later development of the Frog is also very suggestive from an evolutionary point of view. Thus it illustrates in a striking manner the transformation of a purely aquatic gill-breathing

  • Vertebrate into one which breathes largely by lungs, and is capable of

extended existence on land. Third, in the course of its development the Frog shows the origin of practically all of the fundamental Vertebrate systems. Yet in many cases these systems remain in a rather primitive condition, and are thus helpful to an understanding of the complications which are met with in other types. Fourth. the development of the Frog is important bothbecause of the thoroughness with which it has been observed under normal conditions, and also because of the active experimental work which has been and is being done upon it and its near relatives. Lastly, there are also certain practical considerations. The living material is usually available at an appropriate time of year,

it is easy to handle, and the young can be readily cared for under laboratory conditions.

THE REPRODUCTIVE ORGANS or THE ADULT, oc">eENE— SIS, AND THE EXTRUSION or THE OVA

THE MALE ORGAN S

The Testes. —-There are two" testes in the Frog, each one lying in the dorsal region of the coelom, close to the kidney (Fig. 56). Each is enveloped by the peritoneal epithelium, which is fused above the organ into a two-layered sheet of tissue, like a mesentery. This sheet attaches the testis to the body wall and is termed the mesorchium. In appearance ,m

~ THE MALE ORGANS 105

each testis is a white ovoid body. which may be a half inch or. so in length. In some species in which _the sperm are produced continuously, the size of the organ remains fairly

' constant. In others, however, in which ‘ spermatogenesis is chiefly confined to

the breeding seasons, the dimensions vary considerably. This variation is

‘nevertheless relatively small com pared to what always occurs in the ovary.

In structure each testis consists essentially of a mass of seminiferous tubules. These are grouped into lobules and the latter again into lobes separated by thin partitions of supporting or connective tissue. This tissue also covers the whole organ in a coat called the tunica albuginea, outside of which is finally the peritoneum. The walls of the tubules are lined internally with follicle or nutrient cells (Sertoli cells), while between the latter and the lumen of each tubule come groups of germ cells in various stages of development, those in any given group being in approximately the same stage‘ As the cells of a group reach the condition of spermatids their heads are gathered together and the tips embedded in a Sertoli cell. Finally when fully ripe the spermatozoa are liberated into the tubular lumen.

To the anterior end of each testis


Fig. 56.--The male urinogenital system of the adult Frog (Rana pipiens) viewed from the ventral side. The testes in this case are medium sized. The urinary bladder and rectum have been dissected out and reflected posteriorly. Otherwise in the ventral View they would cover the lower part of the reproductive organs. Note the large fat bodies as compared with those in the’ female. Also note'\ the rudimentary oviducts. In many species of Frogs these ducts do not develop so far in the male as in R. pipiens. They have no known function in this sex.

ad. Adrenals. bv. Blood vessel. cl. Cloaca. fb. Fat bodies. 1:. Kidney '(mesonephros). od’. Rudimentary oviduct. ‘r. Rectum. sv. Seminal vesicle. t. Testis. ub. Urinary bladder. ur. Ureter, in the male serving also as a vas deferens. ut’. Rudimentary -uterus. ve. Vasa eflerentia.

otobedrawnonatthistime.

is attached a fat body, composed of a mass of yellow streamers. Its function is uncertain. Inasmuch as the animals do not eat during the breeding season, however, it may serve as an extra supply of nutrient material 106 THE FROG: THROUGH GASTRULATION

The Sperm Ducts. —The tubules of each testis open into about a dozen fine ducts, the vasa eflerentia. These connect with some of the more anterior kidney tubules, which thus function as continuations of the vase eiferentia as well as in excretion. These tubules in turn of course empty into each kidney duct, which therefore acts as both ureter and sperm duct (vas deferens) . The two vasa deferentia are dilated just before entering the cloaca to form the seminal vesicles. In these, the

sperm are stored previous to discharge.

THE FEMALE ORCANS

The Ovaries. —— The ovaries are also paired organs and occupy the same relative position as the testes (Fig. 57). As in the case of the latter, each is


Fig. 57.—The female urinogenital system of the adult Frog (Rana pipiens) viewed from the ventral side. The left ovary has been removed, showing the fat body, kidney and oviduct upon that side. The right ovary full of nearly mature eggs remains in place. Note that the fat body is smaller than in the male, having presumably suffered depletion during the development of the eggs. The urinary bladder and rectum are omitted from the figure, but occur in the same position as in the male. .

inf. Infundibulum. o. Ovary. ad. Oviduct. ut. Uterus. Other abbreviations as in Fig. 56.

suspended from the body wall by a double sheet of peritoneal tissue in this instance called the mesovarium. Unlike the testes, however, the ovaries always vary greatly in size and appearance, depending upon the time of year. After ovulation in the spring they

appear as flattened cream colored organs, about three—quarters of an inch long in Rana pipiens, with a few dark specks scattered through them. As the oiigonia for the ensuing season multiply and presently grow into oiicytes, however, the organs increase immensely in size, and by the end of the summer they occupy a large share of the body cavity. They are now lobulated in form, and exhibit a characteristic black and white speckling, due to the color of the ripe eggs. Under normal circumstances they OOGENESIS 107

remain in this condition throughout the winter. As will be indicated later, however, the eggs are completely developed, and by artificial means such ovaries can be made to ovulate viable ova at any time.

In structure, the ovary consists of a number of compartments, whose outer walls are formed of connective tissue or stroma. Within the compartments the oiigonia may be in the process of multiplication, as suggested above, or if this stage has passed the compartments will be filled with oiicytes. Each of these oéicytes is surrounded by a single layer of flattened cells which constitute its follicle. Outside of this is another layer termed the theca, which serves to attach the ovum to the wall of its compartmert. This theca in turn is divided into an outer layer containing chiefly blood vessels, the theca externa, and an inner layer of smooth muscle fibers, the theca interna.

Attached to the anterior end of each ovary is a fat body similar in appearance, and presumably in general function, to those connected with the testes.

The Oviducts. ———These are long convoluted tubes whose size and convolutions are somewhat increased during the breeding season. They open anteriorly into the coelom by a ciliated funnel, the infundibulum. Posteriorly they open into the cloaca. Throughout the greater part of their length the walls are quite thick, especially during breeding time. This thickening is due to the hyper-development of numerous simple tubular glands which secrete the gelatinous covering of the eggs. The lumen of the ducts is lined by ciliated epithelium. At the posterior end, each duct widens and its walls become thinner and very elastic. These dilated regions, known as the uteri, serve for storing the ova just prior to extrusion. Each duct is covered by a layer of peritoneum and slung from the dorsal body wall in the same manner as are the gonads.

OGGENESIS

The Ofigonia. —— The normal breeding season, as already suggested, occurs in the spring or early summer. At this time the ovaries are emptied of ripe eggs, and the relatively few oiigonia which remain begin to multiply to produce the eggs for the next season. These occur in nests, and in each such nest only one cell is destined finally to become an ovum, the others constituting its follicle. As soon as an ovum has become definitely differentiated as such, and its follicle formed, the period of growth and membrane formation sets in.

‘The Growth Period.-——When this period has been reached the

young ovum or oiicyte, as it may now be called, begins to accumulate A ..Tr..;..- -~


is 5

108

THE FROG: THROUGH GASTRULATION

Fig. 58.--Oogenesis in the Frog (R. temporaria). From Kellicott (Chordate Development). A—E, after Lams. F—I, after Lebrun. A. Primary oocyte in synizesis. B. Primary oocyte with vitelline substance

(yolk) of mitochondrial (chromidial?) origin in the cytoplasm. C. Primary oocyte showing feathery chromosomes and chromatin nucleoli. D. Primary oocyte with ring-like vitelline mass. E. Primary oocyte showing cytoplasm in two zones. F. Nuclear region of primary oocyte after dissolution of the nuclear membrane showing the small chromosomes and large chromatin nucleoli. Egg still in ovary. C. First polar spindle in primary position. From egg in body cavity. H. First polar spindle in metaphase. From egg in uterus. I. First polar body formed and second polar spindle forming. From eggs in uterus.

c. Centrosorne. ch. Chromosomes. f. Follicle cells. g. Contents of germinal vesicle. n.'Chroznazin nucleoli. v. Vitelline substance of mito bd "..Ylk]t.I.F' 1 ‘d1 «.01.. my in 1). zz.°sZc§.L‘f’,§‘o13. sp‘.-’..ai§" °‘ W ‘W “"" ° OOGENESIS 109

yolk. Before this starts nucleoli appear under the nuclear membrane. Also hasophilic yolk-nuclei arise within the cytoplasm, and move first to the cell periphery and second to the nuclear periphery. The yolk then develops as granules just beneath the surface of the oocyte. Though the source of these granules is uncertain, they may be derived from Golgi apparatus, the ground substance of the cytoplasm and the nucleus (Hibbard, ’28) . This layer of granules gradually widens, and the granules or platelets increase in size (Fig. 58, B, D, E). Eventually, the entire cytoplasm is filled with yolk (Kemp, ’53), but the platelets are larger and more concentrated in what proves to be the vegetal half of the egg, thus making the latter telolecithal. What causes this polarity is still unknown. However, it is initiated very early by establishment of a ribonucleoprotein gradient (Brachet, ’4~7b), a movement of the nucleus toward the animal pole, and by the collection of pigment beneath the surface of the animal hemisphere (Wittek, ’52). This pigment soon spreads somewhat below the egg equator, shading in the vegetal hemisphere into a creamy white, thus giving the Frog ovary its speckled appearance. The ovum has meantime been acquiring two membranes. The inner membrane is an extremely delicate and close-fitting envelope secreted by the egg itself. It is therefore a true vitelline membrane, but is so thin that its actual existence is denied by some investigators. The outer covering is thin, but tough, and is formed by the follicle. Hence it is a secondary membrane or chorion.

While the ovum has been growing and acquiring its membranes, the nucleus has been passing through the stages preliminary to the first maturation division. In the female Frog these stages vary somewhat from what has been described as typical. The chief difference consists in the fact that after synizesis (Fig. 58, A), the chromatin threads are less visible so that when the heterochromosomes for -the first maturation division later appear they seem to come from the chromatin nucleoli, but this is unlikely. They probably arise as usual from chromonema threads. (Fig. 58, C, D, E, F).

Before these chromosomes actually form, however, certain other events occur, as follows: The nucleus moves quite close to the animal pole, and the latter becomes slightly flattened. It is also claimed by some that the pigment of this pole withdraws to a certain extent just above the nucleus to form a small light area termed the fovea. The writer has never observed this in normal freshly spawned eggs, but this does not preclude its existence in eggs at the proper stage within the ovary or oviduct. Porter (’39) notes the existence of a small white spot at the 110 THE FROG: THROUGH GASTRULATION

animal pole, with a dark dot within it marking the location of the second maturation spindle. This, however, was in eggs outside the ovary, and he makes no reference to the term “ fovea.” Likewise Rugh and others have noted that a fading of pigment occurs at the animal pole of aging eggs, but this again is in eggs outside the ovary, and probably not in a normal condition. The fovea as originally described therefore is, if it exists, apparently a separate phenomenon. The egg has now reached a diameter of from 1.5 to 3 mm., depending upon the species of Frog, and is ready for ovulation.

As noted, the series of processes leading to this result have taken place during the summer, and are virtually completed before the time of hibernating arrives. The eggs then normally remain in this condition until the period of spawning in the following spring.

OVULATION TO F ERTILIZATION

Ovulation.—When spring arrives the ova are released from the ovary by the process known as ovulation. It was originally thought that the embrace of the male Frog known as amplexus, which occurs throughout spawning, was a necessary stimulus for the ovulatory process. As Rugh (’37) has so ably shown, however, amplexus really has nothing to do with it. This investigator clearly demonstrated that ovulation is brought about by an increase in the secretion of one of the pituitary hormones. Thus by injecting a suliicient number of minced pituitary glands into the body of a female Frog, ovulation can be artificially produced at any time when the ovary contains ripe eggs. Pituitaries from female frogs are more eflective than those from males. However, any pituitary will probably do if properly prepared. The production of ovulation. by this technique has been a great boon to Frog embryologists, since it is now possible to obtain fertiliiable eggs at least nine months out of the year._ The process of ovulation itself may be described as follows: The ovarian follicle breaks, and the ripe ovum is forced out through the epithelial covering of the ovary into the coelom. No matter in what region of the body cavity this act may occur, ciliary action on the peritoneum serves to convey the egg to the mouth or infundibulum of the oviduct. This is also ciliated and the ovum is drawn into the duct.

The First Maturation Division.-—-Before following the progress of the egg further it will be necessary to return for a moment to processes occurring within it. '

At about the time of ovulation the nuclear membrane dissolves, and OVULATION TO FERTILIZATION A 111

shortly afterward the chromosomes of the first maturation figure arise from the nucleoli, as indicated above. As this figure forms, another peculiarity of maturation in the female Frog becomes evident, for neither centrioles, centrosomes nor asters are visible. Out of the fibrillar protoplasm, however, a spindle develops, division of the chromosomes occurs, and the first polar body is pinched off while the egg is in the upper part of the oviduct. This body lies just beneath the chorionic membrane. Immediately following this the spindle for the second division develops, and the division proceeds to the metaphase. In this stage it remains until after fertilization.

The Tertiary Egg Coverings.—As the egg passes down the oviduct from the infundibulum to the uterus the walls of the duct secrete about

it three or four layers of 3]. Fig. 59.—Egg of Frog a short time after

. . . laying and fertilization, showing the swollen bummous mammal whlch c°n' egg membranes. From Ziegler (Lehrbuch,

stitute a tertiary covering. etc-ls after 0- 5°h“1tZe . b. Th h ' bl 1 These layers are hardly d1s- m’é’ume r.fenfhf§£ZT"p."'§i‘;§';Znt§dppiiefii

finct as Such at this time, but tion path of the spermatozoon. r. Polar bod . ' 1‘ ' ‘ ' Hi .1 2 ‘ as mu appear below they be :§§d.¥1’;‘i;3 §:i:¥“§u.?.if.§i.“::...;...1.f;,i§:‘§

come so after contact with the layers of “jelly.” water.

Spawning. ——Within about two hours after entering the infundibulum the egg reaches the uterus where it may remain for a day or two until this portion of the duct is full. The accumulated mass of ova are then expelled into the water, and in the common American Wood Frog a single such act of expulsion usually completes the process of spawning. In some varieties of Frog, however, the expulsive act is followed by another accumulation of eggs, and the spawning period is thus prolonged. Hence, though in American Frogs its duration is usually not more than a few days, in some,European species it may continue for over a week, the process in any case being retarded by cold. As already noted the male remains in amplexus throughout this time, although in those instances where repeated expulsions are the rule, the actual extru112 THE FROG: THROUGH GASTRULATION

sion of eggs generally occurs only in the early mornings of successive days. In this way he is always in a position to discharge sperm over the ova as they emerge. Furthermore, although this act of amplexus has been shown to have nothing whatever to do with ovulation, it is now clear, as intimated above, that it does afford the stimulation for spawning. Without it “ stripping ” of the female is necessary in order to press the accumulated eggs out of her uteri. The total number of eggs spawned in a season varies in different species of Frogs and in different individuals. Thus in Rana Lemporaria it runs from 1000 to 2000, while in Rana esculenm it may be anywhere from 5000 to 10,000.

It is of some interest in this connection to note the factor which is the stimulus for amplexus on the part of the male. It might be assumed to be the presence of the female, or at least of a female with eggs in her uteri. Such, however, is not the case. As again clearly shown by Rugh (’37) this action. on the part of the male Frog is, -like ovulation in the female, entirely conditioned by a secretion of the anterior pituitary. Indeed not only does the secretion of his pituitary cause him to go into amplexus with a female Frog, or any other convenient object, but it also brings about the release of ripe sperm from the Sertoli cells of his testis. Without an adequate increase in this hormone on the other hand, the male shows no interest in a female even though her uteri may be filled with eggs.

The Effect of Water on the Tertiary Membrane. ——After spawning the membrane indicated above of course comes in contact with the water, and by absorbing it, begins immediately to swell. This action progresses rather rapidly at first, so that within two or three minutes the jelly-like covering has increased from one sixth the diameter of the egg to about one half that diameter. In fifteen minutes it generally equals the egg diameter: thereafter the swelling becomes slower. At this point, if fertilization has not occurred the absorption of water by the jelly is said almost to cease. If fertilization has taken place, however, the swelling process may continue for several hours until the thickness of the jelly is as much as twice the width of the ovum.

This thickening reveals more clearly the three or four layers of which the jelly membrane is really composed. The innermost is a thin dense stratum applied closely to the chorion, and sometimes erroneously referred to as the chorion itself. Next comes a rather thick and watery layer, and finally one which is both thick and firm. When a fourth is present it is thin and fibrous; it does not occur outside, butijust beneath the thick firm layer which is always outermost. FERTILIZATION 113

Although some species of Frogs have elaborate habits connected with the care of the eggs, the common Frog does not. When fertilized, the eggs are simply deposited and left to their fate. On this account the thick envelope of jelly which they possess appears to exercise several important functions. In the first place it serves to attach them to each other and to debris, so that they are not readily washed abo.ut. It protects them from mechanical injury, and also appears to be distasteful to water snails and perhaps other animals.

In addition to these functions it has long been claimed that the jelly serves as a lens to concentrate the rays of the sun upon the eggs, and thus to raise their temperature. This it was assumed would be of advantage because it would speed up the otherwise slow development in the cold water of early spring. This particular claim and assumption, however, is an excellent example of the way in which an untested assertion which seems superficially reasonable, may become widely accepted, and yet be entirely without foundation in fact. "Thus to begin with, Hugh (’33) showed that temperatures a little too high will injure the eggs, and we know from other sources (see below) that such temperatures upset the sex ratio. Hence it would appear probable that the risk accompanying such an effect as suggested would more than overbalance any possible advantage. Bethat as it may, Rugh has further shown that the water in which the eggs occur, plus the jelly, which is about 78 percent water, filters out most of -the radiant energy of a heat-producing character. Consequently the light which the eggs receive, even though it is absorbed by the black pigment on their surface, produces relatively little heat. Lastly Cornman and Crier (’4-1) have demonstrated very conclusively that even if there were any heat. in the light passing through the jelly, the latter totally lacks the effect of a lens. Indeed its refractive index is about that of the water in which it occurs, and hence with the curvatures involved would bring the light to a focus far beyond the egg. Thus it would appear that far from raising the tempera ture of Frog eggs the jelly may even act as an insulator to keep them from getting too warm.

FERTILIZATION AND EGG SYMMETRY

FERTILIZATION

The Penetration of the Sperm. —— As the eggs are extruded by the female, the male Frog immediately discharges over them the seminal fluid. This fluid contains thousands of spermatozoa, and hence the eggs 114 THE FROG: THROUGH GASTRULATION

tend to be surrounded by them. Many of these pierce the outer jelly, I

but usually one of them is slightly in advance of its fellows and thus arrives first at the surface of the egg itself. As soon as it has started to enter some change is effected in the egg so that the remaining sperm are unable to pass beyond the jelly. Polyspermy is thus abnormal in the Frog and when it occurs the course of development is interfered with.

The entrance of the sperm always occurs in the animal hemisphere of the egg, and usually, according to some authorities, about 40° from the pole. Aside from these limitations, however, there is apparently nothing which fixes the point of penetration; that is, this point may be located on any one of the infinite number of meridians which may be imagined to pass from one pole of the egg to the other.

The Perivitelline Space.——The penetration of the‘ ovum by the sperm seems to cause the egg to give up a certain amount of its fluid. In any case, whatever its source, fluid does collect at this time between the chorion and the surface of the ovum. It is indeed presumably inside the vitelline membrane if the latter exists, and hence the space containing this fluid is as usual termed the perivitelline space. Its formation releases the egg from the grip of its coverings so that it is free to rotate within them. Under these conditions if the lighter animal pole is not already uppermost it presently becomes so. _

The Entrance Path.—— In the case of the Frog the whole spermatozoan enters the ovum, and it usually requires a minute or two for it to get entirely inside. The tail then disintegrates, and the head and middle piece travel steadily along a path which is generally approximately a radius of the egg, leaving a trail of pigment behind them (_ Fig. 60, A). This is the penetration or entrance path, and as the head and middle piece move along it, the usual rotation of these parts occurs, thus placing the latter structure in the lead. At the same time the head is enlarging to form a typical nucleus.

The Second Maturation Division.—Meanwhile the stimulus of the entrance of the sperm has incited the completion of the second maturation division of the egg nucleus which had paused in the metaphase. After throwing off the second polar body, the egg nucleus withdraws from the surface of the ovum, usually to a position in the egg axis. The sperm nucleus then proceeds toward it.

The Copulation Path and the Fusion of the Egg and Sperm Nuclei.—-—As suggested under the general topic of fertilization, the course followed by the sperm immediately after its penetration of the egg (i.e., the entrance path) may not be directed exactly toward the egg . SYMMETRY or THE OVUM 115

nucleus. In those instances where it is not, therefore, the point where the sperm does start to move directly toward this nucleus is marked by a slight change in its course. The second portion of the sperm path which thus arises, as has already been noted, is then called the copulation path, and like the first portion, in the case of the Frog, it is marked by a trail of pigment (Fig. 60, A).

Proceeding along this second path the sperm nucleus presently meets that of the ovum. Meanwhile the middle piece has initiated the formation of a division-center and aster, and before the meeting of the pronuclei occurs this new center and its aster have divided into two. The division has taken place at right angles to the copulation path, and hence as the nuclei come together the axis joining the division-centers coincides with their plane of union (Fig. 60, A, B).

THE SYMMETRY OF THE OVUM AND ITS SIGNIFICANCE

The causes which determine the symmetry of any ovum and the relation which this symmetry bears to cleavage and to the symmetry of the embryo are subjects of fundamental importance for the understanding of development. They have therefore received considerable attention in different groups of animals, and among Vertebrates the Frog’s egg has seemed particularly well adapted for such study. Hence it appears desirable in the case of this animal to make some mention of the results to which this study has led. It must be noted, however, that in spite of the work which has been done, there still exists some disagreement as to the exact facts, at least as regards certain details. In the interest of clearness, therefore, it seems best merely to state the main features of this phase of development in the Frog according to one view, the accounts followed being chiefly those of Roux and J enlcinson.

The First Plane of Symmetry.———Before the egg is fertilized it is radially symmetrical about an axis passing through its poles. The penetration of its surface by the sperm, however, confers upon it a bilateral symmetry. That is to say, the point of this penetration, together with the polar axis, determines a plane which, save for the possible eccentricity of the egg nucleus, divides the ovum into equal halves. It may be termed, therefore, the sperm entrance point plane (Fig. 60, A). The existence of this plane of symmetry,'determined solely by the egg axis and the sperm entrance point, however, is brief. Other factors presently enter which determine a second plane, often, though not necessarily, closely correlated with the first (see below), and developed in the following manner: ‘ 116 THE FROG: THROUGH GASTRULATION

also cleavage plane sperm entrance l point.

sperm entrance deavagg P°”“ \. / plane

entrance










entrance

path _ copulation

path COP’; pat mitotic spindle 3'3Y crescent A W .n& embryonic crescent axis b d . _ C '3 / entrance point plane em ryonic axis an entrancexpom p ne 5 erm entrance sperm entrance P point

entrance

cleavage path

plane‘ _

8'3)’ crescent

\

cleavage plane



gray

C"°5'5€""v embryonic

entrance point plane axis embryonic axis and entrance

point plane, also cleavage plane

Fig. 60. —— Diagram to illustrate some of the possible relations of the axes in a fertilized Frog egg. In all cases the egg is assumed to be viewed from the animal pole. The arrow indicates the longitudinal axis oi the future embryo, with the head pointing anteriorly. The dash line indicates the first cleavage plane where the latter does not coincide with the longitudinal axis of the future embryo. The dotted line indicates the entrance point plane where this does not coincide with the longitudinal axis of the future embryo. The dot in the center indicates the center of the animal pole.

A. An egg in which the entrance point plane, the entrance and copulation paths, the gray crescent plane, the first cleavage plane and the longitudinal axes of the future embryo all coincide. B. An egg in which the entrance path and the copuIa~ tion path are not in the same straight line. Hence the gray crescent plane and the longitudinal axis of the future embryo fail to coincide with either the entrance point plane or the first cleavage plane. C. An egg distorted by pressure. Notethe consequent orientation of the mitotic spindle as explained in text. This prevents coincidence of the first cleavage plane with any of the others. D. The same situation with the added complication due to the fact that as in B the entrance path and copulation path are not in the same straight line. Note that in all instances the gray crescent plane and that of the longitudinal axis of the future embryo coincide. EGG SYMMETRY OF THE OVUM

Fig. 61.—CIeavage stages and the beginning of gastrulation in the Frog’s egg (Rana pipiens). The shading in this figure indicates the distribution of pigment, except along the lines of cleavage, where as usual it denotes shadow. A. Fertilized egg viewed from the left side in terms of the future embryo. Note the left half of the gray crescent at the right side of the figure, i.e.. its dorsal side in terms of the embryo. B. An egg in which the first cleavage has been almost completed. Since the egg is again being viewed nearly from its left side in terms of the future embryo, the cleavage furrow is virtually in the plane of the paper and scarcely shows. The gray crescent is again to the right as in A, but at this stage the region of the crescent has evidently become whitened and so added to the original light area of the vegetal pole. C. An incomplete four-cell stage, also viewed from the left side. The second furrow has not quite reached the vegetal pole. D. A view of C from the animal pole, with the region of the gray crescent toward the right (dorsal). E. An eight-cell stage. The animal pole is again at the top of the page, and the vegetal pole at the bottom, but the future dorsal region is turned slightly toward the observer, thus showing art of the first furrow. F. An approximate sixteen-cell stage directly mm the left side. The cleavage is obviously somewhat irregular. G. Between a 64- and 128-cell stage viewed from the left side. H. A virtually complete blastula‘ from the left side. Note that the pigmented area is tending to move downward somewhat. I. An early gastrula from the left side. The cells in the animal hemisphere are too small and numerous to indicate separately. The beginning of the blastopore lip is visible as a slight notch in the lower right side of the figure.

117 118 THE FROG: THROUGH GASTRULATION

The Second Plane of Symrnetry'.——As the sperm travels along the first part of its path within the egg, it seems to cause certain disturbances in the egg substance. The result is a more thorough separation between yolk and cytoplasm, and an apparent streaming of the latter in the direction of the sperm. This flow seems to cause a withdrawal of pigment granules from along the border of the pigmented animal hemisphere on the side of the egg from which the How is taking place, i.e., the side approximately opposite to that upon which the sperm entered. The result is the appearance upon that portion of the pigmented border of a lighter strip termed the gray crescent. This crescent is usually quite clear shortly after fertilization and during the first few cleavages. After a little time, however, its outlines become less distinct. Hence its existence is soon detectable only by the fact that the light area extends somewhat higher up on the side of the egg where a definite crescent originally occurred (Fig. 61). The new plane of symmetry, therefore, is one which again passes through the egg axis and also bisects the gray crescent, or the increased area of white which replaces it. It may be called the second or gray crescent plane, and by virtue of its method of formation it will evidently have a decided tendency, as suggested above, to-coincide with that of the sperm entrance point (i Fig. 60, A ). That this is a tendency rather than an inevitable condition, however is due to the following considerations:

It will be recalled that the path of a sperm toward the egg nucleus is not necessarily a straight one. Presumably because of failure to enter at quite he correct angle, the sperm may not at first be headed in the right direction, and hence has to alter its course, thus producing the initial or entrance path and the later copulation path. But, as suggested above, ithtprns put phat tlhe infiuednce of the spelrm in causing the pigment wit rawa is arrve y exerte as it asses a on the entrance ath. Therefore, if the entranoce path does not happen to Ii: in a vertical rilane coinciding with the poles and a radius of the ear , it follows that in such cases the entrance point plane and the gray crgsghent plane also will not coincide (Fig. 60, B).

The Cleavage Plane. —Following the union of the egg and sperm a third plane makes its appearance, i.e., that of the first cleavage. This incidentally is of course an actual plane, not merely a hypothetical one determined by three points. Under normal conditions this plane passes approximately through the animal and vegetal poles, a condition resulting from the following facts:

In accordance with a generalization known as Hertwig’s law the

, .-.,___,.,_._. ,, ........ SYMMETRY OF THE OVUM 119

mitotic spindle always tends to lie so that its longitudinal axis coincides with that of the yolk-free cytoplasm of the cell. Now in the Frog egg this yolk-free cytoplasm ordinarily occupies about the upper third"of the animal hemisphere, and hence has approximately the form of a rather thick plano-convex lens. Therefore the long axis of the spindle may fulfill Hertwig’s law by lying in any direction so long as it is parallel to the fiat surface of the lens-shaped disc of cytoplasm. This will of course also make it at right angles to the polar axis of the egg. Furthermore, since the egg nucleus is in this axis the movement of the sperm and spindle to that nucleus will presently cause the middle of the spindle to coincide with the eggs polar axis. Finally because the plane of cleavage is perpendicular to the length of the spindle at its middle, this plane will also coincide with the eggs polar axis and so pass through its poles (Fig. 60, A, B).

Though Hertwig’s law thus determines that the first cleavage must pass through the egg poles, this law does not determine with which of the infinite number of imaginary radii emanating from the polar axis the cleavage must coincide. There is another consideration, however, which does determine the radial direction of this cleavage. The sperm division center, it will be recalled, divides so as to cause a new mitotic spindle to form at right angles to the copulation path. Hence the cleavage plane should coincide with this path, as well as pass through the poles of the ‘egg. Under most circumstances these are the only factors involved, and such coincidence occurs (Fig. 60, A, B). It should be noted, however, that pressure on the egg perpendicular to its polar axis may distort the lens-shaped disc of cytoplasm so that its periphery is no longer circular. Under such conditions the mitotic spindle, in accordance with Hertwig’s law, will be displaced so that the cleavage plane may not be related to any other (Fig. 60, C, D).

The Plane of Embryonic Symmetry. —This plane is of course the one which divides the future embryo into equal right and left halves. In the Frog it always coincides with the gray crescent plane (Fig. 60), i.e., except when the latter fails to exist (see below}. This coincidence‘ results from the fact that normally the dorsal blastoporal lip develops at the middle of the lower border of the crescent. On this basis one might assume that the median plane is determined by the gray crescent, the latter having been in turn determined by the entrance path of the sperm. Indeed this has been quite generally regarded as true. As parenthetically suggested above, however, it must now be stated that the existence of a gray crescent is not inevitable. Thus the writer has ob120 THE FROG: THROUGH GASTRULATION

served fertilized eggs in which the pigment merely tapered 01? in streamers more or less equally distributed on every side. Yet many of these eggs appeared to develop quite normally. It should be added that these were eggs which had been obtained by stripping pituitary injected females, and which had then been artificially inseminated. Whether this lack of a gray crescent ever occurs in eggs normally produced the author cannot say, but it seems not unlikely that it does. Indeed this seems highly probable in view of the fact that in some Amphibian eggs there is no pigment from which a crescent can be formed, and yet needless to say, these eggs develop an embryonic symmetry.

_In view of these facts, then, the question arises as to what if any relation the gray crescent, when it exists, really does have to embryonic symmetry, since, under some circumstances, the latter can quite evi dently develop without it. The most probable explanation of the situa--_

tion seems to be this: The passage of the sperm along the entrance path causes a rearrangement of materials within the egg with a certain reference to this path. Of this there seems little doubt. Normally, moreover, this rearrangement involves the withdrawal of superficial pigment in the eggs of those Amphibians which possess it, and thus produces the gray crescent. However, the two phenomena, i.e., withdrawal of pigment and‘ rearrangement of internal ‘materials, are not inevitably connected, and it is the latter which is fundamentally significant: Hence it would appear that the entrance path of the sperm is the initially determining factor of embryonic symmetry in fertilized Amphibian eggs. What this factor may be in eggs artificially stimulated to parthenogenesis is at present unknown. Also, what may happen to the initially determined symmetry in eggs later abnormally oriented remains to be stated (see below). Relationship of the Various Planes Summarized. ———There have now been defined ‘four planes, the sperm entrance point plane, the gray

‘crescent plane, the first cleavage plane, and the plane of embryonic

symmetry. Of these four the one most frequently out of line with the others is that of the sperm entrance point. This is because, as shown, the other planes are all related in one way or another to the path, or paths of the sperm, and not essentially to its point of entrance. Thus the gray crescent plane is determined by the entrance path. The cleavage plane in turn is fixed by the copulation path in conjunction with the shape of the yolk-free cytoplasm and its relation to the egg poles. The plane of embryonic symmetry normally coincides with that of the gray crescent, but this is probably not a causal relationship. The really fundamental determiner of embryonic symmetry under normal conditions is CONCLUSIONS FROM EXPERIMENTS

121

probably the path of sperm entrance. In conclusion it may be stated that there will be a considerable tendency for all four planes to coin cide (Fig. 60, A).

CONCLUSIONS DERIVED FROM EXPERIMENTS

It is of interest in connection with the question of the relation of embryonic symmetry to the cleavage and gray crescent planes to note the

results of certain experiments which have been performed upon the two cell stage of the Frog and other Amphibians. It is not possible to kill or re ._ move one blastomere of the egg of the common

Frog without killing the other. It has been found, however, that if a hot needle is thrust into one of the cells, this cell though not dead will fail to divide. Under these circumstances it was long ago discovered by Roux (’88), Morgan (’O2, ’O4), Hertwig (’93) , and others, that when this is done to eggs in which the first cleavage plane has passed through the middle of the gray crescent, the uninjured cell may eontinue to develop. Under these conditions it


Fig. 62.——A half embryo of the Frog produced by thrusting a hot needle into one of the first

two blastomeres. After Roux.

then generally produces approximately the lateral half of an embryo, with the undeveloped hemisphere of material comprising the other blastomere adhering to it (Fig. 62). Another investigator, McClendon (’10), then found that in the case of the tree frog, Chorophilus, it is possible by the proper technique to remove one of the first two blastomeres without injuring the other. When this was done it was discovered that the remaining cell developed not into a half embryo as in the preceding experiment, but into a whole one. Taken together these results might reasonably be interpreted to mean that the failure


Fig 63_ _ Two to develop a whole embryo in the first case was due Frqg €mb1'Y0S simply to the inhibiting presence of the inert blasteEglétfid proléfgéd 12;). mere, and indeed McClendon himself did reach this inverting the tW0- conclusion. Other facts exist, however, which render gfilultifge‘ Aim another interpretation more probable. They are as

follows: It was discovered by Schultze (’94) that if the egg of the Frog is exactly inverted following the first cleavage, andheld in this position, each blastomere will give rise to approximately a whole embryo, the 122 THE FROG: THROUGH GASTRULATION

two animals being united, however, in various degrees after the manner of Siamese twins (Fig. 63). This interesting result was supplemented by an experiment by Morgan (’95) in which he inverted the two cell stage after the manner of Schultze, but in addition inyured one of the blastomeres. Under these conditions the remaining blastomere instead of developing into a half embryo as in the first experiment, formed a virtually whole one, despite the presence of the injured hemisphere. The latter, therefore, cannot be the cause of the half embryos. More detailed observation of what takes place in the inverted cells, however, seems to furnish a possible explanation of the results in all the above cases.

It has been noted by several observers that when the eggs are inverted the contents of the cell or cells becomes rearranged in response to gravity. Thus the materials of the gray crescent can sometimes be seen to become separated into two parts. At the same time the lighter yolk free cytoplasm comes to what is now the top (the former vegetal pole), and the heavier yolk sinks to the former animal pole. With such profound changes going on there is every reason to believe that the critical materials concerned with embryonic symmetry are also rearranged, and probably divided. If this is so it might be expected that with their division two embryos would develop, as in fact they do. As regards McClendon’s isolated, but uninverted blastomeres, it must of course be supposed, according to this hypothesis that a similar reorientation takes place, though in these cases it must presumably occur either as a result of the manipulation of the eggs, or on account of the change in shape of the isolated cells. ’

CLEAVAGE, GASTRULATION, AND THE FORMATION OF MESODERM, NOTOCHORD, AND MEDULLARY PLATE

It has already been suggested that in the Frog the character of the processes indicated is transitional; it serves to bridge the gap between the activities observed in the development of Amphioxus and those in some of the forms which are to follow. Not only is this true, but the character of the Frog’s egg as regards its yolk content is also transitional. The egg of Amphioxus was telolecithal, but the amount of yolk was relatively slight. The egg of the Frog is telolecithal, but the amount of yolk is much greater. Finally, as will be seen, this condition is carried to its extreme in the Fish and Bird. As our study of these forms proceeds it will become increasingly apparent that this parallel ism between the character of early development and the yolk content is CLEAVAGE . 123

not a coincidence. Rather, as intimated in the first chapter, the latter very largely determines the former. The student then should keep this clearly in mind in attempting to understand the stages which follow as compared with corresponding stages in Amphioxus. V

CLEAVAGE

The Early Stages. ——In spite of the larger amount of yolk in the Frog’s egg, segmentation is still holoblastic. Following the second cleavage, however, it is less nearly equal than in Amphioxus (Fig. 61).

As has been stated the first division plane normally passes through the <

poles of the egg, and is thus perpendicular to the egg equator, and vertical if the egg is normally oriented. This means that it divides the ovum into parts which are at least quantitatively similar. The particular meridian cut by the division is determined by factors noted above. The furrow which marks the beginning of this cleavage appears on the upper surface of the ovum about two and one half hours after fertilization and within an hour has extended around to the ventral pole. By the time it has reached this pole, the internal substance of the egg is also divided.

A period of “ rest ” ensues, and then, about three quarters of an hour after the appearance of the first divi.sion, the_furrows of the second become evident. This cleavage is also vertical and at right angles to the first. The furrow in each of the two hemispheres again begins approximately at the animal pole, often exactly so. When the latter is the case the upper ends of these furrows will evidently lie opposite each other and form a continuous line across the pole (Fig. 61, D).

Following the completion of the second cleavage, the third soon starts. It is horizontal, and in each of the four cells it lies about 60° below the animal pole. Hence its furrows form a virtually continuous line around the egg a little above the equator. This is the typical or at least the ideal condition (Fig. 61, E). There-are, however, not infrequent variations.

The furrows of the fourth cleavage are in general vertical, and tend ideally to meet one another at the poles. This tendency, however, is seldom perfectly realized, even in the animal hemisphere. Thus in the latter half, the lines of division usually pass either to one side or the other of the polar center, while in the vegetal hemisphere this and other irregularities are even more marked. The ideal result, however, is sixteen cells, eight relatively small pigmented ones above, and eight larger whitish ones below (Fig. 61, F).

The fifth cleavage, resulting in the formation of thirty-two cells, is 124 THE moo: THROUGH GASTRULATION

still more variable than the fourth. There is a tendency, however, for the furrows to be horizontal, and to form four tiers of eight cells each. In the most regular instances the cells of the two upper tiers are about equal, and are all pigmented. The cells of the third tier are about mid Fig. 64.—-Median vertical sections of four cleavage stages in the Frog’s egg. A. An eight-cell stage. Note the small segmentation cavity or blastocoel. B. A later stage (about 32 cells) which may be called an early blastula. C. A later blastula. D. A still later blastula, showing marked increase in size of segmentation cavity.

way in size between those above and those below them. They are ap proximately on the equator, and contain less pigment than the two upper tiers. The lowest tier is formed of the largest cells, which are mostly without pigment.

The Blastula. — By the time the thirty-two-cell stage is reached it is hardly possible longer to refer to this dividing sphere as an egg. It may now, therefore, be termed the blastula. Within this blastula is the blastocoel or segmentation cavity, which arises as follows: CLEAVAGE 125

From the first the cells into which the ovum has been divided are

pressed rather closely against one another so that their surfaces of contact are flattened. This, it will be recalled, is contrary to the rounded con dition of the very early cleavage cells of Amphioxus. Even in the Frog, however, the inner ends of the cells show some curvature, and by about the eight to the sixteen cell stage these inner ends are sufficiently rounded

so that they are no longer in contact. Thus is produced the blastocoel, which, because of the smaller size of the cells at the animal pole, is

somewhat above the equator of the blastula (Fig. 64). Also up to the beginning of gastrulation the blastocoel is gradually increasing in size, due partly perhaps to the closer packing of the cells, to the secretion of albuminous fluid from them, and to the infiltration of water from without (Fig. 64, A, B). The latter two factors are probably the more important.

Besides this increase in size of the blastocoel, cleavage following the thirty-two cell stage becomes quite irregular, and cells begin to be split off internally. At the same time the relatively yolk-free cells of the animal hemisphere begin to divide _much faster than those of the vegetal hemisphere, and some of the smaller ones tend to migrate toward the equator, thus making the roof of the blastocoel thinner. Regarding the matter of the cleavage rate in general, an interesting fact has been noted by Ting, ’51. He found by crossing different species of Frogs, using both normal and enucleated eggs, that the rate of division up to the time of gastrulation is determined entirely by the egg cytoplasm, whose character was presumably previously determined by maternal genes.

Finally, at what may be termed the end of the blastula period, the following conditions obtain: First the blastula is about one fifth larger than the original egg, the increase in size being mainly due no doubt to the absorption of water noted above. Secondly, the superficial pigment has everywhere extended downward somewhat, thus decreasing

the white area (Fig. 61, H). This extension having been approximately I

uniform, however, the latter region still reaches farther upward upon the side where it was originally augmented by the addition of the gray crescent. Thirdly, sections reveal the fact that on the side opposite to that which was marked by the gray crescent, the wall of the blastocoel is usually slightly thicker than it is elsewhere (Fig. 67, A). Lastly, it may be noted that a split has occurred in the roof of the segmentation cavity, so that this wall is composed of two sheets. The outer is the epi dermal layer; the inner is called the nervous layer because parts of it '

help form the nervous system. 126 THE FROG: THROUGH GASTRULATION


Fig. 65. —Diagrams of the closure of the blastopore in the egg of the common Frog (R. tenzportzria). From Jenkinson (Vertebrate Embryology). In A—E the egg is viewed from the vegetal pole, and in F, speaking in terms of the future embryo, from its ventral side. The dorsal lip is at the top of the figures. In D the ventral lip has just been formed and the blastopore is circular.

.In E the rotation of the whole egg has begun, and in F is complete.

GASTRULATION

External Processes.—Upon the side of the blastula where the white area was increased by the addition of the region of the gray crescent, it has been noted that the pigment is still not quite so far down as upon the side opposite. Nevertheless, even at the former point the pigment extends markedly below the equator, the line between the light GASTRULATION 127

and dark zones being everywhere marked by an area of intermediate shading. It is then midway between the ends of the former crescent region, and toward the lighter and lower side of the shaded area in-this region that the dorsal blastoporal lip first appears. It is thus probably located at approximately the lower border of the original crescent, though the exact relation is difiicult to determine because of changes in pigmentation during cleavage. It is also somewhat below the level of the floor of the hlastocoel. This lip has the appearance at first of a small dent, which soon elongates into a groove following roughly the border of the pigmented area (Fig. 61, I; Fig. 65, A, B; Figs. 67, B and 68, B).

As the process of elongation continues it is accompanied externally by two phenomena. In the first place the groove gradually extends around either side of the gastrula, and as it does so the pigment advances to its edge, i.e., to the lip of the blastopore. This lip thus comes to constitute a sharp boundary between the dark and light areas (Fig. 65, A, B, C). In the second place the blastoporal lip everywhere moves steadily nearer to the vegetal pole. This movement is greatest on the side where the groove first appeared, i.e., at the dorsal lip, and becomes progressively less toward either side. The first process, i.e., that of lateral extension, causes the groove to become curved so that it has the shape of a crescent, and eventually the horns of this crescent meet each other so as to form a complete circle. A continuation of the second process, i.e., the downgrowth of the lip, and hence also of the pigmented area, then results in a rapid diminution of the white region. Thus the latter is soon in the form of a circular spot which is being encroached upon from all directions (Fig. 65, D, E, F).

Epiboly.-——The white region evidently occupies the position of the blastopore. The first appearance of the groove marks the beginning of overgrowth by the dorsal blastoporal lip, while the lateral extensions of this groove indicate the same process on the part of the lateral lips. Finally, as already noted, the ends of the grooves meet one another on the future postero—ventral side of the gastrula, and thus show that there also a slight downgrowth is taking place. This overgrowth of the yolk, or epiboly, by the cells of the blastocoel roof necessarily involves the use of material which can only be supplied by a thinning of this roof due to a rearrangement of its cells (Fig. 67). _

C0n11ergenc.e.——-In correlation with epiboly certain other processes are also occurring, for an understanding of some of which more than mere external observations are required. There is one other, however, 128 THE FROG: THROUGH GASTRULATION

which can also be studied from the outside. Such a study has proven especially fruitful in the case of some of the tailed Amphibians, like Triton, in which the’ egg is relatively colorless. In these animals it is thus possible to put stains upon the outside of the blastula, and so to observe what movements occur there during the ensuing gastrulation. This was done by Vogt (’22, ’25, ’26) and Goerttler (’25) who placed pieces of agar saturated with stains upon the egg membranes. The stains penetrated the membranes and colored the cream tinted surface of the late blastula. The results are depicted in Figure 70, A, B, C, D. From these it appears that there is a streaming of the materials of the dorsal and lateral surfaces of the ea-rly gastrula toward the blastopore. At the same time, as is especially indicated by the later stages (C and D of Fig. 70), there is a shifting of the lateral regions toward the midline. It is this combined type of novement which is now generally described as convergence, and though it has some aspects of the old alleged concrescence it is obviously not the same thing. Thus it is evident that in this case, as in Amphioxus, the lips of the blastopore do not actually constitute the sides of the embryo, or even furnish much of the material for it. However, a good deal of this material does as usual pass over the lips, and for this, and perhaps merely historical reasons, they are sometimes referred to in this animal as the germ ring.

More recently Schectman (’-4-2), Holtfreter (’4-3) and others have made further studies of the movements thus described in an effort to arrive at a more basic understanding of them. Schectman particularly stresses the idea that none of the regions undergoing the movements heretofore indicated act entirely independently. Each has certain autonomous capacities, such as the extension or self-stretching" capacity of the presumptive chordal region of the dorsal blastoporal lip. This region, however, lacks “invagination” (involution) capacity which is conferred oh it by the normally adjacent lateral lips. The combined movements resulting in these regions Schectman therefore calls “correlative.” Holtfreter has sought especially to reach physico-chemical explanations of the gastrulation phenomena. Thus he has suggested that an unfolding of denatured protein molecules is partly responsible. This unfolding, it is thought, causes a spreading of the superficial cells over a substrate with appropriate adsorption properties. The epiboly and perhaps the convergence are hence due to this spreading tendency, which is apparently augmented by a lowering of surfme tension in parts of the spreading cells. It will-be recalled that such a change in surface tension was also referred to in the general discussion of gastruGASTRULATION V 129

lation as a possible cause of involution and invagination. On the basis of these conclusions it is further suggested that all these cell movements may be essentially similar to the cell movements seen in wound healing and in phagocytosis. Additional study is of course needed either to disprove or to confirm and amplify these ideas.

Rotation. — Returning -to more obvious and directly observable matters, we are confronted with a very definite change in the position of the whole gastrula which accompanies the processes just described. The


Fig. 66.-Diagrams of the Frog’s gastruls showing the position of the blastopore at various ages. From Kellicott (Chordate Development). A. Posterior view. B. Lateral view. I-5 indicate the successive positions and forms of the blastepore. The change in position is due both to the actual growth

movements of the blastopore. and to the rotation of the entire gastrula.

movement of epiboly continues until the dorsal lip has passed over an arc somewhat greater than 90°, and the area of white, i.e., the blastepore, is reduced to a small circle. This area, therefore, will be situated rather beyond the original vegetal pole. It is now to be noted, however, that accompanying this downgrowth of the dorsal lip another and quite different movement has also been going on. The entire gastrula has been rotating about a horizontal axislying at right angles to the original median plane of the egg. That is the direction of rotation is such that the dorsal lip is in a sense carried backward in one direction as fast or faster than epiboly moves it forward in the other. The result is that at the completion of both processes the blastopore, formed at approximately the vegetal pole, is posterior, and the morphologically dorsal and ventral lips are actually dorsal and ventral (Fig. 66). From this it also follows that the original animal pole of the egg is to form the antero-ventral. side of the future embryo, while the region formerly marked by the gray crescent is to form the dorsal side.

As regards the events so far described it is evident that gastrulation mes. V.

Fig. 67.—Sagittal sections through Frog’s egg during formation and closure of hlastopore. From Jenkinson t.Vertebrate Enzbryology). A—D. Before rotation. E During rotation. F. After rotation. The arrow marks the egg-axis. its head the animal pole. arch. Archenteron. d.l. Dorsal lip. mes.v. Mesoderm originating at ventral lip (i.e., a very small part of that which is classed as peristomial t. mes.2. Mesoderm originating from the yolk cells pushed into segmentation cavity (i.e., gastral). s.c. Segmentation cavity. 1;.l. Ventral lip. y.p. Yolk plw.

130 “ I9-C

gin’

Fig. 68._—— Semi-diagrannnatir store-o,I__rrams of a l1exui.<e-t-ted Frog hlastula, A, and sttvcessive stages of hcmisected gastrulae, with small curving arrows indicating the directions of L't‘ll movements. Stages in alphabetical order.

.e\rr«_:w.»' an outer uncut surface of the gastrulae, to the right in each figure, show movement of material toward blastoporal lip. Involution of material over lip shown by arrow on cut surface of lip margin. Invagination, of a sort, shown by arrows on cut surface of yolk mass and floor of archenteron. Epibaly, evident from decrease in size of yolk plug. Delamination, in this case gastrular cleavage, shown by extent of splits bracketed under letters g.c. Ingression, being most questionable, not shown. but would be designated by arrows pointing directly from vegetal pole to floor of l)la$I0('0E’l in early stages. Animal pole marked by head of arrow outside each figure. Rotation of entire gastrula shown by changes in the positions of the poles in

E and F.

131 132 THE FROG: THROUGH GASTRULATION

in the Frog is not essentially dissimilar to the same process in Amphioxus. The main differences are due to the presence of the large yolk cells. Thus, to cite one instance, if these were absent the blastoporal lip would bound an opening just as in the former case. Here, however, this opening, i.e., the blastopore, is filled by these cells, which at this point are therefore termed the yolk-plug. As will presently appear, the phenomenon of rotation and various internal peculiarities are also due to the presence of so much inert nutrient material.

Internal Processes. -— While the above changes are apparent from the outside of the gastrula, sections through it at various stages will reveal important accompanying developments within. They are as follows:

Invagination. —- As the external processes of gastrulation begin, meridional sections of the blastula (or early gastrula) bisecting the future dorsal blastoporal lip reveal the fact that the floor of the blastocoel is beginning to move upward. Usually this movement begins on the dorsal side nearest the dorsal lip, and spreads part way around the margins of the blastocoel in company with the external extension of the lateral lips (Figs. 67, B; 68, B). Sometimes, however, the up-pushing is more central, and thus causes the blastocoel to become crescent shaped. In either case the movement is essentially one of invagination, albeit an invagination which is considerably hindered and modified by the mass of material to be moved. This mass of course is the yolk which occupied the vegetal half of the egg, and now occupies the relatively large and numerous vegetal cells. This modified invagina~ tion continues until the blastocoel cavity has been virtually eliminated, except for the narrow slit separating the outer layer of cells, now epi blast from the inner yolk-filled cells, now /zypoblast (Fig. 67; Fig. 68, C, D, E, F ) .2

2 It seems pertinent to mention at this point an observation made upon one of the tailed Amphibians by Schectman (’34l. This investigator stained the vegetal pole of a fertilized Triturus egg and found that by the midblastula stage the stain occupied cells some distance from the surface, and near to the floor of the blastocoel. He did not follow the material in later stages, and refers to its inward movement as “unipolar ingression.” This he properly enough indicates as occurring during “ blastulation ” (cleavage), and does not suggest that it has anything to do with gastrulation. However, he does note that it seems to be involved in the upward movement of the blastocoel floor, in this case at its middle, and this movement, it may be recalled, is one which we have designated as a part of modified imagination. Whcther, therefore, this movement in Triturus is really to be regarded as a kind of premature and greatly modified invagination, and hence a precocious aspect ofgastrulation, is a question for further study. At least it is a possibility to be borne in mind. Finally it may be added that the pfocess in question acquires additional GASTRULATION . 133

As has been suggested this internal process is going on simultaneously with the externally observable process of epiboly. As a result of both a new cavity is being formed which replaces the blastocoel. It is the archenteron, and is lined by hypoblast, a relatively thin layer forming the roof and the main mass of yolk-filled cells constituting the floor. ' ,

Involution.—~It now remains to point out that in addition to the processes so far described there is also a distinct process of involution. This is most active at the median dorsal lip and progressively less so as one passes around either side, until at the ventral lip there is almost none at all. The immediate cause of this movement, as well as of such invagination as occurs, is apparently a change in shape of the cells adjacent to the lips and in the yolk plug.

From this account, the roof and sides of the archenteric interior consist of material originally outside, dorsal and lateral to the blastoporal lip, while the floor is composed of cells originally on the outside of the vegetal region. The latter seem to have moved into their definitive position by an inpushing and inturning of the yolk cells called modified invagination (Fig. 68) . Any inwandering of individual vegetal cells (ingression) , as implied by Sc-hectman and others (see footnote) is denied by Ballard, ’55, who says that only the stain moves in, no cells.

Delamination.—In the general account of gastrulation in Chapter II, it will be recalled that the origin of endoderm by the process of splitting off was said to occur to a slight extent among the Amphibia. It should here be stated, however, that its occurrence is not universally admitted. Those who do describe it (Brachet, for instance) say it takes place in the following manner: _

Reference to the figures will indicate that, as the process of invagination begins, one of the results is as follows: As the yolk cells (hypoblast) about the margins of the blastocoel are pushed upward, they tend, as previously noted, to obliterate the portions of this cavity between themselves and the epiblast. The obliteration, however, is not quite complete, so that between the uprising hypoblast and the epiblast there remains a slight crevice. The upward extent of this crevice is then obviously increased by the continuance of the above processes. By those who maintain the existence of delamination, however, it is held that interest in the light of Peter’s observations on the inwandering of cells in the gastrulation of the Chick (see gastrulation in the Chick). In that case, however, the movement is into the blastocoel from a layer over it instead of from the yolk be neath it. Perhaps, however, in, view of the changed relationships in the Bird, due to excess yolk, this difference is not significant. 134 THE FROG: THROUGH GASTRULATION

besides this upward extension there is also a well marked downward extension. i.e., in the direction of the blastoporal lips. This appears to occur first, but least extensively, in the margin of the blastocoel nearest the dorsal lip, whence it presently extends entirely around the circumference and becomes most extensive toward the ventral lip. Here it apparently serves throughout a considerable region to separate the yolkfilled cells from the epiblast on the definitive ventral side of the gastrula. The significant point, however, is the fact that wherever the process takes place it is due apparently to a splitting apart or delamination of the cells at the bottom of the crevice (Fig. 68, go). But since at all points this crevice serves to separate epiblast from hypoblast, its downward extension in the manner indicated is obviously setting apart these layers by delamination. In this particular situation this separation has also been given the name of gastrular cleavage.

Summary of the Processes. —— To sum up the processes involved in the gastrulation of the Frog, it is found that there are four of them which also occurred in Amphioxus, i.e., epiboly, involution, invagination and convergence or confluence. In addition there seems to be some delamination which appears here for the first time. Though a common method for setting aside mesoderm and notochord, it is not so commonly thought of in connection with gastrulation. As we shall see, however, it is perhaps the only methodin Mammals, and possibly also in Birds.

In connection with these gastrulation processes it may finally be noted that there has been a considerable shifting of the yolk mass, and hence of the center of gravity. It is to these shiftings, apparently, that the rotation of the gastrula is due.

MESODERM, NOTOCHORD AND NEURAL PLATE

The Mesoderm and the Notochord. —— As the archenteron develops the layer which is invaginated, involuted, or delaminated to form its roof has been referred to as hypoblast. It now appears that this hypoblast contains the elements of a part of the endoderm, and all of the mesoderm, including the notochord. The ‘setting aside of these layers occurs as the result of a delamination from the hypoblast. The lower layer of cells thus split off forms the endoderm of the archenteric roof and sides. It is of course continuous ventrally with the yolk cells which l3€t;.0!1‘I.C the endoderm of the floor. The upper layer resulting from this split hes between the newly formed endoderm of the roof and sides and

the epiblast. This in between layerlis mesoderm, while the overlying epiblast may now be called ectoderm. MESODERM,_ NOTOCHORD, NEURAL PLATE 135

It should here be noted that the splitting off of the mesoderm does not occur everywhere simultaneously, but begins on either side and proceeds toward the median line. Here for a time a narrow strip of cells remains connected with the underlying layer. Presently it is separated both from the endoderm beneath and from the mesoderm on either side. It is the notochord (Fig. 69). The mesoderm of the ventral part of the embryo is formed later mainly by a downgrowth of the lateral sheets between the endodermal yolk mass and the ectoderm. Anteriorly it occurs not as a definite layer, but rather as loosely arranged cells, a ‘type of mesoderm generally referred to as mesenchyme.

Presently by the above means the mesoderm comes to exist throughout the greater part of the embryo, as a separate layer between ectoderm and endoderm. As noted,

it is interrupted dorsally Fig. 69.-—Three stages in the differentiation

~ of the roof of the archenteron in the Frog. by fife notochord’ whlle From Jenkinson (Vertebrate Embryology). anterlorly the cells are arch. Archenteron. n.ch. Notochord. mes. Dor very loosely arranged. 5&1 Mewderm‘

Lastly in the region of the blastopore there persists for a time an undifferentiated mass of cells containing the elements of all three layers. These gradually become defined, as the blastopore closes.

The Medullary or Neural Plate and Related Structures.—— It has already been noted that at the end of segmentation the epiblast of the animal hemisphere was split into an outer layer and an inner nervous layer. During gastrulation this becomes true also in the vegetal hemisphere. Thus toward the latter part of that process, a 35?»; of ectoderm exists everywhere except in the immediate,vi&iii;ity"of"tl1di§l‘astoporal lips. Throughout certain regions of the 'la the ner

( ;-t\r-h§‘=b’‘di } 
K J 

it ’ \-,_ _‘;/I -. "~..._......e' ’w._\5:£:h “§ 136 THE FROG: THROUGH GASTRULATION

ectodermal layer then begins to thicken, the thickening being defined’

as the medullary or neural plate. This plate extends forward from the dorsal blastoporal lips as a median band, widening rapidly as it approaches the anterior end of the gastrula. Here it terminates, the extremity having the form of a broad curve (Fig. 77, A).

The thickening process which has given rise to the plate presently grows most marked around its margins, and these become slightly elevated. The elevations which thus occur along the sides of the plate are the beginnings of the lateral neural ridges or folds, while around the anterior end they are continuous with one another as the transverse neural ridge or fold (Fig. 77, B _). Accompanying or immediately following the thickening of the nervous ectoderm which produces the ridges, there is a corresponding thinning of this layer along the midline of the plate. As a result there soon appears here a shallow depression. It is sometimes scarcely evident externally at this stage, but as soon as it becomes so, it is termed the neural groove.

EXPERIMENTAL RESULTS

Some of the most significant work in modern experimental embryology has been done upon the early stages of Amphibian development. There have been two main lines of investigation. One has interested itself in the movements and fate of materials during gastrulation, while the other has sought information concerning the effect of these materials upon one another. Though the aims of these studies have thus been somewhat different, the results, as will presently appear, have largely tended to supplement each other.

Location and Movement of Materials During Gastrulation. — One important method for discovering the movements and fate of materials during this process has been to stain the surface of very early gastrulae with vital stains at certain significant points, and then observe the shifts in these stains in later development. This is possible in some of the Urodeles, such as Triton, which possess unpigmented eggs, and has been done by Vogt, Goerttler, and others, with the external results shown in Fig. 70. Other experiments, noted presently, help prove the reality of the involution of part of this material, as already described, to form the hypoblast of the roof and sides of the archenteron. On the basis of these results Vogt and Goerttler constructed more or less idealized maps showing their views as to the location of this hypoblast

(potential endoderm, mesoderm and notochord) previous to gastrulation. Their conclusions are shown in Figure 71. l

3 EXPERIMENTAL RESULTS

137

More recently the matter has been reinvestigated by Pasteels C42) in another Urodele, Axolotl. and in an Anuran, the primitive Frog, Discoglossus, the results being indicated in Figure 72. It will be noted that aside from differences between the older and newer maps of the

Urodeles there are also some differences between those of the Urodeles and the Anurans. On the whole, however, these are matters of detail, the fundamental patterns being similar in all of them.

Aside from these minor differences show. in the pregastrula maps, there is one alleged postgastrula difference between the Urodeles and at least most Anura which the maps partly sug gest but do not really show, and which is perhaps

worth mentioning. It has to do with the


Fig. 70. ~— Four stages in the development of :1 Triton egg which had been marked with dyes in the early gastrula stage. The changes in shape and position of the colored areas indicate the movements of the materials of the egg during gastrnlation and the formation of the medullary folds. After Goerttler.

A. The early gastrula from the postero-dorsal side. B. A slightly later stage from the same View point. C. A much later stage viewed from the posterior. The neural folds are in evidence, but the blastopore does not show. D. About the same stage as C viewed from the dorsal side.

actual setting aside of notochord and somitic and lateral-plate mesoderm from endoderm, and is as follows: We have already noted that in the common Frog, Rana, the materials for the notochord, somites, dorso-, ateral mesoderm and endoderm are involuted as a single sheet of hypoblast. This hypohlast is then later separated by delamination into notochordal, somitic and lateral-plate mesodermal material above, and the endoderm of the archenteric roof beneath. In the Urodeles, however, this is not true. The involuted hypoblastic roof of the archenteron turns out to be composed exclusively of the definitive notochord and somites with

. perhaps even a little of the dorso~lateral mesoderm. This roof thus lacks 138 THE FROG: THROUGH GASTRULATION

temporarily any endoderm; the latter being presently supplied, not by delamination, but by the upgrowth of endoderm lying lower down on either side.

In concluding this topic there is this further point to note: The external area indicated by these maps as giving rise to the notochord and at least parts of the mesoderm and endoderm is also approximately the area of the gray crescent in those cases where there is one.

The Region of the Gray Crescent as the Center of Organization. ——Turning now to the problem of how the materials affect one

Dorsal





.1: ‘


Mesoblastic M Ventral Rim somites Dom, T ‘I Rim horda 3' 1 I esoblastic mesoderm mlgoedrgrm somites

' Tail mesoderm Dorsal lip

Ventral

Fig. 71.~—Diagrams of a Triton egg previous to gastrulation, showing the supposed location of the materials which, with the exception of the neural plate, are destined to be involuted to form various structures as indicated. A. View from the vegetal pole. B. Side view. After Vogt.

Rim. The region which eventually becomes the lip of the blastopore at the end of gastrulation.

another a long series of experiments might be cited. Only enough will be mentioned, however, to indicate what the trend has been, and the important conclusions which have at present been reached.

As has already been made clear, though the first cleavage in the Frog tends to bisect the dorsal lip of the blastopore, it does not always do so. In some cases indeed it may come as far as possible from this, and lie parallel to this lip. Bracket (’05, ’06) took advantage of this fact to find out what would happen when one of the blastomeres of such an egg was killed, as had previously been done with the blastomeres of more normal cleavages. In the latter case it will he recalled one side of an embryo developed, unless the egg had been so treated as to rearrange materials related to the gray crescent. In the latter event a whole, or nearly a whole, embryo was formed. Now in Brachet’s eggs it is clear that the crescent will be in only one of the two hemispheres, i.e., the one containing the dorsal blastoporal lip. It is perhaps not surprising therefore that when one blastomere of such an egg was killed, the reEXPERIMENTAL RESULTS 139

maining one would only develop when it was the one which contained the crescent. These, moreover, formed better than half of the anterior and dorsal part of an embryo. Thus once again the importance of mate rials connected with the gray crescent region was demonstrated (Fiv. 73).


C V- P- DISCOGLOSSUS V- P- D

Fig. 72.~—Maps of young gastrulas of (A and B) Axolotl, a Uroclele, and (C and D) Discoglossus, an Anuran, showing the location of materials destined for various structures. After Pasteels. The figures to the left (A and C) show ‘the gastrulas from the left side, while the figures todthe right (B and D) show them from the future dorsal si e.

D. Dorsal. V. Ventral. Lat. Lateral. bl. Blastopore. a.p. Animal pole. L‘.P. Vegetal pole. Stippled areas, notochord. Vertically-hatched areas, neural ectoderm. Diagonallycrossed hatched areas, mesoderm. Clear areas toward bottom of page from the mesoderm are endoderm. They contain bars representing material for the future gill slits.

The next step was taken by Spemann and Mangold (’24) . These men took a small piece of material just anterior to the dorsal lip of a Triton early gastrula and grafted it upon the surface of another gastrula. Wherever it was placed, this material was soon covered over by surrounding cells, and the cells which covered it presently formed a medullary plate. Later this plate would give rise to a neural tube, or part of one, as shown in Figures 74, 75. The same experiment was eventually done with the Frog. Also Bautzmann (’26) -performed more detailed experiments to see how far from the blastoporal lip of an early gastrula 140 THE FROG:.THROUGH GASTRULATION

t_he material possessed this power to cause other ectoderm to become neural plate. She found the region extended about 85 degrees anterior to the middle of the lip, and about 80 degrees to either side, the anterior extent decreasing as one proceeds laterally. The effective area thus had the form of a crescent occupying a similar but somewhat wider zone

than that occupied by the gray crescent when the latter exists.

Now normally of course the material transplanted in these experiments reaches a position beneath the ectoderm by being involuted over the lip of the blastopore. Hence involuted material (hypoblast) taken from archenteric roof of a late gastrula should also be expected to stimulate neural plate formation in any ectoderm under which it occurs. Marx (’25) and Geinitz (’25) tested this assumption by transplanting such involuted hypoblast beneath other


Fig. 73.———A Frog embryo produced _ by injuring one Of ectoderm than that which normally produces neural

gllfirirstigwoabliigz plate. The assumption proved correct (Fig. 76) . In where Hie first deed this fact is one of the proofs that involution cleavage pane was parallel to the gray occurs‘

grescent instiad of Though the action of potential chorda mesoderm ' g at ' I an- . . . gig: to sag as is In mducmg neural tube formation has thus been usual. The blasto- .proven, another question still remains. Is all the mere injured was d f bl 1 I C t 1 H

the one which did ecto erm o a astu a or ear y gas ru a rea y ennot contain the tirely equivalent in its potentialities? Though crescent, since oth- h d d -11- d 6 It 1) f temise no develop c or ameso erm W1 1n uce n ura u e orma ion me}1t_ occurs. The in'any ectoderm is it not possible that some ectoéuélyélfziiledcelligfg derm, namely that of the normal neural plate resomewhat more gion, might form neural tube without any chorda than half of the an« d t? A“ t t h.

M301, portion of an meso erm presen . emp s 0 answer t IS quesembryo. After Bra~ tion have been made by several workers, notably Chet‘ Spemann (’I8, ’21) . This worker transplanted small pieces of ectoderm from the prospective neural plate region of a young gastrula to a different region in another gastrula. He also performed the converse experiment of placing ordinary ectoderm in the position of part of the prospective neural plate. In some of the cases, moreover, he made the interchange between different species of Triton having ectoderm of distinctly diflerent shades. Thus it was possible to follow accurately the fate of the transplants in their new environments. The results in all cases showed that at this stage of development the fate of the ectoderm has not

yet been determined. The prospective neural plate material when placed EXPERIMENTAL RESULTS 141

elsewhere did not form neural plate, but ectoderm like that surrounding it in its new location, while the latter when implanted in the midst of the future neural plate became a normal part of the plate and future neural tube. Later Work by Marx (’25), it is true, showed that just before the neural plate appears the ectoderm has become determined, but previous to that time the results are as indicated. These data therefore would seem to prove that in very early gastrulae the ectoderm of the prospective neural plate region has not yet come under the influence of the chorda mesoderm, and that under these circumstances it has the same, or nearly the same, potentialities as in any other location (see below). The Principal of Induction.-—-The action of a substance in thus causing cells to respond by forming some specific tissue or structure is known as induction or evocation. The tissue which responds, on the other hand, is said to have a certain competence. Although such a relationship has received its greatest emphasis in connection with the material in the vicinity of the dorsal lip of the Amphibian blastepore, this particular instance is by no means unique.

Fig. 74.—An embryo of Triton on whose left side an extra neural tube _has been induced. ‘This was done by implanting in the side of this embryo at the gastrula


It occurs in many organisms, and in connection with all sorts of tissues and stages of development, some of the more striking examples of which will be pointed out as we come to them. Because of the

stage a piece of external material from the blastoporal lip of another gastrula.

After Spemann and Mangold.

early discovery and far reaching consequences of the inducing material in the vicinity of the Amphibian blastoporal lip, however, it was especially designated as the ‘organizer.

It must now further be added that the induction and response relationship in general is not always such a completely open and shut one as so far indicated. Some tissues have different degrees of inducing capacities, while the competence of other tissues to respond in a specific way varies considerably as one proceeds away from the site where a particular response normally occurs. Thus even in the original case of

neural plate induction, it now appears possible that, contrary to some

of the earlier results indicated, not all ectoderm is quite alike in its ability to respond. Some areas of ectoderm form neural plate and tube more easily than others, especially if properly oriented (Barth, ’4-1). It should also be stated that when a tissue has once begun to respond in a certain direction, it loses its competence to respond in any other. 142 THE FROG: THROUGH GASTRULATION

The Nature of the Inducing‘ Substance.-—It now remains to add’ a word regarding more recent attempts to analyze the nature of inducing substances, particularly the original one designated as the organizer. The first steps in this direction involved eiiorts at discovering how specific the inducing substance was, i.e., would anything other than material related to the gray crescent region induce neural tube?


pr. neur.

Fig. 75.-—A cross section of the same embryo shown in Fig. 74, at a later stage, showing the two neural tubes.~After Spemann and Marigold.

I. sec. ear. Left secondary ear vesicle. pc. Pericardial cavity. pr. neur. Primary neural tube. sec. neur. Secondary or induced neural tube which because of the orientation of the section appears on the right instead of the left side.

The answer was rather startling. It was found that a very wide variety of materials would work, e.g., pieces of adult liver and kidney as well as certain Invertebrate tissues like. ganglia of Lepidoptera. It has further been discovered that a tissue which normally lacks inductive capacity, such as neural plate, may acquire it by being in Contact with one which normally possesses it, such as chorda-mesoderm. Indeed it is now known that neural tube, having itself been induced, is then capable for a time of inducing tube formation in undetermined ectoderm. It was also shown that tissues need not be alive or recently killed. Tissues would work even after being fixed and imbedded in paraffin as for sectioning. Not only this but in some instances material such as pieces of blastula which normally have no inducing capacity will act as inductors after they have been boiled! Thus it is clear that the substance EXPERIMENTAL RESULTS 143

concerned is non-living, and is fairly widespread. From this point it would seem that with modern analytical methods it should not be too difiicult to trace down the essential chemical involved. Such, however, has proved far from the case. Many workers have attacked the problem, among the most prominent being Spemann in Europe, Needham and Waddington in England and Holtfreter and Barth in the United States. Spemann believed that glycogen might be the substance, but Needham thinks that one of the sterols is responsible. Barth and Grail


Fig. 76.——A. Diagram of Bombinator (a Toad). The small circle indicates the’ blastopore, and the shaded square represents the region from beneath which a piece of the archenteric roof was taken, and transplanted to the blastocoel of a gastrula of Triton. B. An older stage of the Triton to which the transplant from (A) was made. After Geinitz. M. The regular primary neural tube. In. The partial secondary tube induced by the transplant.

(’38) , on the other hand, doubt the possibility of determining with certainty just what the normally acting material may be. The difficulty is that various chemicals and treatments, some of which are probably actually toxic, nevertheless have an inductive effect. It seems unlikely that so many different substances are concerned under natural conditions, and it is certainly unlikely that any of them are toxic. It has been said that these chemicals are not the inductors, but release the latter from the live tissue. Also it is possible that the process may consist of the removal of a blocking substance which has inhibited various

developmental possibilities inherent in the cells acted upon. Finally, the '

reason for different reactions by like material, e.g., the formation from the neural plate of brain in one place a_nd neural tube in another, may be due either to a quantitative or qualitative difference in the inductor produced by different regions of the archenteric roof (Barth, ’53) . Significance of Developmental Concepts.— In concluding this general topic it is well to emphasize first the very great importance of the 144 THE FROG: THROUGH GASTRULATION

fundamental concept of induction. As this concept becomes increasingly established and elaborated we can see, at least theoretically, how a complex structure like an embryo may develop from a specific physicochemical system like an egg. Thus, when the equilibrium of this system is disturbed by fertilization or otherwise, an orderly chain of reactions is started, each one inducing others. Obviously this does not completely explain development. Yet it does reveal a significant aspect of it which will be repeatedly demonstrated as we proceed.

Recently Townes and Holtfreter, ’55, have discovered something which may help to establish another concept. By mixing ectoderm, mesoderm, and endoderm cells from neuralae-gastrulae they have shown that these cells possess certain “ directive movements and selective adhesiveness ” characteristic of each cell type, causing some to move inward, while others spread peripherally, arranging themselves in normal tissue patterns.

A COMPARISON OF GASTRULATION, MESODERM AND MEDULLARY PLATE FORMATION IN AMPHIOXUS AND THE FROG

A comparison of gastrulation, mesoderm and notochord formation, and the development of the medullary plate in Amphioxus and the Frog may now be presented in tabular form, as follows:

Gastrulation AMPHIOXUS Fnoc The processes involved are: in- The processes involved are: vagination, involution, epiboly, modified invagination, involution,

and convergence.

epiboly, some convergence, and clelamination.

Mesoderm Formation

1. Gastrulation is ‘virtually completed before definite setting aside of mesoderm begins.

2. The potential mesodermal material is identifiable in the fertilized egg. It can be traced into the ventro-lateral blastoporal lip of the early gastrula, whence it is carried into its definitive position

1. Gastrulation is completed before mesoderm is set aside.

2. The potential mesodermal material is not visually distin« guishable until after gastrulation, but evidence shows that it exists lateral to the lips of the blastopore. Thence it is brought into its definiCOMPARISON OF AMPHIOXUS AND FROG 145

AMPHIOXUS

by a kind of combined involution, epiboly, and convergence.

3. The setting aside of the mesoderm in the form of somites occurs by a process closely akin to enterocoelic evagination, especially in the more anterior region.

Fxoc

tive position by processes of involution, epiboly, and convergence.

3. The dorsal and lateral mesoderm is set apart as such by delamination. Ventrally, however, it arises to a considerable extent by the proliferation of cells from that already formed.

The N otoc/Lord

The potential notochordal material occurs at the dorsal lip of the blastopore. Thence it is involuted to the archenteric roof from which it is set aside by evagination.

The potential notochordal material lies anterior to the dorsal lip of the blastopore. Thence it is involuted to the archenteric roof. From this roof and from the mesoderm on either side it is then separated by delamination.

The Medullary Plate and Folds

1. There is no split between outer and nervous ectoderm. Dorsally a median strip of ectoderm becomes slightly depressed to constitute the medullary plate. The edges of the ectoderm on each side of this plate presently become separated from the margins of the latter, and then grow together above it. The overgrowing layers so formed thus constitute only the outer half of a true medullary fold. Later, the margins of the plate itself also bend toward one another until they meet and fuse beneath the overgrown ectoderm.

2. In Amphioxus no attempt has been made to demonstrate induc 1. An inner or nervous layer of ectoderm is formed by delamination over the entire gastrula. The medullary plate arises by a thick . ening of this layer in the mid dorsal region. As will appear below, the margins of this plate then come to constitute the crests of true neural folds. This follows from the fact that in this case the sides of the plate are carried upward and together, not later than, but in company with the ectoderm around their edges. Thus no sepa-A ration occurs between the ectoderm of the plate and that surrounding it until’ the crests of the folds meet. _

2. In the Frog experimental procedure has demonstrated that 146 THE FROG: THROUGH GASTRULATION

AMPHIOXUS Faoc

tive action. However, it very prob- the ectoderm is stimulated to form

ably occurs here as in the Amphib- neural plate and tube by the induc ians and other forms. tive action of the underlying chordo-mesoderm.

In concluding this comparison it is well once more to emphasize the fact that the above differences, at least those of gastrulation and mesoderm formation, are chiefly due to differences in relative amount of yolk. It may also be repeated that a further increase in this substance in the Fish and Bird is apparently responsible for the still greater modifications of the above processes in those animals. HE FROG: EARLY OR EMBRYONIC DEVELOPMENT SUBSEQUENT TO GASTRULATION

T H E general condition of the embryo at the conclusion of gastrulation has already been indicated, and there was also noted the origin of the notochord, the mesoderm, the medullary plate and neural folds. Following this there occurs a period characterized by the beginning of elongation and also by the appearance of the rudiments of the main systems and organs. Thus at the end of the time in question, during which the animal has reached a length of from 2:5 to 3 mm., virtually all these rudiments are present. For this reason it will be convenient to carry forward the description of both external and internal development to about this point. We shall then be prepared to describe more clearly the remaining changes which lead to the formation of the adult.

In carrying out this plan it will not be possible to state with any accuracy the age at which a particular size and degree of development is reached, even in the same species of Frog. This is necessarily so on account of the variableness of temperature to which the eggs are subjected. It will nevertheless be helpful occasionally to mention the average age of embryos of a given condition. The student must clearly bear in mind, however, that this is never more than approximate. It is desirable to begin by considering the development of this early period in its external aspects.

EXTERNAL CHANGES

As the embryo begins to elongate certain rather conspicuous features arise as elevations or depressions of the surface. All of these structures are at first more or less connected with the medullary plate, and all of them appear at about the same time. It will be necessary, however, to describe them separately.

External Development of the Neural Tube. —— The neural groove whose beginning has been noted, now becomes much deeper and more prominent (Fig. 77, C) . At the same time the lateral neural ridges or folds begin to increase their elevation and to bend toward one an148 THE FROG: THE EARLY EMBRYO

Sp.

Inf.


Fig. 77.~—Drawings of preserved Frog embryos (Rana pipiens) showing successive stages in the development of the neural tube, the sense plate and the gill plates. A. Antero-dorsal view of a stage shortly after the completion of gastrulatiou, showing the neural or medullary plate. B. Same \-'l(‘W of the next stage, showing the beginnings of the neural folds and the sense plate. C. Same view of somewhat later stage, showing the beginnings of the gill plates. D. Antero-lateral view of same specimen. E. Antero-dorsal view of still later stage, showing neural folds about to fuse.’ The sense plate and gill plates are clearly marked. F. Lateral view of same specimen.

gp. Gill plate. Inf. Lateral neural, or medullary folds. ng. Neural groove. np.

l_‘lt]a§ral, or medullary plate. sp. Sense plate. tn]. Transverse neural or medullary o . EXTERNAL CHANGES ,. i 149

other until eventually their crests meettand fuse; thus is formed the neural tube. Further, as noted above, the neural plate in this case, as in that of all true Vertebrates, is involved in the process from the first. Hence no break occurs along the crests of the folds between their outer and inner layers until after these crests have met (Fig. 80) 1 The phenomenon thus indicated starts somewhat anterior to the middle of the embryo in about the region of the future medulla, and from here the fusion proceeds in both directions. Anteriorly, this lateral closure is further augmented by the back growth of the transverse neural fold. Nevertheless, as will be noted presently, the completion of the process occurs later in the anterior region because of the greater space which separates the folds in this vicinity. The tube which is thus formed soon appears as a prominent ridge along the back.

The Sense Plate and the C-ill PIate.—— During the above process there are also developed certain other structures as follows: Just as the medullary ridges are preparing to fold in, a slight and rather narrow elevation grows outward from the antero-lateral region of each of them, and begins to extend in an antero-ventral direction. This continuesuntil the two elevations meet one another on the front of the embryo some distance below the anterior edge of the transverse neural fold (Fig. 77, B). There is thus formed a relatively narrow band of slightly elevated tissue which traverses the lower anterior region of the embryo in a bro-ucl curve and then ascends on either side until it merges with the edges of the neural folds. It is termed the sense plate. For a time the median area between the inner edge of this semicircular band-like plate and the edge of the transverse neural fold above it remains relatively depressed; i.e., of no greater elevation than theregion outside the plate. Presently, however, the distinction between this median area and the plate which constitutes its ventral and lateral boundary gradually lessens, the central region becoming almost as much elevated as its border. In this manner the sense plate comes to constitute a broad, some-. what shield-shaped region extending across the front of the embryo from side to side, while dorsally it is more or less continuous with the anterior of the neural tube (Fig. 77, E, F).

During the course of these processes anotherevent is taking place immediately posterior to those portions of the sense plate where it joins . the neural folds upon either side. In each of these two regions there is

1 It is to be noted that these literal crests of the folds are not quite identical with the “neural crests” referred to below in Fig. 80, the distinction becoming clear as the tube is about to be completed. 150 THE FROG: THE EARLY EMBRYO 4b.cl. ’ 4b.et. g.p.




OS.

4 br. cl. 2 br. cl.

st. i.

Fig. 78.—-Drawings of preserved Frog embryos (Rana pipiens) from 2-2 to 2-5 mm. in length, showing particularly the changes in the sense and gill plates. A. Right side of a 2.2 mm. embryo. The outpushing of the optic vesicle is just beginning to appear on the dorsal part of the sense plate. The latter is becoming more clearly separated from the gill plate by the rudiment of the hyomandibular cleft, while the posterior boundary of the gill plate, i.e., the rudiment of the fourth branchial cleft, is also becoming more evident. B. Right side of a slightly older embryo than A. The invagination of the left oral “ sucker ” (mucous gland) is visible near the ventral end of the sense plate. C. The same embryo viewed directly from the anterior end. The stomadaeal invagination and the two parts of the developing mucous gland are clearly shown. D. A 2.5 mm. embryo from the right side. The rudiments of the first and second branchial clefts have appeared upon the gill plate. Also, just posterior to the dorsal part of the gill plate the outpushing due to the pronephros is visible, and the external indications of some of the myotomes are beginning to appear.

Ibr. cl. 2br. cl. 4br. cl. Rudiments of the first, second, and fourth branchial (gill) clefts. The arch anterior to each cleft is named in the text. gp. Gill plate. }zy.c[. Rudiment of hyomandibular cleft. my. External indication of one of the myotomes. op. External indication of the outpushing optic vesicle in the upper region of the sense plate. as. Rudiment of oral “ sucker ” or mucous gland in the lower region of the sense plate. prn. External indication of the pronephros. 511. The sense plate,

whose lower portion really represents the mandibular arch. sz.i. The stomodaeal invagmatton. EXTERNAL CHANGES 151

developing another elevation which extends outward from the neural folds approximately parallel with the posterior border of the sense plate. Indeed, each of the new elevations is said by some authors to be merely a part of the original plate separated from it‘ liy the development of a depression. In any event the new raised areas, because of their future development, are termed gill plates (Fig. 77, C, D, E, F).

As the anterior portions of the neural ridges meet one another, a slight protuberance arises upon either side of the dorsal region of the sense plate (Fig. 78, A). These protuberances mark the outpushings of the two optic vesicles (see below). Also at about this time there begins to develop in the middle of the sense plate a rather wide vertical groove extending from near its ventral margin dorsally to about the level of the lower edges of the optic protuberances (Fig. 78, C). This is the stomodaeal invagination, the stomodaeum proper, forming later at its dorsal end. It is evident that the development of this groove results in a division of the sense plate throughout the greater part of its length, so that the raised portions exist only upon either side of the median line. It may now be added that each of these raised areas constitutes the rudiment of one side of the future lower aw or mandible, and hence each such area is designated at this time as a mandibular arch. Lastly, at the ventral end of each of these arches there now develops a small, somewhat elongated, and slightly pigmented depression. These depressions then deepen, while their postero-ventral ends grow toward one another and fuse, thus forming the characteristic V shaped “ sucker ” or mucous gland of the early larva.

It has been noted that the sense plate (now represented by the man’dibular arches) is separated from each gill plate by a slight furrow; it remains to be added that a similar indentation also bounds each of the latter plates posteriorly (Fig. 78). Upon either side the more anterior of these furrows, i.e., the-one between the mandibular arch and gill plate, marks the location of the hyomandibular “ cleft” (in this case

‘never an actual cleft), while the posterior one indicates the approxi mate position of the future fourth bronchial (gill) cleft. There next appear upon the surface of each gill plate itself two more vertically elongated depressions denoting the beginnings of the first and second branchial clefts, the rudiment of the third branchial cleft not developing until somewhat later (Fig. 78, D).

It is now further obvious that, between the depressions just noted, the surface of each gill plate will be relatively raised so as to form ridges which are the external indications of the hyoid and branchial 152


THE FROG: THE EARLY EMBRYO

Inf

Fig. 79.——Posterior ends of a series of young Frog embryos, showing the later history of the blastopore, and the relation of the neural folds to it. The embryos are viewed obliquely from the postero-lateral aspect. From Kellicott (Chordate Development). After F. Ziegler. A. Blastopore nearly closed, neural folds just indicated. B. Blastopore becoming divided into neurenteric and proctodaeal portions, lips between fusing to form primitive streak; neural folds becoming elevated. C. Neuronteric canal forming; neural folds"closi_ng together. D. Neural folds in contact throughout. E. Neural folds completely fused; tail commencing to grow out.

b. Blastopore, containing yolk plurr. b1. Rudiment of neurenteric canal (dorsal part of blastopore). be. Rudiment of proctodaeal pit (ventral part of hlastopore). brz. Branchial arches. g. Neural groove. nf. Neural folds. np. Neural plate. p. Proctodaeal pit. 5. Rudiment of oral “sucker.” t. Rudiment of tail. 9:. Neural folds roofing the blastopore and establishing the neurenteric canal. y. Primitive streak. EXTERNAL CHAN GES 153

arches. The most anterior portion of the plate which lies between the hyomandibular cleft and the fi1‘SlZ hranchial cleft is the hyoid arch, while the portion lying between the first and second branchial clefts is the first branchial (gill) arch. Since the third branchial cleft has not yet appeared, the portion of the plate posterior to the second branchial cleft really represents both the second and the third branchial arches.

The Closure of the Blastopore. ~—~As the above events are transpiring anteriorly, certain processes are also occurring posteriorly, as follows: As the medullary folds begin to move toward one another, the lateral lips of the blastopore also draw together, so that the latter is no longer round. Instead it has the form of a short vertical slit (Figs. 79, B and 80). Presently, moreover, these lips fuse with one another for a certain distance midway between their dorsal and ventral ends. As a result there may appear in this region for a time a slight vertical groove connecting the dorsal and ventral openings which temporarily remain. In the present case this is the primitive streak. In it, ectoderm, mesoderm, and endoderm meet in one mass, and from this mass, cells for all three layers are budded as the embryo increases in length. It is very important to note that this primitive streak is homologous with the similarly named structures which are to be described in connection with the next two forms. It is also probably comparable with the primitive streak of Birds and Mammals, and with the structure similarly defined in the general discussion in Chapter II.'Tl1is question will be discussed more fully in connection with the Chick.

The opening which remained at the ventral lip closes presently, but only the ectoderm and endoderm are involved. Hence the wall is thin at this point, and a slight pit remains. It is the procIo(z'(m11.r7r (Figs. 79, D and 80). The dorsal opening of the blastopore persists for a somewhat longer time. It disappears externally, however, because the neural folds which extend on either side of it fuse at this point as elsewhere, and thus roof i.t over. This process will be further noted in connection with the nervous system.

Other Changes. —— Besides the features already mentioned there are a few other external alterations which usually become apparent by the time the embryo is from 2.5 to 3 mm. in length. In the first place, in con nection with its slight elongation, the animal has begun to lose its spher- ‘

ical form, so that the convexly curved line of the back (Fig. 77, F) becomes first straight and then actually concave (Fig. 78). Secondly, just posterior to the dorsal region of the gill plate there may often he noted a slight swelling, the outward indication of the internal growth of the 154 THE FROG: THE EARLY EMBRYO

B medullary plate C









neural fold


notochord

blastoooral part or future neurenzenc . - neural

- tube

rcgmnaf

future neuranzernc canal

fug:urc_ prumluve streak region {Iervcus

ayer of eccodzrm

mesoderm

reeonscru - cl median sagitral section

lmfargln of oral evaglnallon


mesoderm

blastcporal

part of future neurcnteric canal



nervous layer of ectoderm


neural tube reglan of future neurenteric canal ‘

proccodaeum

E X section I22 F X secuan I27

Fig. 80.———A median sagittal section reconstructed from serial cross sections, and a stereogram of a hemisected total neural groove stage. A, B, C, D, E, F. Selected cross sections as shown by serial numbers, at levels indicated by vertical lines on the sagittal section. The neural folds have not yet closed posteriorly to- form the neurenteric canal and the primitive streak. THE NEURAL TUBE AND RELATED PARTS 155

pronephros or embryonic head‘ kidney (see below). Also along the dorso-lateral region posterior to the gill arches and just above the level of the pronephros, > shaped markings arise giving external evidence of the myotomes. Lastly the embryo by this time is partially covered by cilia whose motion causes it to rotate slowly within its membranes.

Under average outdoor conditions the stage thus described is generally reached at about the end of the second day after fertilization. Let us now turn to a consideration of the internal processes which have been going on during the same period..

INTERNAL CHANGES: THE NERVOUS SYSTEM THE NEURAL TUBE AND RELATED PARTS

The Neural Tube. —- This structure, as its name suggests, possesses an internal, laterally compressed canal termed the neurocoel or neural canal. From the manner of its formation, the lining of this canal is obviously the former outer ectodermal layer of the medullary plate, while the present outer wall of the tube was previously the inner or nervous layer of that plate. Thus the floor of the tube is relatively thin, since it occupies the position of the former medullary groove where the inner or nervous layer was least developed. The lateral walls, on the contrary, are thick because they are constituted of the well developed nervous layer on either side of the groove. The roof is evidently formed as the edges of the two folds meet one another and fuse, and, like the floor, it is thin as compared with the sides. As will appear below, this is due to the fact that not all of the nervous layer along the line of fusion becomes involved in that process. Finally it should be added that as the tube is thus made complete, the meeting of the folds likewise makes continuous the ectodermal wall above it.

The Neural Crests.»-—As just noted, not all of the nervous layer of the medullary plate is used up in the formation of this tube. The lateral edges of the plate, i.e., the neural ridges proper, although carried up to the region of dorsal fusion are not included in the walls of the tube. Instead, these ridges of nervous tissue are partially constricted off from the main part of the nervous layer. Each of the two ridges is thus semi-independent, and occupies a position well up in the angle between the sides of the tube and the ectoderm of the body wall (Fig. 80, no) . These are the neural crests, which presently become out up into successive segments. In the head and branchial region the crests are quite prominent, but more posteriorly they are obscure and difficult to detect. 156 THE FROG: THE EARLY EMBRYO

In general they are concerned with the development of the cranial and spinal rranglia although those in the head and branchial regions have n 7 _

been fdaund also to furnish material for some of the visceral arches. Their respective fates will be discussed in more detail in connection with the development of the nervous system.

THE BRAIN REGION AND SENSE ORGANS

The Brain Region.—In the anterior region the complete closure of the neural tube is somewhat delayed because of the greater breadth of the medullary plate at this point. Indeed, the process here might be still slower were it not that the growing together of the lateral edges is accompanied by the backgrowth of the transverse ridge. At the place where this ridge and the lateral folds are about to fuse there exists for a brief time a small opening; it is the neuropore, and is homologous with the similar structure in Amphioxus.

At the time the medullary plate first appeared, the embryo was still virtually in the form of a sphere, and the plate followed its curvature. As the neural tube begins to form, however, the embryo, as already noted, starts to lengthen out, the line of the back becoming straight, and then slightly concave. During this pm;-cess, net-ertl‘.-eless, the original curvature in the foremost portion of both the neural tube and the notochord not only ersists but even increases. it thus happens that these parts are bent ldownward so that the anterior and sornewl‘zut expanded extremity of the tube has the aspect of the bulbous closed end of a chemical retort. This bending‘ is termed the cranial flexure. Hence it comes about that the roof of the tube in this region is actually anterior, and in the midst of this anterior wall is the recently closed neuropore. This point is marked by a slight iuvagination, both exterinally and in the brain wall, and by a small thickening in the nervou:-: layer of ectoderm (Fig. 81). V

Elementary Divisions of the Brain. —The constrictions which divide the brains of most vertebrate embryos into fore-brain, mid-brain, and hind-brain have not become evident in a 2.5 mm. Frog larva. These divisions of the brain may be roughly defined at this time, however, by reference to the following landmarks: Just opposite the curved anterior region of the notdchord, the posterior wall of the brain, as suggested above, also curves, and the most anterior point on this curve may be designated as the tuberculum posterius. Directly across from this on the anterior brain wall is the invagination already noted as marking the closed neuropore, and immediately dorsal to this is a distinct inTHE BRAIN REGION AND SENSE ORGANS 157

ward bulge formed by a mass of cells termed the dorsal thickening

(Fig. 81). Using these points as places of reference the brain may now_

be divided into its three fundamental regions: I. The fore-brain or proscencephalon extends from the anterior ex notothord










endoderm

neural plate

beginning of future neurenterl: canal

pm‘mdaeum_ transverse neural ridge rectal evagination

mesodermal layer nervous layer of ectoderrn

pldermal layer

tuberculum potterlus dorsal thickening

neurenteric canal

Iosed neurbpore

infundlbulum

1- primordium of anterior pituitary

proczosiaeum

stamodaeum

mesoderm of future pericardium

beginning of mucoux gland pharyngeal reglon

nervous layer 0! ettoderm


\§ cm.-‘

liver evaginatlon

Fig. 81.—A. Sagittal section of neural groove stage. The remains of the blastecoel is not often seen so late as this. In this case the region between the beginning neurenteric canal and the proctodaeum (primitive streak_l has been occluded by the fusion of the sides of the blastopore. B. Sagittal section of neural tube stage. The proctodaeum does not usually have so large a cavity connected with it, but did in this case. The rectal evagihation which meets the proctodaeum is unlabeled.

tremity of the tube, i.e., the lowest part of the bent region, to a plane joining the tuberculum posterius with a point between the dorsal thickening and the closed neuropore. »

II. The mid-brain or mesencephalon extends from the posterior boundary of the proscencephalon to another plane which joins the tuberculum posterius with a point slightly back of the dorsal thickeninv.

III. The hind-brain or rhombencephalon. extends from the posterior boundary of the mesencephalon insensibly into the spinal cord. 158 THE FROG: THE EARLY EMBRYO

It is thus evident, as indicated above, that the fore-brain is chiefly below and in front of the end of the notochord, the mid-brain is anterodorsal to the end of the notochord, while the hind~brain lies entirely

‘over the notochord.

Within the divisions of the brain thus defined there is very little differentiation of any sort as yet. In the most ventral portion of the forebrain, however, there does appear at about the end of the time we are considering, a slight rather broad and vaguely delimited posterior outpushing. It is the rudiment of the infundibulum, which will become the posterior part of the hypophysis or pituitary body. The anterior part of this important endocrine gland: also appears at this time, and it is therefore convenient to describe it here, though unlike the posterior infundibular part it is not a brain derivative at all. At this stage it is more clearly defined than the infundibulum, and arises as a tongue of ectodermal cells of the nervous layer extending dorso-posteriorly from the dorsal margin of the stomodael invagination. It lies therefore just beneath the forebrain, and is growing backward in such a way as eventually to meet the infundibulum (Fig. 81, B).

With the mention of these structures it becomes necessary to digress for a moment in order to make clear the way in which we shall use the terms applied to them and their parts. This is because the definitions of these terms have been considerably confused by various writers, especially as they have been employed in connection with some of the lower animals. Strictly speaking the organ referred to as the hypophysis or pituitary in human and other mammalian anatomy includes two main parts from the point of view of origin. One is derived from an ingrowth from the stomodaeum, and includes the pars distalis (anterior lobe proper), pars intermedia and pars tuberalis. The other main part is called the pars nervosa, which is derived from the larger portion of the infundibulum, the smaller remainder forming the stalk of the hypophysis. The pars nervosa is also frequently referred to as the posterior lobe (Gray’s Anatomy, 24th edition). Even here, however, there is confusion since the “posterior lobe” according to some authors (Maximow and Bloom, 5th edition) seems to include not only the pars nervosa. indubitably of infundibular origin, but also the pars intermedia which is indubitably from the stomodaeum (Hegre, ’46) . As a matter of simplification, and for the purposes of this text, the writer will term all parts of the hypophysis derived from the stomodaeum simply the anterior part, and all parts derived from the infundibulum, i.e., the pars nervosa, the posterior part. Finally it should be understood that in the Amphibia THE BRAIN RF:GION AND SENSE ORGANS 159

the position of the anterior part as here defined is really posterior to the pars nervosa or posterior part. The parts are nevertheless designated in this way because in adult avian, human and other mammalian anatomy the anatomically and embryologically homologous parts do actually. occur in the anterior and posterior positions.

The Sense Organs.—Before the anterior or brain region of the medullary plate has closed, there appears on either side a patch of pigmented cells (Fifi. 82). As a result of the closing process, these

outpushtng

Pom“ of optic vesicle

neural crest


Fig. 8Z.—Cross section of a 2 mm. Frog embryo through the anterior end of the neural groove, showing optic vesicles starting to push out. Note the pigment spot on the inner side of each vesicle. The epidermal and nervous layers are thicker because they are cut

tangentially due to the curve of the embryo in this region.

patches presently come to occupy positions on opposite sides of the interior of the fore-brain. The area of the brain wall including and immediately surrounding each patch now begins to push out or evaginate toward the external ectoderm of the head (Fin. 85, A). These evaginations are the optic vesicles. Presently each vesicle reaches the ectoderm in the dorso-lateral region of the sense plate, and by its pressure here soon causes a slight external protuberance noted above. Meanwhile the regions of the vesicles nearest the brain begin to become slightly constricted to form the optic stalks (Fifi. 85, A).

The sensory portions of the ears, unlike the above parts of the eyes, do not develop from any region of the brain itself. Instead they arise from the dorso-lateral walls of the head. The rudiment of each appears during this period as a thickened patch of the nervous‘ layer of ectoderm opposite the hind-brain. These thickenings in part constitute the auditory placodes (see below under ear).

At about the same time in another region of the head two other thick160 - THE FROG: THE EARLY EMBRYO

enings of the nervous ectoderm develop. In this case each is within the area of the sense plate a short space beneath, and, median to, the corresponding optic protuberance. These are the beginnings of the olfactory organs, and are termed the olfactory placodes (Fig. 83). Though later each is indicated externally by a pit, these markings are usually not in evidence at this stage (see below). Figure 83, however, is of a slightly later stage (3.5 mm.), which accounts for their appearance in that case.



olfactory placo

nervous layer

f d optic vesicle O CCIO erm



‘tii:'LT?'9n mandibular arch

' yomandibular cleft (3 hyoid arch

‘em, lst branchtal cleft ’ H‘

infundibulum ' i‘



|Vth branchial cleft

undivided mesoderm

Fig. 83.—Frontal section of a 3.5 mm. Frog embryo through the olfactory pits, optic vesicles, and visceral clefts and pouches.

Experimental Results. —— In connection with the discussion of gastrulation a good deal was said about the principle of induction, and it was indicated that further illustrations of it would be noted as occasion arose. Three excellent examples are afforded with respect to the origin of the oral mucous glands, the nasal capsules and the optic vesicles.

In the Urodele, Amblystomaf it happens that in place of the mucous glands there occur leglike projections called balancers on which the ani 2 The writeriis aware that the correct generic name for this animal is Ambystoma rather than Amblystoma. However, the latter has become so firmly fixed in the literature, particularly the embryological literature, that it seems best to use it in

this text. This is made even more advisable in view of the fact that the latter spelling is the one used in all the articles cited. THE N EURENTERIC CANAL 161

mal rests. Schotte and Edds (’40) found that Frog ectoder'm from regions which would not normally produce mucous glands, would do so when transplanted to the head of Amblystoma at the site of, and in place of, the latter animal’s balancer producing ectoderm. This shows two things. It indicates first that the formation of either mucous glands or balancers is apparently due to the inductive action of the underlying mesoderm. Secondly, it shows that though the Frog ectoderm is thus acted upon by the Amblystoma inductor, it can, nevertheless, only form the kind of organ for which it has competence, namely, mucous gland, not balancer.

In the case of the nasal placodes Zwilling (’4«O) has shown among other things that apparently they may be induced in the nervous ectoderm by the roof of the‘ underlying archenteron. Also the olfactory pit can be induced in the epidermal ectoderm by the layer of nervous olfactory ectoderm underlying it.

Finally in the case of the optic vesicles Adelmann (’30, ’37) and others, by the usual transplantation experiments, have demonstrated two points. First, the inherent capacity (competence) of the head ectoderm to form these vesicles at all is considerably reinforced by the inductive action of the underlying prechordal plate (potential notochord). Secondly, this inductive action causes two vesicles to form where there would otherwise be only one (cyclopia) .

THE NEURENTERIC CANAL

While the above developments have been taking place in connection with the anterior end of the nervous system there has also been a change posteriorly. It was noted in describing the externals that as the neural folds close in this region, they roof over the dorsal part of the blastepore. As stated, however, this portion of the blastopore, though no longer communicating with the outside, still remains open. It thus constitutes a temporary connection between the enteron and the neurocoel. As in Amphioxus, this connection is termed the neurenteric canal (Fig. 81). It should be noted in this case that the canal is seldom if ever demonstrable as an actual open tube, and its existence has therefore been denied by some. Usually in fact it appears merely as a line of pigment. In good specimeliis which the writer has examined, however, the clean cut character of the cells bordering the path of the “ canal ” in all probability indicates a definite line of cleavage. Indeed it seems clear that what amounts to a “ probe patency ” certainly exists, were it possihle to use a probe on so small a structure. 162 THE_ FROG: THE EARLY EMBRYO

INTERNAL CHANGES: THE ENTERON THE FORE—GUT

The anterior region of the archenteron is enlarged and lies in front of the mass of yolk cells which form the floor -of the middle region. This anterior portion is therefore termed the fore-gut, and a little later will be differentiated into the pharynx, esophagus, stomach, and liver. These parts are as yet scarcely distinguishable. Nevertheless, during the period under discussion, the fore-gut as a whole gives rise to certain rudiments as follows:

The Pharyngeal Region.——In the antero-ventral region beneath the fore-brain there is an outpocketing in the direction of the invaginated ectoderm, though the two walls are not yet in contact. It is called the oral evagination and may be considered as the extreme anterior end of -the pharynx (Figs. 81; 85, B). Immediately posterior to this in the region of the fore-gut which is destined to hecome the pharynx proper there have already been noted the external rudiments of certain of the visceral clefts; i.e., the hyomandibular, and the first, second, and fourth branchials. Considering now the internal development of this region at a corresponding stage, the following condition is to be observed. Opposite the invaginating ectoderm which marks externally the rudiments of the above mentioned clefts the endoderm of the pharynx is beginning to push outward upon either side to form the corresponding pairs of

' hyamandibular, and first and second branchzal or gill pouches. It

should further be added that although these vertically elongated pharyngeal evaginations are called pouches, they do not actually appear as such. This is because the anterior and posterior walls of each outpushing are at this time fused together, so that no pouch cavity really exists. Thus it may be noted that each pouch resembles rather a two layered sheet of endoderm, extending from the fore-gut toward the ectoderm (Figs. 33, 102).

The Liver.—In the extreme ventro-posterior part of the general pharyngeal region there is evident a slight posteriorly directed pocket beneath the anterior end of the yolk mass. This represents the rudiment

" of the liver (Fig. 81, B). THE MID-GUT

The portion of the enteron following the fore-gut lies, as noted, above the main mass of the yolk cells which thus form its floor. Its lumen is THE VISCERAL ARCHES 163

, relatively small with a thin roof, and sides which thicken ventrally. It

is the mid-gut, and is destined later to develop into the intestine.

A peculiar and transitory structure developed in connection with this region is the hypochorclal rod. It arises at about 2.5 mm. as a longitudinal string of cells constricted oil‘ from the dorsal wall of the mid-gut, between it and the notochord. Appearing first slightly posterior to the pharyngeal region it later extends even into the tail. It soon becomes separated from the gut by the development of the dorsal aorta, and shortly after hatching it disappears entirely.

THE HIND~GUT

Posterior to the mid-gut just in front of the neurenteric canal the en teron enlarges slightly. This region is termed the hind-gut, and is destined to form the rectum.

THE MESODERM AND REIATED STRUCTURES

Shortly following gastrulation, the condition of the mesoderm is as follows: Ventrally and laterally it exists as a continuous sheet extending up to the notochord on either side. In the head and most of the pharyngeal region it is represented only by scattered cells, while posteriorly it reaches to the blastoporal region, which continues to bud it oil. During the period we are now discussing the mesoderm thus indicated begins to give rise to various structures in the following manner:

THE VISCERAL ARCHES

It will be recalled that in the pharyngeal region at this time) the hyomandibular and the first two pairs of branchial or gill pouches are developing as solid vertically elongated evaginations of endoderm. As these evaginations push out to the ectoderm, it is obvious that the mesoderm in the way of each will be thrust to either side. In this manner such mesoderm becomes more or less concentrated in the regions of the future visceral arches which are to alternate with the pouches. Indeed, it may at this time be said to represent their rudiments, whose external appearance has already been described, as having the form of raised areas between the incipient clefts. Thus in front of the first or hyomandibular pouch is the mesodermal rudiment of the mandibular arch (apparent externally as the lower portion of the sense plate upon either side of the stomodaeum) , while between the hyomandibular and first branchial pouch is the rudiment of the hyoid arch. The first bran» 164 THE FROG: THE EARLY EMBRYO

Fig. 84.—-Sections through Frog embryos (R. sylvatica) illustrating the formation of the pronephros. From Kellicott (Chordate Development). After Field. A. Through the anterior body region of an embryo at the commencement of its elongation. B. Through the anterior end of the pronephric rudiment of an embryo in

which the neural folds are just closed together. C. Through the second nephrostome of an embryo of about 3.5 mm.

c. Coelom. ca. Rudiment of pronephric capsule. cc. Communicating canal. ec. Ectoderm. en. Endoderm. g. Gut cavity. mp. Medullary plate. my. Myotome. n. Notochord. nc. Rudiment of neural crest. ne. Nephrotome. 5. Pronephric nephrostome. sc. Spinal cord. sn. Subnotochordal rod (hypochorda). so. Somatic layer of mesoderm (in A the reference line points to the rudiment of the pronephros). sp.

Splanchnic layer of mesoderm. t. Pronephric tubule. v. Vertebral plate of mesoderm.

chial arch then follows the first branchial pouch, and the second branchial arch follows the second branchial pouch. Since, however, the third branchial pouch is scarcely formed as yet, the mesodermal ele ment of the second branchial arch is not at this time very clearly distinguishable from the tissue posterior to it. '

THE SEGMENTAL PLATES AND THE LATERAL PLATES

Along either side of the notochord ‘posterior to the pharyngeal region, the mesodermal sheet thickens into a relatively narrow band which THE SEGMENTAL AND LATERAL PLATES _ 165

is termed the segmental or vertebral plate. The remainder of each sheet below this region is then called a lateral plate. Ventrally the two lateral plates are continuous with one another (Fig. 85, D).

Formation of the Coelom. — In its dorsal region each lateral plate now begins to become split into two sheets. The outer sheet next to the



beginning of auditory vesicle llnd placode

beginning of perlcardial cavity

Fig. 85.——Four selected cross sections from a series of one 2 mm. (neural-tube stage) Frog embryo. A. Through the optic vesicles and rudiment of anterior pituitary. B. Through auditory vesicles and oral evagingtion. C. Through pharynx in region of III neural placodes and crests, and the future heart. D. Through anterior part of mid-body region, showing liver evagination and nephrotomes.

ectoderm is the somatic mesoderm (somatopleure), while the inner sheet next to the enteron is the splanchnic mesozlerm (splanchopleure) (Fig. 85, D). Between them a space presently becomes evident which is the rudiment of the coelom. Upon either side, this coelom then gradually extends downward through its respective lateral plate. During the period we are describing, however, these two extensions do not reach quite far enough to meet one another beneath the gut. Thus in this region the coelomic cavity in each plate is temporarily separated from the one on the opposite side. Besides this downgrowth of these cavities 166 THE‘ moo: THE EARLY EMBRYO

Fig. 86.—Transverse section through the sixth mesodermal somite of a 5 mm. larva of R. temporaria, illustrating the arrangement of the mesoderm. From Kellicott (Chordate

Development). From Maurer Harulbuclz, etc.).

c. Cutis plate. ch. Notochord. D. Gut wall. In. Myotome (muscle plate). me. Nerve cord. p. Lateral plate. scl. Sclerotomal cells. 12. Ventral process of myotome and cutis plate.

( I-lertwig’s

there is also an upgrowth into

the mesoderm of the segmental"

plates (Fig. 84). Here the slight spaces which last but a brief time are termed the myocoels.

The Somites. —— Meanwhile the segmental plates are also undergoing other changes. Just back of the pharynx each plate is being divided transversally into sections termed somites. During the period under consideration, about four pairs of these. somites are thus formed, development proceeding posteriorly. Shortly after its formation each somite loses its connection with the lateral plate, and exists as a separate mass of cells. Within each somite so isolated the myocoel may persist for a brief time, not at the center of the mass, but just beneath the outer surface. Because of its previously supposed subsequent history (see below) the thin layer of cells forming this outer surface is termed the cutis plate or dermatame. For the same reason the remaining inner part of

the somite is called the myotome. The differentiation between these parts is often indistinct at this time (Fig. 85, D), but is usually clearer

at a later stage (Fig. 86). 4 THE NEPHROTOMF

Along the dorsal border of each lateral plate, just at the line of sepa ration between lateral plate and segmental plate, is a narrow strip of i PERICARDIAL CAVITY AND THE HEART ‘I67

somatic mesoderm which is destined to form both the larval and adult excretory systems. This strip is termed the nephrotome, and becomes evident as such very early (Fig. 84, B; Fig. 85,’ D) . Indeed, even before separation of the above plates this region begins to proliferate cells between itself and the ectoderm. In this way the nephrotome becomes a thick band of tissue attached along its inner border to the dorsal edge of the lateral plate, whose side it overhangs slightly, like the cave of a roof. At the very first, as segmentation appears in the vertebral plate, it also‘ extends slightly into the nephrotomal band. Thus the single nephrotome tends to become divided into a series of nephrotomes. This division, however, is very transitory in the Frog and disappears without further significance. As the coelomic split ‘begins to appear in the lateral and segmental plates, spaces also start to form in the nephrotome from about the second to the fourth somites (Fig. 84, C). This

marks the beginning of the pronephros, the evidence of -whose presence has already been noted in the description of the exterior.

THE BEGINNING OF THE MPERICARDIAL CAVITY AND THE HEART

In the region of the pharynx it has been indicated that laterally the rather loosely arranged mesoderm is involved in the formation of the gill arches. In the floor of this region, however, uniting the ventral ends of these arches, there is a sheet of mesoderm coextensive posteriorly with the fused lateral plates. It will be recalled that at this period the downpushing coelornic spaces in these plates have not reached to the ventral side of the animal. Anteriorly, however, in the ventral portion of the mesodermal sheet which lies beneath the pharyngeal floor, there may now appear a. slightly indicated pair of independently developing spaces (Fig. 85, C). Each space lies within the sheet upon either side of the mid-line, the two spaces being separated from one another by a narrow median strip of the mesoderm which remains undivided (Fig. 85, C). These spaces are the rudiments of the pericardial cavity, whose walls are termed the pericardium. The outer or parietal wall is in dicated at present by the lower of the two mesodermal sheets. It is cons —

tinuous, both now and in the completed organ, with the inner or visceral wall which arises from a portion of the upper sheet, and which eventually forms a closely adherent covering for the heart muscle. (See Fig. 85, C and D; cf. also Fig. 107.)

Just above the median strip, between it and the endodermal floor of the pharynx, there may also appear at this time a few scattered cellsl 168 ‘THE FROG: THE EARLY EMBRYO

These cells have been regarded as having originated like the dorsal mesoderm of the lateral plates, i.e., by a splitting off from the endodenn which in this case lies above them. It now appears, however, that they are derived entirely from mesoderm which, in Amblystoma at least, as shown by staining experiments of Wilens, ’55, has migrated from between the ear anlage and the hind-brain. The scattered cells are destined to form the endothelial lining of the heart, or endocardium, while the remainder of this mesodenn forms other heart and pericardial tissue to be described in the following chapter (Fig. 85, C). Though all parts of the heart are thus apparently mesodermal in origin, there is evidence that the overlying endoderm does have an organizing efl'ect on their development (Bacon, ’45).

Before leaving the development of the heart at this early stage, it is of interest to note what happens when these heart-forming elements are manipulated in various ways as was done by Copenhaver (’26) on Amblystoma. Thus if a moderate amount of the median region is removed, the lateral parts will grow down and replace it so that a single complete heart develops. If, however, a piece of foreign mesoderm is substituted for the removed part, the lateral parts will form two separate hearts with mirror image symmetry. Removal of an anterior or posterior half does not prevent the formation of a complete heart if it is done early enough, but the anterior and posterior parts are apparently irreversibly determined considerably sooner than are the lateral

' parts. Not only, however, is it true that parts may form whole hearts,

but two wholes if properly united may form single hearts. Thus if a second layer of heart-forming mesoderm from one embryo is superimposed by transplantation upon the heart-forming mesoderm in another embryo, the two layers will fuse and a single normal heart develops. On the other hand, as might be anticipated from the previous statement about anterior posterior determination, this only happens at

the stage in question if the second layer is normally orientated. If the latter is reversed with respect to its antero-posterior axis, fusion is im perfect. Also, since heart pulsation is initiated at what is at first the

posterior end of the organ, in this latter case disharmonic pulsation results. "HE FROG: LATER Oli LARVAL DEVELOPMENT

IN the last chapter the development of the embryo was discussed up to the point where it had reached a length of about 2.5——3 mm., and acquired the rudiments of most of the chief systems and organs. We shall now continue the history of the animal from this point to the adult condition, having regard to both the external and internal changes. The former will be considered first, under the head of three rather obvious stages which ‘will become apparent as the description proceeds.

EXTERNAL DEVELOPMENT TWO AND ONE—HALF MILLIMETERS T 0 HATCHING

During the first week or two, depending on the temperature, elongation progresses to a considerable extent, largely as a consequence of the outgrowth of the tail region posterior to the blastopore. Concurrent with this process, the > shaped depressions marking the boundaries of the myotomes not only -become evident throughout the body region, but appear also upon the sides of the tail. At the same time just back of the gill plates the pronephric swellings increase in size. In the head the outpushings due to the optic vesicles become somewhat more pronounced, but in a slightly different position from the one which they first occupied, i.e., less upon the front of the head and more upon the side. This last mentioned change is really due to the beginning of a forward growth of the region anterior to them, which continues gradually for some time, and results in the eventual location of the eyes some distance from the tip of the snout. Meanwhile the stomodaeum proper forms at the dorsal end of the elongated stomodaeal invagination, while upon each sense plate, slightly dorsal and to one side of the stomodaeum, appears a small depression, the olfactory pit. Each gill plate, on the other hand, now develops upon its surface another slight vertical groove lying between the rudiments of the second and fourth branchial clefts. This new indentation is the beginning of the third bronchial cleft, so that theypositions of all four branchial clefts are now indicated (Fig. 3br. cl. 2br.cl. spr.


1 br.cl:

°PC .cl.

- by sti.

Fig. 87.—-Drawings of preserved Frog embryos and larvae (Rana pipiens) from 4 mm. to 14.5 mm. in length. For the sake of keeping correct the relative size differences of the drawings in this figure it has been necessary to make them on a smaller scale than those in figure 78. A. Right side of a 4- mm. embryo. It will be noted that the tail has just begun to grow out, that the positions of all the visceral clefts are apparent, and that the olfactory pits are present. The oral “suckers,” being now entirely ventral, are not actually visible from this point of view. The myotomes in this embryo and in B and C are very slightly indicated externally. B. Right side of a 6 mm. embryo. The external gills of the first and second branchial arches have begun to develop, concealing the second and third hranchial clefts. The stomodaeal invagination is deepening, and is slightly visible from the side. C. Right side of a 9 mm. embryo. ‘The external gills have grown considerably, and developed several lobes. From the posterior border of the lower portion of the hyoid arch, the operculum is just starting to develop, and thus covers slightly the region of the first branchial cleft. The stomodaeal invagination, scarcely visible from the side, has almost given rise to the mouth. D. Left side of a 14.5 mm. larva. The external gills have been covered by the operculum, and the gill chamber opens to the outside only through the spiracle. The eye is formed, the mouth is opened into the pharynx and its‘ lips are covered with raspers. The hind limb buds have appeared, and the tail has developed a finely veined memhraneous edge or fin.

a. Anus. I br.d. 2 br.cl. 3 br.cl. 4 br.cl. Rudiments of the first, second, third, and fourth branchial clefts. The corresponding arches and their positions are indicated in the text._ e. Eye. 1 eg. 2 eg. First and second external gills. hl. Hind limb buds. hy.c_l. iludiment of hyomandibular cleft. In. Mouth. ol.p. Olfactory pit. op. External indication of optic vesicle. ope. Edge of operculum. as. Oral “sucker.” pm. External indication of pronephi-os. spr. Spiracle. :t.i. Stomodaeal invagination.

170

ol. p. FROM HATCHING TO METAMORPHOSIS . 171

87, /1). Lastly, a short time before hatching there appears upon the upper part of the first and second branchial arches of each side a small lobed outgrowth; the rudiments of two pairs of external gills (Fig. 87, B).

The embryo (6-7 mm.), which is now ready to hatch, presently wriggles its way out of the surrounding jelly. From this time on it may be referred to as the larva or tadpole.

FROM HATCHING TO METAMORPHOSISV

Early Larval Life. —- For a few days after hatching, the young tadpole. which is a dark brownish color, lies on its side or remains attached to some convenient object by its V-shaped mucous gland. During the first part of this period the mouth is incompletely formed, and the animal is still dependent on the yolk for its nourishment. Meanwhile the two pairs of external gills develop rapidly, the original lobes of each gill putting forth several longer minor lobes or filaments (Fig. 87, C). There furthermore arises upon each third branchial arch a rudimentary third gill. This gill, however, never develops far, and is overlapped and concealed by those anterior to it. Aside from these features it will also be noted that the body and particularly the tail have increased in length, while the optic protuberances are still further back, as a result of the continued outgrowth of the snout. Upon the center of each of these protuberances, moreover, there frequently appears at this time a slight depression marking the external beginnings of the actual eyes which are soon clearly visible.

In another week or somewhat less (9-10 mm.),- certain further changes occur as follows. The mouth is opened and appears as a small round orifice armed with a pair of horny jaws and with lips covered by horny rasping papillae. At the same time the above mentioned mucous gland begins to atrophy, and the larva giving. up its fixed existence swims actively about in search of food. This consists of either animal or vegetable debris which it can scrape loose with its horny aws and lips; in captivity it will feed readily on any sort of cereal. In connection.with this change of. nourishment, the digestive organs are rapidly developed so as to give the body a full rounded appearance. This is particularly due tothe great increase in the length of the intestine which can be seen through the ventral body wall looking like a coiled spring.

As the above alterations occur in connection with the alimentary tract, certain changes also take place in the respiratory system, of which the following may be regardedas exterior. Posterior to the first and second branchial arches the incipient second and third branchial clefts be172 THE FROG: LATER on LARVAL. DEVELOPMENT

come opened into the pharynx by way of the corresponding pouches as actual clefts or gill slits. The first and fourth branchial depressions then presently become true clefts in a similar manner. Concurrent with these events there is also developing from the posterior border of each hyoid arch a fold of integument called the operculum. These opercula then grow backward on each side, covering the gills as they progress. They also grow toward one another ventrally until they meet and fuse. Thus a closed bronchial or gill chamber is formed which opens externally on the left side only, through a short funnel between the body wall and operculum, known as the spiracle (Fig. 87, D). It should finally be noted in this connection that as the closure of the branchial chamber is completed, the external gills start to atrophy and are replaced by internal gills upon the edges of the gill slits. These new organs will be

' more fully described in the discussion of internal changes.

Later Larval Life.—-After the attainment of the above condition during the first two or three weeks of larval life, development proceeds somewhat more gradually to the time of metamorphosis. During this

- interval, which may last for two or three months or sometimes over the

following winter, the larva increases considerably in size.‘ It also loses its brownish color and becomes more or less green dorsally, and white ventrally. Perhaps the most striking external feature, however, is the growth of the legs which begins at about the end of the first month. The fore legs develop first, but are not visible because they are covered by the operculum. The hind legs are easily seen as they arise at the base of the tail, and by the end of the second month they begin to show joints.

Experimental Results.——In connection with leg development a considerable amount of experimentation has been done to discover when the antero-posterior and dorso-ventral axes are determined, and what factors may be involved in the process. These experiments have been made on Amblystoma rather than the Frog, but it seems likely that results would be quite similar in the latter animal. The procedure consisted in reorientating the forelimb rudiment either in the normal (orthotopic) location or in some abnormal (heterotopic) location. Thus Harrison (’21) found that if in an embryo with a small tail bud (stage 29) this limb rudimentwere implanted dorsal side down in its normal place it would develop a limb with the dorsal side up, but with the antero-posterior axis reversed. Eventually of course a stage would

1 The larval condition is said to be prolonged by a cool season or a scarcity of

food. Also the larva of certain species, i.e., the Bull Frog, Rana catesbiana, normally passes through the winter before metamorphosis.

,»__?_R:,.,,.... METAMORPHOSIS 173

he reached where the dorso-ventral axis also could no longer adjust itself following inversion, but that obviously occurs at a later period. Other workers have confirmed and amplified these conclusions. Thus

Swett (’37, ’39, ’4l) showed that subsequent reversal of the dorso ventral axis of.a previously inverted limb is apparently due to factors in "the flank region, since inverted rudiments implanted in the region of the myotomes remain inverted. Also it appears that the effect of these flank factors may be blocked if tissue dorsal to the limb rudiment is included in the inverted implant.

It should be realized of course that all these cases are again simply

illustrations of special instances of the effect of one part upon another, i.e., induction.

METAMORPHOSIS

Usually under normal conditions the tadpoles of most species begin to frequent the surface of the water during the third month. Here they expel bubbles and gulp in air to supply the developing lungs. This is one of the signs that metamorphosis is near at hand, and at about the end of this month the final changes to the form of the adult Frog generally occur with relative rapidity. .

These changes are both internal and external. The former will be described more fully later. They involve, however, a complete development of the lungs accompanied by certain changes in the circulatory system. There is'also an enlargement of the stomach and liver, and at

- the same time a great shortening of the intestine. This change is appar ently correlated with the carnivorous habits assumed by the adult. Externally the alterations are no less fundamental,.and perhaps even more striking. The larval skin is cast 0H, and with it the horny jaws. The frilled lips likewise disappear and the mouth instead of being round becomes very wide. The tongue enlarges, and the eyes grow more prominent. The fore legs become visible by being thrust through the operculum. The left appears first because.it extends through the respiratory funnel on that side, while the right is forced to break through the opercular wall. At the same time, in company with the development of the lungs, the gills dry up and the gill slits opening into the opercular chamber are closed. The hind limbs, which have long been visible, increase greatly in length, and the tail is rapidly absorbed. Sexual differences both internal and external now become clearly evident. There are other minor changes, but those cited comprise the more prominent and important ones. . 174 THE FROG: LATER OR LARVAL DEVELOPMENT

The changes just described have of course been known for a very long time. It is only within recent years, however, that some of the activating factors have been uncovered by numerous experimenters. By appropriate removal, transplantation, and injection operations it has been pretty thoroughly demonstrated that as in the case of so many other bodily functions the prime mover of metamorphosis, so to speak, is the pituitary gland. This small, though extremely important, endocrine gland starts to hypertrophy as the time of change approaches. It, or more specifically the anterior part of it, then secretes a hormone which in turn activates the thyroid. The latter responds by secreting the thyroxin whichin this case brings about the various metamorphic

changes characteristic of the particular tissue and the specific animal concerned, B. M. Allen (’32), Atwell (’35), Atwell and Holley C36), Etkin (’36), Etkin and Huth (’39), Figge and Uhlenhuth (’33) and others.

In addition to this evidence as to the internal secretions involved in metamorphosis there have also been numerous experiments indicating how different tissues respond to the change in general body environment brought about by the endocrines. Thus Helfi (’29, ’30) has shown that tail muscle transplanted to the back atrophies at the time that the rest of the tail disappears, and the same has been demonstrated for the tail skin by Lindeman (’29). This might be anticipated, but it is more significant that back muscle and skin transplanted to the tail does not atrophy with the latter. Instead it simply moves up onto the back. An even more striking example of this is the case of eyes transplanted to the tails. In several successful operations the eye alsowmoved up at metamorphosis, and appeared on the rear end of the Frog (Schwind, ’33, Fig. 88).” These are situations with respect to tissues occurring within a single species. Another revealing result is obtained when Frog tail buds are transplanted to Amblystoma larvae. At metamorphosis, when the Amblystoma loses its tail fin, though not of course its tail, the well-developed Frog tail entirely disappears (as reported by Goldsmith at A. A. A. 5. meeting ’33) .

Thus in all these cases it seems clear that the fundamental bodily condition brought about by the endocrine secretions is similar. What differs is the kind of tissue. Indeed different tissues in the same animal obviously must differ in this respect, else a general condition causing

2 Though not stated, it is scarcely possible that these eyes were functional, even though pieces of brain were present in some cases. Hence these remarkable specimens were probably not blessed with both foresight and hindsight! Fig..88.—Photographs of stages in the metamorphosis of a Frog tadpole which had had an optic vesicle transplanted from another larvato the region of the tail at the tail-bud stage. Both tail and vesicle developed normally. Then when the tail was absorbed, the fully formed eye persisted, and was moved forward to the posterior end of the animal. Why was the eye not also absorbed? See text.

175 176 THE FROG: LATER OR LARVAL DEVELOPMENT

the atrophy of one would cause the atrophy of all. In that Went 110$ only would the tadpole tail disappear at metamorphosis, but the whole tadpole would vanish like the famous cat in Alice in Wonderland. Evidently likewise the difference in the behavior of similar structures, e.g., the tails in the Frog and in Amblystoma, is due to specific tissue differences in these structures.

These activities, it may be noted, are in some sense different from the inductive effects which have previously been cited as playing so fundamental a part in development. The difference, however, is probably not very significant. It must be assumed that in the case of endocrine activities the effects are, or may be, produced on tissues at some distance from the source of the inducing agent, in such instances called a hormone. In the cases of induction previously noted one must likewise assume the production of some chemical substance which produces its characteristic effects. Only in these latter instances the inducing agent “hormone” is only active upon tissues in contact with or very close to its source. There are further striking illustrations of the latter type to be found in connection with metamorphosis. One of these is the case of the histolysis of the opercular skin over the outpushing forelimb. This skin is partly broken by the pressure of the limbs. However, Helfl (’26) has shown that the histolytic action which aids this breakthrough is produced by the atrophying gills in the immediate vicinity.

We have now finished our survey of the external changes in the embryonic development of the Frog. In the description of internal changes, it will be most convenient, in so brief a discussion, to complete entirely the history of one system before taking up the next. In the case of each, however, as many references as possible will be made to the stages noted in the account of the exterior. With this aid the student is urged to correlate as often as possible the condition reached by one group of organs with that reached by another, as well as with external changes. Only in

this way is it possible to obtain a true conception of the growth of the animal as a whole. INTERNAL DEVELOPMENT: THE NERVOUS SYSTEM THE BRAIN

When last mentioned, this organ had been somewhat artificially divided into fore-, mid-, and hind-brain, and within the fore-brain the rudi ment of the infundibulum was vaguely outlined. Further development in the three divisions now occurs as follows: THE BRAIN 177

The Prosencephalon. —Somewhat previous to hatching, at about 4 mm., certain structures _,have developed which are characteristic of Vertebrate brains at early stages, and which are clearly evident in median sagittal sections as follows: To begin with the rudiment of the infundibulum already noted has become somewhat more pronounced. Proceeding anteriorly around the ventral side of the fore-brain, we encounter next a slight thickening, separated from another more anterior thickening by a narrow region where the wall is thin, giving the -effect

posterior boundary of mid-brain

scomodaeum


anteflor pituitary

mmeus any-locus endadnellmn

Fig. 89.——Median sagittal section of the anterior end of a 4- mm. Frog embryo. FB. Fore-brain. MB. Mid-brain. HB. Hind-brain.

of a depression. The posterior thickening next to the infundibulum is

the rudiment of the optic chiasma, though of course no nerve fibers are a

present in it at this time. The thin region anterior to it is the optic recess, and the more anterior thickening is the torus transversus. Continuing up unto the anterior wall of the fore-brain, we see a distinct thought-narrow outpushing slightly dorsal to the end of the notochord. It is the epiphysis (Figs. 89, 90).

As regards other developments in this region of the brain we-find that at about the time of hatching there grows out from the anterior end of the fore-brain a thin-walled vesicle, which represents the rudiment of the cerebrum. Presently its sides become thickened, and somewhat later (12 mm.), it is partially divided in two by a median longitudinal invagination of the anterior and the dorsal wall. The laterally compressed cavities of the resultant halves, or cerebral hemispheres, are then known as the lateral ventricles. Posteriorly they communicate with the main l '78

THE FROG: LATER OR LARVAL DEVELOPMENT

(1 Atrium. ao. Dorsal aorta. b Gall bladder. bk. Basihyal cartilage. c. Cavity of rudimentary cerebrum. e. E ithelial plug closing the oesophagus. ep. Epiphysis. g. Glottis. h. Hypoph sis. H. ind-brain. hr Cerebral hemisphere. ht. Horny “ teeth.” i. Intestine. if. In undibulum. 1'. Lower jaw. 1. Liver. ly. Laryngeal chamber. m. Mouth. M. Mid-brain. mb. Oral membrane (oral septum). n. Notochord. 0. Median portion of opercular cavity. oe. Esophagus. p.~ Pharynx. pb. Pineal body. pc. Pei-i~ cardial cavity. pd. Pronephric (more posteriorly mesonephric) duct. pt. Pituit

body. 'pv. Pulmonary vein. pIIL Choroid plexus of third plexus of fourth ventricle. r. Rostral cartilage. ro. Optic recess. s.

ventricle. pIV. Choroid Stomodaeum. su.

Sinus venosus. t. Thyroid body. ta. Truncus arteriosus. tp. Tuberculum posterius. v. Ventricle. vc. ‘Inferior (posterior) vena cava. of this region becomes folded and hangs down into the cavity of the

THE BRAIN 179

cavity of the fore-brain, or third ventricle, by a pair of openings, the foramina of Monro. During the remainder of larval life the hemispheres continue to grow forward and their walls to thicken. Their anterior ends become slightly constricted away from the main portion of the hemispheres as the olfactory lobes. At first these are separate, but later they become fused. Thus at metamorphosis when the cerebrum is virtually mature, it comprises half of the entire brain. Furthermore, on account of this cerebral increase and the direction of the growth, the relative proportion of the parts of the brain is so altered that the cranial flexure appears to vanish. As a matter of fact, however, it is actually unchanged. I Somewhat after the first appearance of the cerebral rudiment, i.e., at about 9 mm., a change occurs in the antero-dorsal wall of the third ventricle just below and slightly in front of the epiphysis. The thin roof‘

ventricle. Later these folds become very vascular. and are known as the anterior choroid plexus (Figs. 90; 91, B).

With the appearance of this final structure of the prosencephalon, it is possible further to subdivide this region as follows. Suppose a plane to be passed transversely through the third ventricle from the anterior side of the choroid plexus, to the anterior side of the optic recess between it and the torus transversus. The portion of the ventricle anterior to this plane is then termed the telencephalon, and the portion posterior to it, the diencephalon. On this basis it is evident that the cerebral hemispheres arise from the telencephalon and the anterior choroid plexus from the anterior part of the diencephalon.

Although the pituitary body, as already noted, is not strictly a part of the brain, its further history may best be described at this point. The backward growth of the anterior (stomodaeal) part of this organ continues, and at about the same time that the choroid plexus appears, it loses its connection with the stomodaeal ectoderrn. At the same time it acquires a cavity, and presently becomes united with the posterior (infundibular) part of the hypophysis, which retains its connection with the brain through the hollow infundibular stalk. Later the posterior portion of the anterior part of the hypophysis becomes convoluted and tubular. As regards terminology, it is to be remembered that the actu__al=G ,_ .. positions of the above mentioned “ parts ” are reversed in all adult_,§i!l§ ,- 4\ “*). phibia so that the anterior or stomodaeal part is really behind tl;éfipqsterior or infundibular part. Lastly in this connection it is of -i erést

_ , a to note that experiment has shown that neither the stomodaea gxlpu-"“"hb‘ i 180 E THE FROG. LATER OR LARVAL DEVELOPMENT

rm, I

Fig. 91.—Median sagittal sections through the brain of the Frog. From Von Kupfier (Hertwig's Handbuch, etc.). A. Of a larva of R. fusca of 7 mm. in which the mouth was open. B. R. esculenta at the end of metamorphosis.

c. Cerebellum. ca. Anterior commissure. cd. Notochord. ch. Hahenular commissure. cp. Posterior commissure. cpa. Anterior pallial commissure. cq. Posterior corpus quadrigeminum. ct. Tubercular commissure. cw. Optic chiasma. d. Diencephalon. dt. Tract of IV cranial nerve. e. Epiphysis. hm. Cerebral hemisphere. hy. Hypophysis (pituitary body). .7. Infundihulum. M. Mesencephalon. Ml. Myelencephalon. Mt. Metencephalon. p. Antero-dorsal extension of diencephalon. pch. Choroid plexus of third ventricle. R. Rhombencephalon. rm. Recessus mammillaris. ra. Optic recess. :22. Roof diencephalon. t. Telencephalon. tp. Tuberculum posterius. tr. Torus

transversus (telencephali). vc. Valvula cerebelli. vi. Ventriculus impar (te1encephali) (third ventricle). -,u—-n-gum-u~v———--—..-..._....._.......

THE BRAIN 181

fundibular part develops normally in the.absence of the other (Smith, ’20) . .

The Mesencepha1on.——The structures of the mesencephalon or mid-brain are not so numerous as are those of the fore-brain. Its chief features are the crum cerebri and the optic lobes. The former ‘arise gradually as a pair of ventro-lateral thickenings composed of nerve fibers connecting this portion of the brain with the fore-brain. The latter, i.e., the optic lobes. appear at about 9 mm. as a pair of swellings in the dorso-lateral regions of the roof. They attain their full size at about the time of metamorphosis, and their complete development is apparently dependent on the presence of normally developing eyes (Kollros, ’53). The cavity of the mid-brain serves to connect the cavities of the fore- and hind-brains, and is termed the aqueduct of Sylvius.

The Rhombencepha1on.—The rhombencephalon or hind-brain includes the metencephalon and the medulla oblongata. The principal development of the metencephalon immediately behind the mid-brain is quite limited in the Frog, the most prominent part being its roof which at about 9 mm. gives rise to a thickened transverse ridge, the cerebellum (Fig. 91). The medulla, on the other hand, is more extensive with a

thin roof. The latter always remains thin but at the same time that the .

cerebellum starts to develop it begins to become folded. Soon blood vessels extend down into these folds, and thus is formed the posterior choroid plexus (Fig. 90, B). The floor and the ventro-lateral walls of the hind-brain become thickened as nerve tracts. Its cavity connecting anteriorly by way of the aqueduct of Sylvius with the third ventricle,

and posteriorly with the neural canal, is called the fourth ventricle.

The Spinal Cord. -——'-Posterior to the brain region the neural tube gradually assumes the character of the adult spinal cord. The laterally compressed neural canal is, as already noted, lined by cells which were originally external. These are non-nervous and ciliated, and are known as ependymal cells. The relatively thick nervous layer which constitutes the bulk of the lateral walls gives rise to both supporting or gl_ia cells, and to neuroblasts or primitive nerve cells. The latter. lie relatively near the central canal, and comprise the so-called gray matter. The fibers which arise from them, however, course up and down

through the more superficial parts of the cord, helping still further to .

thicken it, and constituting the white matter. This thickening occurs first in the dorsal-lateral regions, thereby causing the neural canal to lie temporarily very near to the ventral side 132 THE FROG: LATER OR LAEVAL DEVELOPMENT

(Fig. 92, A). Gradually, however, the growth of cells and fibers spreads dowhward so that eventually the canal lies practically in the middle of the cord..The ventro-lateral growth, moreover, is slightly greater than

Fig. 92.—Transverse sections through the spinal cord of R. fusca. From Von Kupfier (Hertwig’s Handbuch, etc.). A. Through the anal region of a

larva of 7 mm. B. Through the anterior body region of a larva during metamorphosis.

a. Spinal artery. c. Central (neural) canal. d. Dorsal column (white matter). dw. Dorsal root of spinal nerve. dz. Atrophied dorsal cells. g. Gray matter. oz.

Ventral cells. w. Dorso-lateral and ventro-lateral column (white matter).

that exactly along the mid-ventral line. Thus a shallow depression occurs here in which runs the spinal artery (Fig. 92, B).

Posteriorly the neurenteric canal becomes severed even before hatching, and the nerve cord continues straight out into the tail. This portion of the cord is of course lost at metamorphosis.

THE PERIPHERAL NERVOUS SYSTEM

The Cranial Nerves. — In discussing nerves in general, it is quite customary to divide them into afierent or sensory nerves, and efierent‘ THE PERIPHERAL -NERVOUS SYSTEM

183

F Fig. 93.-—Sections through young Frog embryos (R. fusca), illustrating the development of,the crest segments (“ ganglia”) and plaoodes. From Kellxcott (Chor~ date Development). After Brachet. A. Transverse secuon through the neural plate of an embryo before elongation begins. B. Sagittal section to one slde of the mulline, through an embryo of the same age as A. C. Sagtttal sectmn, to one s1de_o{ the mid~line, through an embryo just beginning to elongate. D. Transverse section througll; an emklirylorslightlfy older thanf that ocf‘ A 231.1:-ltd B. _ E. Friongal iafectfirri thrtglgh an em ryo wit 1; ee or our pairs 0 meso erm somxtes. , , . ee usverse sections through an embryo just beginning to elongate (same age as C ), showingl the) trigeminal, acustico-facial and glossopl1aryngeal~vagus crest segments ‘ gang ia . af. Acustico-facialis crest segment (" ganglion ’’l. c. Notochord. en. Endoderm. g. Gut cavity. gl. Glossopharyngeal crest segment (‘f gan_g11on”). gv. Glossophary1igeal-vagus crest segment (“ganglion”). l. Llver dxvernculum. m. Mesoderm. mp. Primitive medullary plate. mpd. Definitive medullary plate. ’r’u:. Neural crest. s. Mesodermal somites. tg. Trigeminal. crest segment (‘_‘ ganglion 3- mt. vagus (pneumogastric) crest segment (“ ganglion”).

mw».,.,_ Fig. 94.—Portions of sections through the head of the Frog (R. fusca), illustrating the formation of the placodes and the history of the crest segments (“ganglia ”). From Kellicott (Clzordate Development). After Brachet. A. Transverse section through the trigeminal crest segment (“ ganglion ”) of an embryo of 3 mm. B. Transverse section through the trigemmal crest segment (“ ganglion ”) andplacode of an embryo with three or four pairs of mesodermal somites. C. Transverse section through the facial ganglion and auditory placode of an embryo of 2.8 mm.

ei. Inner or nervous layer of ectoderm. en. Endoderm. eo. Outer layer of ectoderm. m. Mesoderm. mpd. Definitive medullary plate. n.-Nerve cord. pa. Auditory placode. pf. Facial placode. ptg. Trigeminal placode. r. Spinal prolongation of ganglion. tg. Trigeminal crest segment (“ ganglion ”).

Q THE PERIPHERAL NERVOUS SYSTEM 185

or motor nerves. In describing both the cranial and spinal nerves, however, it is convenient to add a third category, i.e., mixed nerves, which contain both afferent and efferent fibers. It is understood that all these nerves occur in pairs, but it will be necessary to describe the development only on one side.

Purely A flerent Nerves. ——There are three cranial nerves which are purely afferent; namely, the I or olfactory nerve, the II or optic nerve, and the VIII or auditory nerve. The first two are of a rather special nature, and are also very closely connected with the development of the sense organs which they supply. It will therefore be more convenient to describe them later in connection with those organs. The VIII nerve on the other hand arises in such close connection with the mixed nerves that it will be described under that category.

Mixed Nerves and the Auditory Nerve.——The nature of the neural crests has already been indicated, and ‘it was noted that each crest becomes divided into segments. In the brain region there are three such segments on each side of the head. Considerably before hatching (3-4 mm.) , moreover, the nervous layer of ectoderm on the inside of the head opposite the segments becomes thickened into patches termed placodes, one opposite each of the first two segments, and two opposite the last. It is then from certain nervous or ganglionic elements of these structures, i.e., the crest segments and placodes, that the ganglia of the V, VII, VIII, IX, and X nerves (Fig. 93) and their afferent fibers develop in the manner indicated below. The efferent fiber origins of all mixed nerves will be noted separately. It remains to state that the strands of cells attaching the crest segments to the brain merely contribute to the sheaths of the nerves whose origins are being described.

The V or trigeminal nerve ganglion develops from dorsal and superficial cells (the ganglionic element) of the most anterior crest segment along with cells derived from the inner or ganglionic portion of the corresponding placode (Fig. 94-, B). The anterior part of the ganglion arises almost entirely from the anterior portion of the placode, and produces the afferent fibers of the ophthalmic branch of the V nerve. The posterior part consists of both crest and placode elements, and is sometimes distinguished as the trigeminal ganglion proper, or Gasserian ganglion. This part produces the afferent fibers of the maxillary nerve which are derived from the placodal element, and afferent fibers of the mandib ' ular nerve which seem to come from the crest element (Knoufi, ’27).

From both parts of the ganglion a common bundle of fibers also grows inward to the medulla constituting the sensory element of the V nerve


.;.,;,,,.-.,..,.....,.,.‘..,._~;._._..; .,—.-Q_,... —,.o_-. 186 THE FROG: LATER OR LARVAL DEVELOPMENT

root. The ophthalmic branch of the nerve is destined for the skin of the snout, while the mandibular and maxillary branches supply the lower and upper jaw. As all of these branches start to develop previous to hatching, in a 9 mm. tadpole they are well established. It may be added that the non-nervous part of the crest segment, in this instance the major part, grows ventrally and contributes to the mesenchyme of the mandibular arch. The superficial (outer) non-nervous part of the

placode, on the other hand, disappears. It is how believed that the sensory elements of the VII or facial gan glion and nerve come exclusively from the second placode, while the sheath cells are both crest and placodal in origin. At least this has been proven for Amblystoma (Yntema, ’37) , and seems likely to be true also in the Frog. As before, some of the fibers which issue from this ganglion proceed inward to the medulla, forming the sensory element of the root, while others grow outward as the aiferent fibers of the nerve. Before hatching, the latter have divided into the hyoid and palatine branches. Here also the considerable non-nervous part of the crest contributes in this case to the mesenchyme of the hyoid arch. No part of the placode in this instance, however, disappears. One portion is utilized as just described, while the remainder goes to form the ganglion of the VIII nerve and the auditory apparatus, as indicated below.

The IX and X or glossopharyngeal and vagus (pneumogastric) ganglia arise from the ganglionic portion of the last cranial crest segment in conjunction with the inner, i.e., ganglionic part, of the third and fourth placodes respectively. In these cases both crest and placode contribute neurons as well as sheath cells. Fibers from these two ganglia enter the medulla as a single root. Peripheral outgrowths from the IX ganglion supply the first branchial arch, While branches from the X pass to the remaining branchial arches. The vagus ganglion also sends branches to the viscera and to the lateral line organs (see below), the nerves to these parts being entirely placodal in origin. At least this appears true for Amblystoma (Yntema, ’4-3) , the situation in the Frog not having been so extensively investigated. Both of these ganglia with their nerves develop quite early, and in a 9 mm. larva all the main branches of the vagus nerve are present. In this case the non-nervous part of the crest segment is not large, but, so far as itdexists, it goes to form mesenchyme. The superficial non-nervous portions of the placodes disappear.

It may now be added that the efferent fibers (axones) for each of these four nerves (V, VII, IX, and X) grow out from neuroblasts in the THE PERIPHERAL NERVOUS SYSTEM

walls of the medulla. They pass out of the brain along with the sensory root fibers of the respective ganglia, and having passed through these ganglia they enter the outgoing branches of the mixed cranial nerves.

The VIII or auditory nerve is, as already noted, entirely sensory, and its ganglion arises from the ganglionic portion of that part of the second placode which is not involved in the formation of the ganglion of the VII nerve. The more superficial portion of this placode as usual is not included in either the VII or VIII nerve ganglion, but nevertheless, as suggested above, it does not in this instance disappear. Instead it remains in close contact with the latter ganglion, and develops later into the so-called inner ear, as described below. Because of the prominent part which the major portion of this second placode then plays in connection with the auditory apparatus, it is frequently referred to as the auditory placode (Fig. 94, C l, already noted in the account of an earlier stage (Camphenhout, ’35) . The roots of the VII and VIII nerves are indistinguishable from one another previous to the opening of the mouth (9 mm.). '

Purely E flerent Nerves. — The III, IV, and VI nerves are all motor ocular nerves which innervate the muscles of the eye. Their development is imperfectly known,

187

Fig. 95.—_-Transverse section through 8.6 mm. larva of R. escalenta, illustrating the relations of the sympathetic cord and spinal nerve. From Kellicott (Chordate Development). After Held.

a. Dorsal aorta, c. Spinal cord. d. Dorsal (sensory, aEer ent) root of spinal nerve. m.,

Myotorne. n. Notochord. r. Ramus communicans. sc. Sympathetic cord. sg. Spinal ganglion. sn. Spinal nerve trunk. 11.’ Ventral (motor, efferent) root of spinal nerve.

but they seem to arise from neuroblasts in the mid-brain and medulla. .The III appears first, just before hatching, the others slightly later. The Spinal Nerves.—The ganglia of the spinal nerves, unlike those of the cranial nerves, arise entirely from the neural crests, no placode elements in this case being involved. The division of the originally continuous crests of this region into the segments which eventu188 THE FROG: LATER OR LARVAL ‘DEVELOPMENT

ally become the ganglia is apparently conditioned, moreover, by the previous segmentation of the myotomes (Lehman, ,2?! Detwilers ’37)Also if more or fewer myotomes are experimentally produced the related ganglia are correspondingly increased or decreased in number (Detwiler, ’34-). From each crest segment, fibers grow inward and con nect with the dorsal part of the cord. These are known as the dorsal

root: of the spinal nerves (Fig. 95). At the same time other fibers grow outward to the skin, and other sensory organs; as in the head, all of these ganglion fibers are afferent.

~ While this is occurring dorsally ventral nerve roots also arise (about 4mm.) . Each of these roots consists of a bundle of fibers (axones) originating from neuroblasts in the ventral part of the spinal cord. This has

. been confirmed experimentally by removing parts of the cord while leav ing the crests, in which case the ventral roots are absent (Taylor, ’44) . At or just beyond each dorsal root ganglion the fibers of the respec tive ventral bundle mingle in a common sheath with the outgoing fibers

of the'ganglion. Thus, since the ventral root fibers are all efferent, each

'nerve sheath containing both sorts constitutes a mixed nerve (spinal

nerve trunk) as in the cases of a similar condition in the head. This trunk soon divides into a dorsal and a ventral branch, each of which now contains both afferent and eflerent fibers; the former pass to the various sense organs and the latter to the muscles.

The problem of how these and other fibers are directed to their proper destinations has long been of interest, and is not yet completely solved. There does appear to be a tendency, however, for outgrowing nerves to proceed toward certain kinds of tissue more than toward others. Thus Detwiler (’36) has shown that whereas transplanted pieces of brain failed to attract such nerves, transplanted limb, eye, and nasal placode do so in the order indicated. Even so the attraction is apparently not very specific, i.e., certain nerves are not inevitably attracted to their normal muscles, as shown by somewhat displacing the sources of the nerves (Piatt on Amblystoma, ’4-0). The nature of such general attraction as there may be is not known, but may_ be tentatively assumed to be both mechanical and chemical in character. Finally it may be noted that there is also a question as to what causes more anterior parts of the spinal cord to contain more nerve cells than the relatively caudal parts. There has been some evidence that what a given segment contains is dependent to some extent on the character of the part anterior to it. Thus if a piece of spinal cord were substituted for the medulla this might be expected to lead to fewer cells and fibers in the cord posterior to the ORGANS OF SPECIAL SENSE 189

implant. Such, however, seems not to be true in this case, thus suggesting, to a certain degree at least, an inherent developmental capacity in various levels of the cord (Detwiler on Amblystoma, ’37).

The Sympathetic System. —- In the sympathetic system the neuroblasts have been shown to originate both from the neural crests and the neural tube, while the sheath cells come entirely from the latter. At least

this has been demonstrated experimentally for Triton by replacing part ' of its neural tube by easily distinguishable material from Axolotl (Ra audltory _ I vesicle 4 Wm ° u‘.,;,‘|;, pigment layer < . “"4 <79“ of r ' ‘ lens _ rudiment P°"=l°|‘ 0'


oral "~ ~ ’ . : T ev-«mm « 


Fig. 96.——Cross sections of 4 mm. stage of Frog embryo. A. Section through the optic cups starting to form the vesicles. B. Section through the auditory vesicles and extreme anterior of the heart rudiment. This section also passes through the pharyngeal region at the level of the third visceral or 1st hranchial arch.

ven, ’36), and it is probably true of other Amphihia. The cells of the sympathetic system first appear, however, in small collections upon the spinal nerves at about the level of the dorsal aorta, a position in which they may be noted shortly before hatching. Presently they migrate to the aorta, along each side of which they give rise to a sympathetic cord. From these cords, nerve fibers later grow backto the spinal ganglia, as the rami communicantes. Still other fibers proceed to the viscera, and along these, cells migrate to form the various peripheral sympathetic ganglia.

ORGANS OF SPECIAL SENSE

The Eye. —- When the rudiments of the eye were last considered the optic stalks were just beginning to be defined as such, owing to a slight constriction between the optic vesicles and the brain. This process is now rapidly completed so that the stalks are clearly indicated. It is then evident that they do not join the vesicles exactly at the centers of the latter but nearer to their ventral sides. There then begin certain changes ‘in connection with the vesicles themselves as follows: 19o '_ THE FROG: LATER OR LARVAL DEVELOPMENT

The wall of each vesicle next to the ectoderm is flattened and then pushed inward. By this process the cavity of the optic vesicle is obliterated, and at the same time a double-walled cup is formed, the optic cup (Figs. 96, 97, 98). It must be noted, however, that the direction of this imagination is not exactly horizontal. It begins rather in the ventrolateral region and proceeds obliquely upward. This fact, together with

T the original relation of the vesicle and stalk, means that the latter will necessarily be attached to the cup at its ventr-al edge. The rim of the cup now grows outward, particularly in its ventral and lateral regions, these being the regions which, as a .result of the direction of invagination, are further from the ectoderm. This outward extension of the sides of the cup leaves between their ventral edges a slight fissure extending inward to the optic stalk. This is the choroid fissure, whose length is somewhat further increased by the continued outgrowth of the sides of the cup. Furthermore, concurrent with this outgrowth the entire rim begins to bend toward the center of the cup’s aperture, thus obviously decreasing its diameter. This aperture,


Fig. 97.--Plastic figure of hemiseoted optic vesicle, lens and optic stalk of the Frog. From Kellicott (Chardate Development) .

f. Choroid—fissure. l. Lens.

pc. Posterior (Vitreous)

chamber of eye. pl. Outer or pigmented layer of optic cup. rl. Inner or retinal layer of optic cup. s. Optic stalk. v. Orig which faces the ectoderm, is the pupil, from whose ventral edge the choroid fissure runs back to the optic stalk.

ma] cavity °f °Pfi° V‘i5i°1°' Meanwhile, about the time of hatching, a

thickened portion of the inner ectoderm on the wall of the head opposite the pupil becomes constricted off as a solid rounded mass of cells (Fig. 98). This is sometimes, though erroneously, called the visual placode. It presently acquires a central cavity, which is soon obliterated, however, by the thickening of the cells on the future retinal side. This mass now moves in to the center of the pupil, and becomes the lens. The invagination of the ectoderm to form the lens appears to be induced by the adjacent optic vesicle (Beckwith, ’27), though the competence of all ectoderm so to respond has been questioned. Thus, in this as in some other cases, the ability of ectoderm to react specifically at a given stage seems to depend upon its earlier subjection to another inductive agent, e.g.,’ the mesentodenn (Liedke, ’51). On the other hand, under ORGAN‘-5 OF SPECIAL SENSE 191

Fig. 98. — The development of the eye in the Urodele, Siredon pisciformis. From Kellicott (Chordate Development). After Rab]. A. Of embryo with about twenty-five pairs of somites, showing the thickening of the lens rudiment. B. Invagination of the lens and formation of the optic cup. C. Lens separating from the superficial ectoderm in an embryo of about thirty-five pairs of somites. D. Thickening of the inner wall of the lens. E. Shortly before hatching; differentiation of the rods and cones in the retinal layer.

a. Anterior chamber of eye. c. Cavity of primary optic vesicle. co. Cornea. e. Ectoderm of head. f. Choroid fissure. i. Inner or retinal layer of optic cup. ir. Rudiment of iris. Ic. Optic stalk. 1. Lens. 0. Outer or pigmented layer of optic cup. p. Posterior (vitreous) chamber of eye. 192 THE FROG: LATER OR LARVAL DEVELOPMENT

certain conditions it is known that if at the neural fold stage a lens has been removed it can only be replaced by cells derived from the dorsal rim of the iris (see below).

Shortly after hatching the cells in the walls of the optic cup begin to differentiate. The inner wall thickens and develops into the retina, its outermost. cells becoming the rods and cones. Its inner cells, i.e., those toward its cavity, form neuroblasts which send axones over the inner surface just beneath the thin internal limiting membrane, which is produced from fibers growing out from non-nervous cells deeper in the retina. The axones, leaving the cup through the inner end of the choroid fissure, grow within the substance of the ventral wall of the optic stalk to the ‘brain, where those from opposite sides cross to form the optic chiasma. The ventral wall of the stalk, thickened by its axones, soon obliterates the stalk lumen, the other stalk cells disappear, and the neural sheath is formed of connective tissue, the axones and sheath cells together constituting the II or optic nerve. The outer wall of the cup adjacent to the rods and cones develops pigment, and hence is called the pigment layer of the retina.

Slightly before hatching the lips of the choroid fissure begin to fuse, and shortly this fusion becomes complete everywhere except next to the optic stalk, where the blood vessels and axones leave the cavity of the cup. At the edge of the pupil the ‘closure is marked by a thickening, the choroid knot, from which arise the cells of the iris. This closure of the fissure is said not to occur in the absence of the lens (Beckwith, ’27) .

’The vitreous humor is formed in the cavity of the cup by cells budded from the retinal wall and from the inner side of the lens. It is thus entirely ectodermal. The choroid coat of the eye is laid down outside the pigmented layer, and outside of all is the tough sclerotic coat. Both the choroid and sclerotic tissues are derived from mesenchyme. Opposite the lens the ectoderm of the head becomes transparent, and, again with the addition of mesenchyme, forms the cornea. The detailed development of the eye is not entirely completed until metamorphosis.

The Eat.

The Inner Ear or Membranous Labyrinth.-—Just before hatching the superficial part of the auditory placode, i.e., the part not involved in the formation of the VII and VIII nerve ganglia, moves in slightly from the ectoderm. At the same time it invaginates to form a closed membranous vesicle, the auditory sac or otocyst. By appropriate transplantations it was shown that the differentiation of this sac is induced ORGANS OF SPECIAL SENSE

VIII‘

Fig. 99.—The development of the auditory organ in the Frog and Toad. From Kellicott (Chordate Development). A, B, F. After Krause. C, D, E. After Villy. A. Section through the auditory vesicle of an embryo just beginning to elongate. B. Section through the auditory vesicle that has very nearly separated from the superficial ectoderm. C. Transverse section, somewhat oblique, through the auditory organs of a 12 mm. R. temporaria. D. Slightly more advanced stage than C. E. Section through the auditory organs of a 25 mm. R. zemporaria. F. Membranous labyrinth of the Toad (Bufovulgaris).

a. Auditory sac. zztz. Anterior ampulla. ac. Anterior vertical semicircular canal. b. Pars basilaris. d. Dorsal outgrowth of primitive auditory vesicle (rudiment of endolymphatic duct). e. Endolymphatic duct. g. Ganglion of auditory (VIII) nerve. hc. Horizontal semicircular canal. Z. Lagena or cochlea. pa. Posterior ampulla. pc. Posterior vertical semicircular canal. 5. Saccule. ss. Sinus superior. u. Utricle. VIII. Auditory nerve.

193 194 THE EROG: LATER OR LARVAL DEVELOPMENT

by the presence of the medulla, and also to some extent by the roof of the archenteron. This seems to be true even when the medulla is from a different species of Amphibian. As in the case of lens induction, however, it again appears that ectoderm near the normal site is more competent to respond in this manner than that from elsewhere (Albaum and Nestler, ’37, and- Zwilling, ’4-1). From the dorsal wall of the otocyst a small evagination now appears which is the rudiment of the endolymphatic duct (Fig. 99, A, B). An oblique partition then (10-12 mm.) begins to grow across the cavity of the otocyst in such a way as to divide it into a lateral and ventral portion, the saccule, and an upper and median portion, the utricle. These cavities remain connected by a small poie in the membrane (Fig. 99, D). .

During the growth of the above partition there appear upon the inner surface of the wall of the utricular portion of the otocyst, two pairs of ridges. One pair is vertical and anterior, the other horizontaland lateral, upon the side nearest the ectoderm. Presently (15 ’mm.), there is added another pair which is posterior and vertical. The edges of each pair of ridges now fuse with one another along their entire length, thus giving rise in each case to a tube open at each end into the cavity of the utricle. The tubes thus formed are the rudiments of the three semi-circullzr canals. From the manner of their formation these tubes or canals evidently lie upon the inside of the utricular wall. Shortly, however, each canal pushes outward and presently becomes constricted away from the wall of the utricle except at its ends. The canals which thus come to lie outside of the utricle now continue to grow, and so reach the adult condition. During this latter process, however, each canal acquires an enlargement at one of its ends termed an ampulla. These ampullae are not developed from the canals themselves, but are added to them through a further constricting off of portions of the utricle (Fig. 99, E, F). A

Meanwhile the saccule in the course of its separation from the utricle has become the part of the otocyst which receives the endolymphatic duct. The two ducts, one from each side of the head, then grow up over the brain; during this process their ends become enlarged (at about 20 mm.) to form the endolymphatic sacs. By the time of metamorphosis, these sacs have increased greatly in size, have become very vascular, and fused with each other. In the adult they form a considerable vascular covering for the myelencephalon. It is also stated by Wilder (’09) that in all the Anur-a an ‘outgrowth from each endolymphatic sac extends down along the side of the dorsal nerve cord outside the dura ORGANS OF SPECIAL SENSE 195

mater. Where each spinal nerve root emerges an extension from these outgrowths also emerges, and forms asmall pocket partially wrapped around the respective spinal ganglion. These pockets are filled with calcareous material, and it is this whitish substance seen through the pocket wall that one observes when viewing the “ ganglia ” in a gross dissection of the Frog.

In larvae of 15-20 mm. the saccule is also giving rise to two other structures as follows: From its upper portion the lagena or cochlea arises as a postero-ventral evagination, while a similar and slightly more dorsal outpushing, in close connection with the first, constitutes the basilar chamber (pars basilaris) (Fig. 99, F).

Sensory patches develop on the inside of the epithelial walls of the utricle, saccule, cochlea, and ampullae, and these are connected with branches of the auditory nerve which proceeds from its ganglion. The

. entire membranous labyrinth thus formed is eventually encased in car tilage and bone arising from the surrounding mesenchyme. The casing follows the contour of the membrane, and constitutes the auditory capsule. There is a slight space between the capsule and membrane, the perilymphatic space, and this is filled with perilymphatic fluid.

At this point experimental procedures have again been applied which show that not only is the membranous otocyst produced by induction, but that it in turn induces the formation of the cartilaginous capsule around it (Kaan, ’38). Apparently not quite any mesoderm is competent to react in this way, but at least that of the head region and some of the somites will do so. Kaan also noted a reciprocal action in that a normal capsule was necessary to induce the membranous otocyst to go on and develop a normal membranous labyrinth. Thus we see a good illustration of the continuous actions and reactions in a developmental system that has once been set going. _

The Middle Ear. —This portion of the auditory organ develops chiefly during and after metamorphosis, as follows: The vestigial visceral pouch between the mandibular and hyoid arches, i.e., the hyomantlibular, produces from its dorsal end a rod of cells with a terminal knob. This rod grows out until the knob reaches a position between the inner ear and the wall of the head. A cavity then develops in the knob and in the rod of cells. The cavity in the knob is the tympanic cavity, and that in the rod the Eustachian tube, which connects the cavity with the pharynx. The tympanic cavity, or cavity of the middle ear, increases in size until its outer wall fuses with the ectoderm. The membrane thus formed is the tym panic membrane or ear drum, separating the tympanic 196 ‘THE FROG: LATER OR LARVAL DEVELOPMENT

cavity from the exterior. This membrane it may be noted has a special histological character, and Hellf (’28) has proven that this Character is induced by the presence of two pieces of cartilage. One is the annulus tympanicus, a ring-shaped structure which surrounds the membrane at -its periphery and supports it. The other will be indicated presently. Hellf (’34)- has shown further that rings of cartilage cut from the supra scapula have a slight tendency to produce changes in the ectoderm similar to those produced by the annulus tympanicus. He has also shown that rings cut from the palato-quadrate cartilage (see account of skeleton) will act just as well as the annulus tympanicus itself. This last fact is significant for the following reasons: In the lower Vertebrates the palato-quadrate forms a part of the upper jaw, and it has long been suspected that a small part of it survives in the higher members of this group as a bone of the middle ear. Such a hypothesis is obviously

strengthened by this observation of the similar peculiar inductive quali- ties possessed by both palato-quadrate and annulus tympanicus. Continuing with the history of the middle ear, we find that opposite to the tympanic membrane the wall of the tympanic cavity contacts the auditory capsule. Here there is an aperture in the latter, the fenestra ovalis, opening into the perilymphatic space. In this aperture there develops a cartilaginous plug, -the operculum. Across the roof of the tympanic cavity there is also formed a cartilaginous rod connecting the operculum with the tympanic membrane. It is the plectrum or columella, and is thought to be a vestige of the upper part of the hyoid arch. It will be recalled that the histological character of the tympanic membrane is due to two pieces of cartilage one of which is the annulus tympanicus. The other is the columella, without which the peculiar yellow fibers of the membrane are not formed (Helff, ’31). Finally, at the close of metamorphosis, the columella separates from the dorsal wall of the tympanic cavity, so that it stretches freely from the tympanic membrane to the operculum. The columella and operculum then fuse, and the latter and part of the former become ossiiied. Interestingly enough in the larvae of some Frogs a temporary so-called bronchial columella connects the inner ear and the lung (Witschi, ’55). This is suggestive of the ossicles connecting the air bladder and the inner ear in some Fish. There is no outer ear, the tympanic membranes appearing on the outside of the F rog’s head. « _ The Olfactory Organ.—In the account of the external developments, we have already referred to the olfactory pits, which are evident, »even in a 2.5 mm. larva. Each is situated slightly above and anterior to ORGANS OF SPECIAL SENSE 197

Fig. 100.—-The development of the olfactory organ in R. fusca. From Kellicott (Chordate Development). After Hinsberg. A, B, C. Sections through the olfactory pit and organ of 5 mm., 6 mm., and 11 mm. larvae, respectively. D.” Medial View of a model of the olfactory organ of a 31 mm. larva. The dotted line marks the limit

between the sensory and non-sensory portions of the epithelial lining of the olfactory cavities.

c. Hypophysis. ch. Internal nares (choanae). d. Dorsal lumen. dc. Dorsal sac. en.

External nares. g. Olfactory pit. 1'. Cut edge of integument. in. Internal nares (choanae). I. ‘Elongation toward the mouth. la. Lateral appendix. m. Mouth cavity. n. Inner or nervous layer of ectoderm. ns. Part of chamber lined with non-sensory epithelium. p. Olfactory placode. r. Ridge marking the limit between middle and ventral chambers. s. Superficial layer of ectoderm. se. Part of the chamber lined with sensory epithelium. st. Stomodaeum. t. Telencephalon. v. Thickened bands of superficial ectoderm cells (possibly the vestige of a primitive sense organ). ,vc. Ventral sac. zig. Ventral nasal gland attached to Jacobson’s organ. x. Elevation~around

external nares. y. Canal leading to olfactory cavity. z. Fold around internal narial opening. I'_ 11 " LA _

"y'TV'f I r .. 1.5!’: . > _, !‘;"’)::

a. Auditory vesicle (in A, its rudiment). b. Basement membrane of epidermis. ch. Notochord. g. Gut. gV. Trigeminal ganglion, of V cranial nerve. gVIIl. Acoustic ganglion of VIII cranial nerve. gX. Vagus ganglion. gXl. Ganglion of lateral nerve (branch of the vagus). i. Intersegmental thickenings of epidermis (ectoderrn). I. Rudiment of lateral line nerve. lp. Lateral plate of mesoderm. my. Myotomes. 1:. Inner or nervous layer of epidermis (ectoderm). nc. Nerve cord. 12. Pigment in epi dermis. 5. Superficial layer of epidermis (ectoderm). si. Inner sheath cells of lateral

line organ. sn. Sensory cells of lateral line org'an. 50. Outer sheath cells, of lateral line organ.

198 ORGANS OF SPECIAL SENSE 199

the side of the mouth. As these pits form, the superficial epithelium in this case disappears, while the inner invaginating layer thickens. These thickenings, which thus constitute the walls of the pits, are the olfactory placodes already indicated (Figs. 83, 100). Compare with Figure 88 of the exterior for general location.

A little after hatching there grows inward and downward from the floor of each pit a solid rod of cells. These rods presently become connected with the buccal cavity just at the posterior limit of the stomodaeum, and in tadpoles of 12 mm., each has acquired a lumen. Their openings into the cavity thus constitute the internal nares.

Somewhat later the olfactory lobes develop from the cerebrurn, as indicated above. From each of these lobes, cells are then proliferated, which mingle with other cells derived from the placodes. The two strings of tissue thus constituted seem to become the sheaths of the I or olfactory nerves. The actual fibers of these nerves, however, arise from neuroblasts in the placodes, and grow backward to the lobes.

Meanwhile the pits are enlarging as the nasal cavities, and the remainder of the placode cells line them as the nasal epithelium. In the course of growth the cavities are removed somewhat from the surface

' of the head, but remain connected with it by tubes whose outer open ings form the external nares. Changes in the shape and the proportion of the head alter from time to time the direction of the olfactory tracts. Thus these tracts first become vertical rather than horizontal, and later during metamorphosis develop a sharp flexure, due to the backward movement of the internal nares. At this latter period, also, each of the nasal cavities becomes greatly modified by complex evaginations and foldings. Of the former the most prominent arises ventro-medially from each cavity. The two bodies thus produced are the organs of Jacobson; they later acquire glandular masses at their medial ends.

The Lateral Line Organs. —At about 4 mm., a small dorsolateral portion of the vagus ganglion of each side separates from the remainder and unites with a part of the most posterior or fourth placode. The placode then grows backward through the epidermis until, just before hatching, it reaches the tip of the tail (Fig. 101). At intervals along this cord there meanwhile arise groups of sensory cells which push their way to the surface and develop hair-like processes. These organs are innervated by a branch from the X nerve ganglion constituting the ramus lateralis (lateral line nerve). Other similar sensory organs develop in rows on the head,_and are innervated by branches of the VII, IX, and X nerves. All these organs disappear at metamorphosis.

.4 7”"”'\ 200 THE FROG: LATER on LARVAL DEVELOPMENT

INTERNAL DEVELOPMENT: THE ALIMENTARY CANAL ’ AND DERIVATIVES '

THE MOUTH

‘When last described, the endoderm in the antero-ventral part of the pharyngeal region of the fore-gut had pushed out an evagination toward the ectoderm. The ectoderm had also “pitted in ” toward this evagination to form the stomodaeum already noted. The stomodaeal wall now meets and fuses with the endodermal wall in this region forming the oral plate or oral membrane (Fig. 90, A). A few days after hatching (about 9 mm.), the oral plate becomes perforated, and henceforth the stomodaeal cavity or mouth communicates freely with the pharynx. The margins of the small larval mouth are formed fundamentally of the mandibular ridges, i.e., the outer edges of the mandibular arches. Outside of these ridges, however, the skin is drawn forward to form the dorsal and ventral lips.

The dorsal lip of the larva soon develops three medially incomplete rows of “teeth.” Each of these teeth is formed from a cornified ectodermal cell which is periodically replaced by a similar cell pushing up from beneath. The ventral lip has four rows of such teeth; these rows, however, are complete. At the base of each lip, parallel with the rows of teeth, is a hardened ridge or jaw, also formed of cornified ectoderm.

At metamorphosis the horny teeth and jaws are lost, the adult jaws being of course much wider than those of the larva and formed largely of elements derived from the mandibular arch (Marshall). The permanent teeth occur only on the upper jaw, and are similar in their general structure to mammalian teeth. The tongue develops at this time from a proliferation of cells in the floor of the pharynx.

THE FORE-GUT AND ITS DERIVATIVES

The Visceral Arches and Pouches. —The beginnings of the first three pairs of pouches arising as solid vertically elongated evaginations of endoderm have already been indicated. The most anterior pair are the rudiments of the hyomandibular pouches, whereas the second and third pairs are the rudiments of the first and second branchial pouches. There presently arise three more pairs of these solid rudiments, making in all six pairs, one hyomandibular and five branchial, the last pair, however, being mere vestiges. The condition of both pouches and arches at hatching may be summed up in the following manner (Fig. 102): THE FORE—GUT AND ITS DERIVATIVES

With the exception of the sixth and last, the pbuch rudiments, as noted, push out until they finally reach and fuse with the ectoderm of the corresponding clefts. They thus divide the

mesoderm into the following bars or 3

visceral arches: (1) the mandibular arch in front of the first or hyomandibular pouch; (2) the hyoid arch between the hyomandibular pouch and the first branchial pouch; (3) the first branchial arch following the first branchial pouch; (4) the second branchial arch following the second branchial pouch; (5) the third branchial arch following the thirdbranchial pouch; (6) the fourth branchial arch, poorly defined, and following the fourth branchial pouch. There are thus six arches in all, beginning with the mandibular arch in front of the hyomandibular pouch, and ending with the fourth branchial arch in front of the last vestigial fifth branchial pouch.

The further development of the gill slits and gills has already been partially described in the account of the exterior. Nevertheless, it will be well at this point to recall the main features indicated, and to add certain details.

It will be remembered that, at about the time the mouth opens, the pharynx was said to be placed in communication with the exterior by means of the. four pairs of branchial clefts and pouches.

Fig. 102. — Diagram of a frontal section of a Frog larva at the time of hatching. From Kellicott (Chordate Development). After Marshall (modified). (Vertebrate Embryology, courtesy of Putnam’s Sons.)

c. Coelom. d.’ Pronephric duct. F. Fore-brain. i. Infundibulum. in. Intestine. n. Nephrostome. a. Base of optic stalk. ol. Olfactory pit (placocle). p. pharynx. t. Pronephric tubules. II. Hyoid arch. III—VI. First to fourth branchial arches. 1. Hyomandibular pouch. 2-6. First to fifth branchial pouches.

The changes in the solid pouches which make this possible, however, remain -to be noted. Shortly after hatching, cavities appear in the first four pairs of branchial pouches, and these cavities become continuous with that of the pharynx. The cavities of the second and third pairs of branchial pouches then acquire openings to the outside by breaking through the points of fusion between the invaginated ectoderm and the endoderm, 202 THE FROG: LATER OR LARVAL DEVELOPMENT

Fig. 103.—-Semi-diagrammatic sections through the branchial region of tadpoles of R. esculenm, showing the development of the gills and the history of the aortic arches. From Kellicott (Chordate Development). After Maurer. A. 4 mm. larva showing the continuous first branchial aortic arch. B. 5 mm. larva showing the anastomosis between the afferent and efferent portions of the aortic arch. C. 6 mm. larva with vascular loops in the external gills. D. 13 mm. larva. On the left the opercular chamber is closed and the external gill is beginning to atrophy, while on the right -this chamber is still open and the external gill well developed and projecting through the opercular opening. E. 17 mm. larva. Vessels of the second branchial arch. External gill represented only by a minute pigmented vestige.

a;. First branchial aortic arch. ab. Afferent branchial artery. ao. Root of lateral dorsal aorta. au. Auditory organ. c. Conus arteriosus. e. Epithelioid body. eb. Efferent brarichial artery. eg. External gill. i. Internal (anterior) carotid artery. ig. Internal gills. n. Nerve cord. 0. Operculum. p. Pharynx. pc. Pericardial cavity. r. Gill rakers. 5. Oral “sucker.” v. Velar plate of floor, roof plates not visible here. .1. Anastomosis between afierent and efferent branchial arteries.

and the cavities of the first and fourth presently do likewise. The two hyomandibular pouches never develop any real cavities, however, and the tissue which composes them later disappears. Since, likewise, there

are no cavities in the fifth vestigial branchial pouches, there are formed altogether but four pairs of actual gill slits.

It has been noted that after the external gills are covered by the operTHE F ORE—GUT AND I'l‘S DERIVATIVES 203

culum they soon atrophy and are functionally replaced by the internal gills. On the first three pairs of branchial arches these consist of a double row of filaments situated just ventral to those which are disappearing, but upon the posterior side of each arch, rather than upon its outer face. There is also a single row of filaments upon the anterior side of each of the fourth branchial arches. It is due to the fact that these new gills are upon the sides of the arches instead of upon their outer faces

anterior pituitary "ab: infundibulum Vth nerve

internal carotid artery _ . fragment of audimry capsule ‘ ' internal jugular vein

cular cartilage endolymphatic lining






muscle

hypobranchial Plate palate-quadrate

velar plates

gill rakers

external jugular vein '~'

gill chamber anterior fragment

of main coelom

Fig. 104. -— Cross section through the head of a late 10 mm. Frog larva in the region of parts of the 1st, 2nd, and 3rd hranchial arches. The arches are cut trans versely because of their diagonal courses. Only the extreme anterior portions of the auditory vesicles appear.

that they are termed internal. Nevertheless, they are still ectodermal rather than endodermal, and project well into the branchial (opercular) chamber. Thus, save for the fact that they are covered by the operculurn, the term internal as applied to them is something of a misnomer. Meanwhile during the development of these structures other changes have been taking place, as follows: First, owing to the inequalities in growth, there has been a considerable ventral shifting of the two branchial regions, accompanied by a marked dorso-ventral flattening of the pharyngeal cavity, so that the extent of its strictly lateral walls is greatly reduced. Thus instead of being situated on the sides of the pharynx the gill arches soon come virtually to occupy its floor, upon_either side of a median strip which is relatively wide anteriorly and narrow posteriorly. Hence the new gills do not project laterally, but tend to hang 204 THE FROG: LATER OR LARVAL DEVELOPMENT

Fig. 105.~———Diagrams of derivatives of visceral pouches and arches in Frog. From

Kellicott (Chordate Develop ment). After Maurer, with Greil’s modification. A. Lateral view, Frog larva. B. Lateral view, after metamorphosis. C. Transverse section through gill of Frog larva. D. Transverse section through gill region just after metamorphosis; gills still visible.‘

a. Afierent branchial arteries. c. Carotid gland. d. Dorsal gill remainder. e. Epithelioid bodies. pg. Internal gills. In. Middle gill remainder. o. Operculum. s. Suprapericardial body. t. Thyroid bod . th. Thymus bodies. 11. Ventral gill remainder. I—IV. Visceral arches. I. Mandibular arch. II. Hyoid arch. IIl~VI. 1st to 4th branchial arches. 1—.6. Visceral pouches. (1. Hyomandibular pouch. 2-6. 1st to 5th branchial pouches).

downward into the opercular chamber (Fig. 104). Furthermore, the direction of the arches is not at right angles to the long axis of the pharyngeal floor. Instead they run diagonally backwards and outwards from the somewhat triangularly shaped median strip to the sides. From the borders of this strip which run almost at right angles to the gill arches, flaps of tissue now grow postero-laterally so as to cover these arches at their inner and more anterior ends. The two flaps, moreover, become continuous with one another at their posterior and median extremities, so that actually only a single V shaped flap exists, whose posteriorly directed apex is attached to, and overlaps, the narrowest region of the median strip-. At the same time on each side a somewhat lesser flap develops from the lateral and dorsal wall of the pharynx along a diagonal line parallel with, but slightly posterior to, the respective side of the flap arising _from the floor. These dorsolateral flaps then grow anteriorly, medially and slightly downward, and because of the present close approximation of the pharyngeal floor and roof, they almost meet the lateral portions of the outgrowth from the former. The single ventral, and two dorsalateral flaps, thus. indicated are termed velar plates, and their arrangement is obviously such that only a narrow slit on either side leads from the pharynx to the gill chamber. It is these plates, together

"with toothlike processes on the inner sides

of the gill arches, called gill rakers, which tend to prevent the escape of food, while allowing the free passage of water. Finally at the time of metamorphosis the gill pouches and the gill cavity are filled by THE FORE——GUT AND ITS DERIVATIVES 205

proliferated cells, while the mass thus formed is later absorbed leaving the gill slits closed.

Structures Derived from Vestiges of the Gill Pouches.——Just before hatching, proliferations of cells occur from the dorsal ends of the hyomandibular and first branchial pouches. Those from the hyomandibular pouch presently disappear, but those from each of the first branchial pouches form a cell mass. These separate from the pouches (about 12 mm.), and eventually take up their position back of the auditory capsules near the surface of the head. They are the thymus bodies (Figs. 105, 106).

From the ventral ends of the first pair of branchial pouches there occurs, at about the 9-10 mm. stage, a proliferation of cells. These cells, together with the anastomosis of the proximal ends of the


Fig. '106.—Diagram of the branchial pouch derivatives in

afferent and efferent blood vessels of the first branchial arch (see below) form the so called carotid glands. Though long usage has apparently firmly fixed the title of gland upon these structures, they are not glandular in histological appearance or in function. They consist rather of a spongy network which. performs an im ‘VI. First to

the Frog. From Kellicott (Chordate Development). After Maurer, with Greil’s modification.

cg. Carotid gland. e1, e2, ea. Epithelioid bodies. th. Thyroid body. lml, tmz, Thymus bodies. ub. Ultimobranchial body. Isixth visceral pouches (I. Hyomandihular II~ VI. First to fifth branchial pouches).

portant service in helping to secure a rela tively aerated blood supply for the internal carotid artery of the adult Frog. While the ventral ends of the first branchial pouches thus help to form the carotid glands, cells from the ventral ends of the second and third branchial pouches give rise to what are known as the epithelioid bodies.

The fifth pair of branchial pouches never actually develop as such but become mere masses of tissue known as the ultimobranchial bodies (suprapericardial) .

The Thyroid.-—This organ appears before hatching as a median longitudinal evagination from the floor of the pharynx in the form of a solid rod. Later (about 10 mm.), this separates entirely from the phar206 THE FROG: LATER 011 LARVAL DEVELOPMENT

ynx, and divides into two lateral parts which eventually become vascular.

The Lungs.——They appear just after hatching as a pair of solid posteriorly directed proliferations from the ventral side of the pharynx just back of the rudiment of the heart. The pharynx at this point is later depressed, and partially constricted off from the part above it as the larynx. The opening left between the pharynx and larynx is the gloztis (Fig. 90). The lungs soon acquire cavities, and as they grow, become spongy and vascular. Part of their tissue is derived from the splanchnic mesoderm, only the inner lining being endodermal.

In connection with the origin of these organs it may be noted that there have been two general theories concerning their phylogenetic history. One school has regarded the lungs as coming from a modified swim bladder, while the other has considered them as developments of what were once a seventh pair of gill pouches. The latter notion at least has the merit of preserving a continuity of function in the forerunner of the respiratory organs of air breathing Vertebrates.

Further Development of Liver. —- The liver rudiment has already

' been noted as a small endodermal diverticulum extending back slightly,

beneath the yolk mass. The anterior wall of this diverticulum becomes folded and thickened, partly by the addition of scattered mesoderm and yolk cells (Fig. 90). This is the liver proper, the posterior part of the original outgrowth becoming partially constricted away from it as the gall bladder. The original connection with the fore-gut remains as the bile duct. These organs become well developed during the larval stage.

The Pancreas. —— At the posterior margin of the opening of the bile duct into the fore-gut, a pair of outgrowths arise connected with the gut by a single piece of tissue, the future pancreatic duct. The free ends of these outgrowths then grow forward and fuse in front of the bile duct. Later they are joined by a mass of tissue which originated from the dorsal wall of the gut, and the three elements thus fused constitute the pancreas. Eventually the pancreatic duct comes to open into the bile duct very near to the point where the latter joins the gut, instead of directly into the gut itself. _

With respect to the histogenesis of this organ, it appears that the islets of Langerhans in many species of the Frog at least, arise first from the endodermal cells of the primitive pancreatic anlage. Later these are added to by cells from the ductules. During metamorphosis some of the acinous cells degenerate, while the remainder persist as the THE FORE—GUT AND ITS DERIVATIVES 207

cells of the pancreatic tubules. The islet cells, on the other hand, become more aggregated, and develop two characteristic types with respect to staining capacity (lanes, ’38) .

The Esophagus and Stomach. — Shortly subsequent to hatching, the portion of the fore-gut between the future glottis and the opening of the bile duct elongates, and the anterior part of it becomes the esophagus. For a brief time the aperture between the latter and the pharynx is closed, but reappears at about the time the mouth opens. The posterior part of the above fore-gut region dilates slightly and assumes a transverse position as the stomach. This organ remains inconspicuous, however, until the time of metamorphosis, when it enlarges somewhat.

THE MID-GUT

The mid—gut is that portion of the archenteron lying above the large yolk mass at the time of hatching. After hatching, the yolk, and some of the cells of its floor are rapidly absorbed, and it begins to elongate. The front portion extends across the body in the form of a loop, the duodenum, which with the remainder is soon thrown into a double spiral. The coils of this spiral have a total length about nine times that of the body, but this is shortened about one third during metamorphosis.

THE HIND—GUT

The Rectum. ——This terminal part of the gut originates with a rela tively slight amount of growth from the small portion of the archenteron remaining between the yolk mass and the posterior body wall. It will be remembered that the endoderm of this region had come into contact with the ectoderm which had become invaginated to form the proctodaeum. About a week before hatching a perforation occurs at the point- of contact forming the anus, while the rectum itself becomes slightly dilated. In this connection it is of interest to note that the proctodaeal portion of the blastopore which in the Frog closes with the rest of this orifice, and later reopens, in the Salamander always remains open. Thus the temporary closure in the former animal is probably a secondary or non-primitive characteristic.

The Postanal Gut.——As the tail region develops, the notochord

‘and nerve cord extend into it, but since the proctodaeal region does not move backward, the neurenteric canal is drawn out into a small tube‘ beneath the posterior end of the notochord. Somewhat before hatching

it breaks away from the neural tube and persists for a brief period as the postanal gut. 208‘ THE FROG: LATER OR LARVAL DEVELOPMENT

The Cloaca and Urinary B1adder.—The general region where the endoderm of the rectum joins the ectoderm of the proctodaeum constitutes a chamber called the cloaca.‘It has been said that the cloaca. is in fact all ectodermal and therefore proctodaeal, but this seems to the writer highly doubtful and extremely difficult, if not impossible, to prove. The reason for this doubt is that the pigment which at first marks the ectodermal cells, later becomes rather diffused, and the exact boundary of the original fusion of rectum and proctodaeum is obliterated. At all events the point at which the rectum may be judged to end, i.e., to open into the cloaca, is technically the anus. The dorsal walls of the cloacal chamber also receive the urinogenital ducts. Finally at metamorphosis the ventral part of the cloaca gives rise to an anteriorly directed outgrowth within. the body cavity; this becomes the urinary bladder. In the higher animals this bladder is endodermal, and although as indicated above it is impossible to be certain, it seems highly probable that it is so here. One difference between Amphibians and some of the higher forms which is evident, however, is the fact that in the Frog and its relatives, as noted, the above ducts do not open into this bladder, but into the dorsal wall of the cloaca.

INTERNAL DEVELOPMENT: THE FURTHER DEVELOPMENT OF THE NOTOCHORD AND MESODERM

THE NOTOCHORD

When last indicated the notochord was merely a rod of undiHerentiated cells with a considerable curvature at its anterior end to conform to the cranial flexure of the brain. By the 4« mm. stage, however, the cells of this rod have become vacuolated, intercellular vacuoles have also appeared, and the anterior curvature so far as the rod is concerned has almost vanished (Fig. 89). At the same time around the notochord there presently develop two sheaths. The outermost, known as the primary or elastic sheath, is formed from the most superficial chorda cells.

The secondary or fibrous sheath lies within the latter and is formed of the chorda epithelium. ' '

THE SOMITES '

I When last considered, the segmental plates had divided into four pairs of somltes. This process continues posteriorly until there are thirteen such pairs, extending from just back of the auditory capsules to the THE SOMITES 209

base of the tail. Within the latter organ the number is much larger and somewhat variable. Thus in a 5.5 mm. larva there may be all told as many as forty-five. Sometime after hatching, however, the first two pairs disappear, and those in the tail are of course all lost during metamorphosis; there thus remain eleven well-defined somites in the body region. Meanwhile, as these somites are formed they have been undergoing certain changes, as follows:

Each somite it will be recalled consists of an outer layer of cells called the cutis plate, and an inner larger mass, the myotome. From the inner and ventral edges of the myotome‘. (about 5 mm.), loose sclerot0nzal cells are proliferated (Fig. 86). "these cells then migrate medially and dorsally between the rows of myotomes on the one hand, and the notochord and nerve cord on the other. Eventually they thus form a layer about the latter structures known as the skeletogenous sheath. This ultimately (see below) gives rise to the cartilage and finally the bone which forms the centra of the vertebrae together with their transverse processes and neural arches. Thereaare nine vertebrae thus formed in such a way that they alternate with the myotomal elements of the somites. The skeletogenous elements of the last two of the eleven somites have a somewhat dilierent history, as will be indicated later.

At about the same time that the sclerotomal tissue is being prolifer , ated, there are developing, within the myotomes, muscle fibrillae, which

are to form the muscles of the back. Also from the outer ventral edges of the myotomes and from the ventral edges of the cutis plates or dermatomes, outgrowths extend down next to the ectodermal wall. These are to form the ventral body musculature, and in the region of the limbs, their musculature as well. The main part of each cutis plate breaks up and some of the cells from these plates form the dermal layer of the dorsal region, while others migrate between the myotomes to form connective tissue. It would appear that the dermis of the ventral regions is not derived from the dermatomes at all, but from part of the somatopleure, as has been demonstrated for the Chick (see below). Partial continuation for this View has been furnished for the Amphibia by the e:~;perime.nts of Detwiler ("37) already cited. He has shown that although absence of somites ( including the dermatome) prevents spinal ganglion formation, the dermis of the operated side is present as usual. It might also be noted here that virtually all, if not all, pigment in the Amphibia is ectodermal in origin, that of the later stages coming mainly from the neural crests. This is true not only for pigment in the epider ~mis, but for that in the dermis and viscera as well (Dushane, ’38). 210 THE FROG: LATER OR LARVAL DEVELOPMENT

Finally, as indicated above, the mesoderm in the region where the segmental plate separates from the lateral plate constitutes the nephrotome, and is concerned with the formation of the excretory system. This will be described later.

THE GENERAL COELOM

The beginning of the coelomic spaces in the two lateral plates has already been described. These spaces continue to extend downward, until in a short time they meet one another beneath the gut and fuse. Thus in the trunk region, the coelom or splanchnocoel becomes continuous ventrally from one side of the embryo to the other.

Dorsally, the lateral plates of mesoderm on each side press up and in, between the dorsal wall of the gut and the notochord, until they meet. The splits in these plates then follow, but never quite reach each other, and hence the splanchnocoel never becomes continuous dorsally; there is always a thin but double-walled sheet of cells separating the right and left cavities. This is the dorsal mesentery. The gut as it develops is therefore slung from the dorsal wall by this mesentery, and completely encased in the splanchnic mesoderm.

INTERNAL DEVELOPMENT: THE CIRCULATORY SYSTEM

THE HEART AND PERICARDIAL CAVITY

The Primitive Cardiac Tube. —— It will be recalled that when last mentioned the heart consisted merely of a few scattered endothelial cells lying between the endodermal floor of the pharynx and the mesoderm. It will also be remembered that upon either side of the mid-line this mesoderm had developed within itself a space which was designated as a rudiment of the pericardial cavity (Fig. 85, C). These spaces now enlarge, and the mesoderm forming their uppermost walls presses up and around each side of the above mentioned endothelial cells so as to separate them from the overlying pharynx. Meantime these cells have become arranged in the form of two parallel tubes (Fig. 107, A) , which very shortly become more or less completely fused into a single tube (Fig. 107, B) extending throughout the region. Presently the in-pushing mesoderm from either side meets and fuses above this tube, so as entirely to surround it (3-6 mm), (Fig. 107, B, C). The latter with its covering now represents the complete rudiment of the heart. The endothelial portion, as noted, forms its lining, the en.docara'ium, while the THE HEART AND PERICARDIAL CAVITY 211

mesodermal envelope gives rise to the muscular wall, or myocardium, and the close fitting covering of the latter, the visceral pericardium. From the method of its formation, it is evident that this tubular heart will at first be attached to the walls of its pericardial cavity by both a dorsal and ventral sheet of mesodermal epithelium, or mesocardium. The dorsal sheet was formed like that which suspends the gut, by the fu


Fig. 107.—— Sections showing the formation of the heart in the Frog. From Kellicott (Chordate Development). A. Section through pharyngeal region of R. temporaria. After Brachet. B, C. Sections through the same region in older embryos of the smaller Frog, R. sylvatica.‘ A. 3.2 mm. embryo. Endothelial cells becoming arranged in the form of a double tube. B. Embryo of about 3 mm. C. Embryo of 5—6 mm. The single heart tube established; dorsal mesocardium still present.

(1721. Dorsal rnesocardium. e. Cardiac endothelial cells. en. Endoderm. g. Wall of gut (pharynx). p. Pericardial cavity. so. Somatic layer of mesoderm (future parietal wall of pericardial cavity). sp. Splanchnic layer of mesoderm (future myocardium plus visceral wall of pericardial cavity).

sion of the sheets of mesoderm pushing in from each side. The ventral sheet, on the other hand, has existed from the start as the median strip separating the two pericardial rudiments. Thus the pericardial space remains temporarily divided along this middle line. Meantime, as indicated above, the lateral coelomic spaces in the trunk region have extended ventrally, and now each side of the pericardial cavity communicates posteriorly with these spaces. The next step involves the entire disappearance of the ventral mesocardium, followed very soon by the disappearance of the dorsal mesocardium also, except at its anterior and posterior ends.

At this point it is worth pointing out that all Vertebrate hearts develop in essentially the same manner, except for some of the later de-V 212 THE FROG: LATER OR LARVAL DEVELOPMENT

tails involving the development of septa and orifices. That is, they all start with a pair of straight tubes which shortly fuse into one, as has been described, and this tube then develops in the manner about to be indicated to arrive at the adult condition. Since this is true it would he





'" truncus arteriosus "umus

arteriosus

_ bulbus ventricular

P°"tl°" ventricular

portion


snino-atrial portion

.. SIl'lUS venosus . ii -Ventride DORSAL DORSAL VENTRAL A B C sinus venosus atrium sinus venosus atrium truncus ventricular portion l l



.1 truncus - - _ bulbus bulbus


sino- atrial i truncus

. t ' l portion an°"°s"s Egritirciiiu ar - I ventricle RIGHT SIDE RIGHT SIDE RIGHT SIDE

sinus venosus /anterior vena cava


atrium .


truncus

RtGHT SIDE VF-l§ITRAL

Fig. 108.—Stages in the development of a Ve1'tch1'ato heart. These figures are primarily of the Frog heart, but would apply almost equally well to that of the

Chick or Mammal (see text). The earliest stage is /1, and that of an essentially

adult heart is D. There are two views of each stage as indicated on the figure.

well for the student to follow the ensuing be sure that it is clearly understood.

As the already-mentioned mesocardia disappear, the tubular heart be gins to increase in length, and hy so doing becomes twisted in the fol lowing manner. The straight tube first develops a marked bend to the right (Fig. 108, Al. The broad apex of th

posteriorly and slightly to the left. Up

description carefully, and

e bend then moves ventrally, on completion of this movement THE HEART AND PERICARDIAL CAVITY 213

we find that what amounts to a loop has been thrown into the originally straight tube (Fig. 108, 1?). The posterior limb of this loop extends ventrally and then curves outward to the right to form the wide apex, From the latter the ascending limb proceeds dorsally, slightly anteriorly and leftward into the median plane. Thus the two ends of the loop, an. terior and posterior, are still in essentially the same straight line. An. teriorly the ascending limb of the tube divides at its upper extremity into certain vessels which pass dorsally into the visceral arches. These will be described presently. At the posterior end, on the other hand, the tube comes into immediate and close contact with the anterior surface of the yolk mass which is in process of developing into the liver (Fig. 90, A). In connection with the latter certain vessels are forming which will also be discussed more fully below.

It is now possible to indicate how the parts of this twisted tube give rise to- the adult structures for which they are destined. As will imme~ diately become apparent, not all of them belong to the heart proper. Nevertheless, because of their very close connection and simultaneous development it is convenient to describe them together.

Sinus Venosus Vitelline Veins and Atria. ——Beginning at the posterior end it has just been noted that the heart tube abuts against the developing liver. Forming on the antero-ventral surface of the latter organ are two vessels, the vitelline veins, which become continuous antero-dorsally with the posterior end of the heart tube. The fused region of their entrance to the tube later becomes dilated to form the sinus venosus, while just anterior to this another enlargement occurs. This latter enlargement is the atrial portion of the heart proper, and presently there grows down from its roof :1 sheet of tissue dividing it into right and left chambers.“ These chambers are the atria of the Frog heart, and the sheet of tissue is the inter-atrial septum. It is further to be

There has been considerable confusion over the definition of the terms auricle

and atrium. According to the virtually universal usage of American medical men in human anatomy the two upper chambers of the heart are “ atria” which have earlike appendages or “ auricles ” attached to them. In many of the lower animals including the Frog, however, there are no such appendages, i.e., there are in the strict sense no auricles, only atria. It should be noted that among British medical men the term auricle is frequently more loosely used to include all of each upper chamber, though they do sometimes refer to the auricular appendages of the atria. Also among zoologists the terms auricle and atrium are used as essentially synony mous. Nevertheless, there is good historical and logical precedent for the strict definition of these terms adopted by American human anatomists. Hence, sincemany

students of embryology are sure to he premedics, the present writer intends to try

to save them future confusion by adherence to the more precise definition of atrium and auricle throughout this text. ’ is this region which sets the pace for the

214 THE FROG: LATER OR LARVAL DEVELOPMENT

noted in this connection that the growth of this septum occurs in such a manner that the sinus venosus comes to open into the right atrium. The left atrium, on the other hand, eventually receives the pulmonary veins (see below).

The Ventricles, Bulbus and Truncus Arteriosus. —— While these events are taking place in the postero-dorsal extremity (atr gion) of the looped tube, the curved apex of this tube connecti descending limbs is expanding. As it does so, it incorporates into itself the ventral part of the descending limb not involved in forming the atria. This expanded portion of the tube constitutes the ventricle. In the case of the Frog, of course, it contains no dividing septum. Its wall, nevertheless, becomes greatly thickened by the development of muscular tissue, some fibers of which traverse the ventricular chamber itself forming partial partitions. These, in connection with other factors, are said to help prevent the mixture of the two classes of blood received from the respective atria (Fig. 108, C).

Later, as a result of a rotation of the whole structure about an axis passing transversely between the atria and ventricle, the ventricle assumes its definitive posterior position. Finally the ascending limb of the original tube, also as a consequence of this rotation, comes to run more or less anteriorly from the ventricle across the ventral side of the

atria. It is not strictly part of the heart, but constitutes a thick walled vessel with two enlargements in it. The one nearer the vent

bulbus, and the more distal less prominent one the truncu (Fig. 108, D). Within the latter extendin

ial re ricle is the

5 arteriosus g throughout its length there

With respect to the initiation of functioning of the parts of the heart

tube the following may be said: pulsation in all Vertebrate hearts so

far as known begins long before any innervation, it being the nature of this particular type of muscle to contract rhythmically.

ave moving from This point is shifted backnd as might be expected it rate of heat. This has been

the posterior point of initiation anteriorly. ward as the length oflthe tube increases, a -sq

BLOOD VESSELS AND CORPUSCLES 215

clearly demonstrated for Arnblystoma by Copenhaver (’39, ’45) by cut. ting the tube at various places and times so as to show the inherent rates of the separated parts. By such experiments he has made clear that the posterior part of the tube, i.e., the region where the pulsation ultimately starts has a faster inherent rate than more anterior parts. Not only is this true, but interchange of posterior parts between species having different heart rates causes the imposition of the rate of the transplanted posterior part upon the anterior part of the host heart with which it has fused. In view of these facts it is not surprising to= find that in the completed heart the beat is initiated and its rate determined in the sinus, which arises from the posterior end of the original tube. However, in the adult organ the situation is altered to this extent: though the beat is always initiated in the sinus, its inherent rate is modified by nervous control to meet the demands of changing conditions.

Isolation of the Pericardial Cavity.—~l\/lost of the above processes take place in the deyeloprnent of the heart before or shortly after the tadpole hatches (7-12 mm.). One step which remains until considerably later, however, the separation of the pericardial cavity from the general coelom which lies posterior to it. This is accomplished by the outgrowth of folds of peritoneum (epithelial lining of the coelom) from the lateral coeloznic walls, in company with the cluctus Cuvieri (see below). The partial transverse wall thus formed is then augmented medially by the splitting off of peritoneal tissue from the anterior face of the liver. The entire partition is not completed until metamorphosis, when it is known as the septum z‘.ran.sversum.

DEVELOPMENT OF BLOOD VESSELS AND CORPUSCLES

The blood vessels develop out of the rnescnchyme and the splanchnic rnesoderm by a rearrangement and differentiation of the cells to form a flat endotheliurn which constitutes the inner lining of all the vessels. It is entirely similar to, and continuous with, the endothelial lining (endocardium) of the heart which has just been described (Figs. 89 and 107) . The muscular and connective tissue coats are likewise differentiated from mesoderm and added later, the muscle being much more abundant in the arteries and the connective tissue in the veins. In connection with these processes it should be emphasized that the early endothelial tubes do not originate as such at some one place, e.g., the heart, and simply grow outward from there as immediately continuous structures. They rather appear as disconnected sections or vesicles which grow toward each other until they are united. However, though it is true that the ves216 THE FROG: LATER OR LARVAL DEVELOPMENT

sels do not originate at one point, the procedures indicated do occur first in the more proximal regions of the embryo, and particularly in the vicinity of the heart. It is important to bear these facts in mind whenever the development of blood vessels is referred to, not only in the Frog, but also in any other Vertebrate for the method of formation is the same in all.

The corpuscles are formed chiefly from patches of splanchnic mesoderm on the ventral side of the yolk mass, from whence they find their way into the developing vessels. These patches are called blood islands. It appears, however, that the corpuscles produced by the islands do not last long, but are replaced by corpuscles from other blood-forming centers, particularly the spleen under stimulation by the liver (Goss, ’28; Cameron, ’4-1; Copenhaver, ’43). In Salamanders a diffusible substance from the endoderm seems to aid haemoglobin formation, at least in the island corpuscles (Finnegan, ’53).

The Arterial System. —-A few days before hatching (4~5 111311.}, the dorsal aorta develops as stated, just above the gut, and in the pharyngeal region is divided into two lateral dorsal or suprabra/Iclzial aorzae.

The Visceral Arch and Gill Circulation. —— At about the same time the

blood vessels of the visceral arches also develop in the following manner:

dorsally with the corresponding suprabranchial aorta. Presently similar connections are also established by the other two pairs. Thus complete loops or aortic arc/Les are formed in all but the mandibular and hyoid arches. Here no real aortic arches ever develop, though certain transitory vessels appear for a time.

As the external gills now begin to form, the following changes occur in the first, second, and third hranchial arches: A second looped vessel appears external to the primary aortic (branchial) vessel, the new vessel being attached to the primary vessel dorsally and ventrally (Figs. 103, C; 109, B). This new loop now extends out into the tissue of the corresponding external gill, where the two sides of the loop are con loop and its capillaries. The greater part of the blood, however, takes the latter course. Hence it passes out from the truncus arteriosus along the more ventral and external side of the gill loop, which is therefore aflerent, and back along the dorsal side, which is therefore eflerent. BLOOD VESSELS AND CORPUSCLES 217

When the external gills disappear, the ventral limb of the external loop (i.e., the section ab) remains to form the afierent vessel of the in. ternal gills (Figs. 103; 109). The efferent vessel, with which it then beComes connected by capillaries, is the more ventral part of the original primary loop (section x) . iV'leanwhile, this primary loop breaks its main ventral connection at the point where the external loop branched off from it. Thus during the remainder of larval life all the blood in the arches has to go through the internal gill capillaries. Since the fourth


Fig. 109. — Diagrams of the second aortic arch of the adult Frog and tadpole. From Kellicott tChar(late Development). After .\laurer. A. The continuous second (main systemic aortic arch of the adult; showing the parts corresponding with the larval vessels, 8. External gill and associated vessels in young tadpole. C. Internal gill and associated vessels in the tadpole after the disappearance of the external gills.

ab. Afferent hranchial artery. e. Epithelioid body. eb. Efferent hranchial artery. eg. External gill. ig. Internal gill. o. Operculum. .r. Direct connection hetween afferent and efferent" hranchial arteries, i.e., ventral part of primary loop.

arch never develops external gills, the vessels related to these particular structures never appear in it. Otherwise the history of the blood system within this arch is essentially similar to that just described in those anterior to it.

Changes in. Gill Circulation at Metamorphosis. ———The gills and their capillaries, including the major part of the afferent or external loops, gradually degenerate. At the same time the original primary loop vessels re-establish their ventral connections with the proximal parts of the afferent gill vessels. The primary vessels in the four pairs of branchial arches then undergo the following changes.“ The vessels of the first pair

4 It is to be noted in this connection that at least in some Frogs, as indicated in a preceding paragraph, no genuine aortic loops are formed in the mandibular and hyoid arches (Marshall and Bles on R. temporaria). In many other Vertebrates or their embryos, however (see the Chick), complete arteries do exist in these arches at one time or another, as well as in the four branchial arches. Thus in such cases the third aortic loop of the entire series is homologous with that in the first branchial arch referred to in the following account. 218 THE FROG: LATER OR LARVAL DEVELOPMENT

VI l VI V‘ IV 111


Fig. 110.—Diagrams of the branchial blood vessels in Frog larvae. From Kellicott (C/zorrlate Development) . After Marshall. ( Vertebrate Embryology, courtesy of Putuam’s Sons.) /1. A 7 mm. larva (shortly after hatching). The vessels supplying the external gills are removed, only their roots being indicated. B. A 12 mm. tadpole. The vascular loops in the gills are omitted.

rz. Atrium. ac. Anterior (internal) carotid artery. am. Anterior commissural artery. eo. Dorsal aorta. (rp. Anterior palatine artery. b. Basilar artery. c. Anterior cerebral artery. cg. Carotid gland. cv. Posterior (inferior) vena cava. dC. Ductus Cuvieri. g. Pronephric glomus. h. Hepatic veins. /Ly. Hyoidean vein. 1. Lingual artery. in. Mandibular vein. 1). Pulmonary artery. ph. Pharyngeal artery. pm. Origin of posterior commissural artery. pp. Posterior palatine artery. pv. Pulmonary vein. s. Vein of oral sucker. t. Truncus arteriosus. u. Cutaneous artery. 1:. Ventricle. I~~4. First to fourth afferent branchial arteries. 1, II. Efferent arteries of the mandibular and hyoid arches. II1'—VI. First to fourth efferent brauchial arteries. VI I. Lacunar vessel of the fourth branchial arch. BLOOD VESSELS AND CORPUSCLES 219

oi branchial arches retain their dorsal connections with the respective dorsal aortae, and with them form the proximal ends of the internal carotids which run forward into the head (Fig. 110). The vessels of the same arches are joined at their ventral ends by the external carotids or lingual arteries which have grown back from the floor of the mouth. Almost at the junction of the external and internal carotids on each side, the latter develops an enlargement consisting of spongy tissue. This is the carotid gland already referred to. It arises from a slight anastomosis between the proximal ends of the afferent and efferent aortic vessels of the first branchial arch, with the addition of some epithelial cells from the ventral end of the first branchial pouch.

The vessels of the second pair of branchial arches also retain their dorsal connections with the lateral dorsal aortae, while the latter disappear anteriorly be- Fig. 111.——Diagram of the tween this point and the first branchial “mic ‘“°h°5 ‘md ‘heir Chief


arches (disappearance not shown in Fig. branches 1" an adult Frog110). Thus the vessels of the second branchial arches become the main 3 /slemic arteries. The vessels of the third branchial arches disappear. The vessels of the fourth

From Kellicott (Chordate Development). Ventral view.

an. Dorsal aorta. c. Carotid artery. cg. Carotid gland. cu. Cutaneous artery. 1. Lingual artery. p. Pulmonary artery. .9. Systemic arch. sc. Subclavian

branchial arches; having already given off ‘m°"V' " T““‘°“5 ‘“‘e‘i°5“5' _ v. Vertebral artery.

branches to the lungs and skin, become the

pulmocutaneous arteries. The portion of each of these vessels connecting it with the respective lateral aorta disappears after metamorphosis. Thus all the blood going to these aortic arches must henceforth pass to the lungs or skin.

It may be noted that in most of the air-breathing Vertebrates not all of the section of the fourth arch between the origin of the pulmo-cutaneous artery and the dorsal aorta, known as the ductus Botalli, completely disappears. Instead it remains as a vestigial strand. Among the Amphib~ ians this is true of many of the Urodeles. but not of the Anura.

In conclusion the functions of certain of the rather special structures of the Frog heart whose development has been described may be briefly indicated. It will be recalled that muscle fibers in the undivided ventricle tend to act as partial partitions and to keep the kinds of blood in it

V 1,: - .t_._..._ ___.__....__..,a.-_....M.-..........._.

i T l 220 THE FROG: LATER on LARVAL DEVELOPMENT

separated. The spiral valve in the truncus arteriosus then assists in guiding these different kinds to the proper pairs of arches. Thus the relatively unaerated blood leaves the heart first, and goes into the fourth arch on the way to the lungs and skin. Then the mixed blood is guided into the main systemic arches and external carotid. Lastly, the relatively aerated blood is forced through the carotid “gland” and into the internal carotid to the upper part of the head and brain (Fig.

111 .

0)ther Arteries.——The pharyngeal arteries develop at about 9 mm.‘ from outgrowths of the suprabranchial aortae, which at first connect with transitory vessels in the mandibular arches. At about the middle of each main systemic aortic arch a large branch is given off to the fore limb; it is the subclavian. The suprabranchial or lateral aortae come together to form the single dorsal aorta at about thelevel of the pronephros (see below). Throughout the remainder of its course this artery gives off several lumbar arteries to the body wall, as well as larger branches which supply the viscera (mesenteric arteries), and the hind limbs and adjacent regions (iliac arteries).

The Venous System.

The Hepatic and the Hepatic Portal Systems. —- In discussing the development of the heart, it was noted that almost from the first two veins entered it posteriorly, i.e., the vitelline veins. Just at the point of entrance to the heart their fusion resulted in the formation of a common chamber, the sinus venosus. Between this point and the liver a further fusion of these veins occurs not long after hatching, and the result is for the time being the hepatic vein. (F ig. 110) . Although first mentioned in

V connection with the heart, the vitelline veins actually appear first on

the ventro-lateral sides of the yolk mass, whence they pass along the

sidesof the yolk and liver to the heart. As noted, fusion early occurs an- i

terior to theliver, but posterior to it the vitelline veins remain separate. The right vein within this region then disappears, and the left becomes the hepatic portal vein. It remains connected with the anterior hepatic vessel only through capillaries within the liver substance, while posteriorly it sends branches to the digestive tract. This vein with its branches and liver capillaries constitutes the hepatic portal system. BLOOD‘ VESSELS AND CORPUSCLES 221

each of these connections there presently develops a sinuslike vein, the ductus Cuvieri. These veins do not run horizontally from the sinus ve nosus to the body walls, but obliquely upward. At the points of union with the respective wall each ductus then gives rise to an anterior and a posterior branch within the wall itself. These are the anterior and pos atr1o- ventricular aperture





external jugular





internal jugular anterior

smo


externai iugular atria. “"3 ‘"3 . Lj subscapular aP°m"° “"31 . innominate portion of internal heart . lU8'-"3" . . ‘ ubclavian sinus venosus ‘T lg‘ d,U=l_uS nth”; , ‘ uviera \_ , _muszulocutaneous he zitics , P left posterior anterior part ol cardinal sinus posterior vcna cava spam liver portals r‘ ht oster' « ' . cfidinpal '°r left posterior - _ v osterior vena cava cardinal vein hdney ' 7 (mesonephros substance of forming , rnesonephros

Fig. 112. — Figure A is areconstrnction in ventral view of the chief veins of a 10 mm. Rana pipiens larva made from serial cross sections, and enlarged 22.5 times. The ventricle of the heart is omitted, and _the mesoncphros is of course shown only diagrammatically to indicate its relative position. Figure B is a semi-diagrammatic representation in ventral view of the veins in an adult Frog which are derived from those shown in A, with the addition of the abdominal vein, as described in the text. The entire heart is omitted from this figure, and the dotted lines merely outline where the posterior cardinal sinuses would be if, they were still present. It should he noted, as indicated by the labels, that the aperture in figure A is a completely difierent one from that represented in figure B. Also, it is to be emphasized that since figure B is near natural size, the two figures are on nowhere near the same scale. As usual, relative degrees of growth of parts account for many of the differ - ences, especially in connection with the development of the anterior vena cavae.

terior cardinals. Presently there grows anteriorly from the base of each ductus Cuvieri a vein which extends into the lloor of the mouth, the inferior (external) jugular. This situation is clearly in evidence at 10 mm. or earlier (Fig. 112, A). Later at about the point of origin of each inferior jugular there also grows toward the region of the respective future shoulder another vein which becomes the subclavian. At approximately the same time, so far as is known, the base of each ductus Cuvieri becomes extended somewhat, thus separating the place of origin of the respective inferior jugular and. subclavian from the sinus venosus. The 222 THE FROG: LATER OR LARVAL DEVELOPMENT

short new section of vessel thus added to the proximal end of each duc~ tus is then known as an anterior vena cava. The remaining portion of each ductus between the origin of the respective inferior jugular and the origin of the respective anterior cardinal, the posterior cardinals having meanwhile disappeared (see below), is henceforth called an innominate. Thus each anterior cardinal itself now becomes a superior (internal) jugztlar. At about the junction of each innorninate vein and the respective superior jugular a backward curving vessel arises which is a subscapular (Fig. 112, B).5

Turning now to the posterior veins, each posterior cardinal will be found proceeding from the junction of the ductus Cuvier and anterior cardinal (superior jugular) backward through the pronephric region. Here it has the form of a broad sinus which more or less surrounds the pronephric tubules (see below). Posterior to this region, it turns sharply toward the median line and continues along the median side of the respective pronephric (Wolffian) duct to the cloaca (Fig. 112, A) Along its course, each of the cardinals receives branches from the body wall, and at their posterior extremities the two veins unite and receive the caudal vein which brings the blood from the tail.

At about the 10 mm. stage in Rana pipiens, modifications in this arrangement begin as follows: Along the median dorsal surface of the liver a new vein forms which empties into the hepatic vein anteriorly, and posteriorly unites with the right posterior cardinal just caudal to the pronephros (Fig. 112, A). At approximately the same time, or slightly later the posterior fusion of the posterior cardinals proceeds anteriorly in an intermittent manner into the region of the developing mesonephroi, and eventually it occurs throughout the extent of those organs. Thus is produced a median cardinal vein which, due to the manner of its formation, is continuous anteriorly with the new vein connecting the right cardinal with the hepatic. With the disappearance of the pronephros, the right cardinal, anterior to the point where the new vein has joined it, and all of the left cardinal, also disappear. The single median vein which results is called the posterior vena cava. It is to be noted that its posterior portion is really simply the former median cardinal vein, while its extreme anterior part is merely the old hepatic vein which receives branches from the liver. As the latter vein thus he comes part of the posterior vena cava opening into the sinus venosus,

indicatecl as arising subsequent to 10 mm. actually develop, though the early larval and the adult conditions are of course well known.

5 There is no very complete description of just how some of the branches just -a.... . _. , ,_ . ..s.,,-r........ - ~/«<>~4 . . ............._..,..«,.,..e_a~...s.«a-.,.,.,,

BLOOD VESSELS AND coaPUscLEs' 223

the branches which it receives from the liver substance become the permanent hepatic veins (Fig. 112, A). Meanwhile it is to be noted that as the posterior cardinals fuse and the mesonephroi develop, there arises along the lateral border of each of these organs a new vein. Each of these veins then becomes connected with the rriedian vein (posterior vena cava) by numerous channels through the mesonephric substance (Fig. 112, A). Indeed according to some accounts (Shore, ’01) the cardinals simply fuse, and then are partially divided by the mesonephroi into three main parts, a median and two lateral, the undivided remnants constituting the connecting channels (Figs. 112, A; 113, A, B). Though this is Shore’s description of the process, it seems to the writer that three fairly separate channels exist before the mesonephros has developed to any extent. The mesonephric (pronephric) ducts are of course present, however, and it appears that they may help to split off a lateral channel from each of the fusing, more medially placed, cardinals. It also appears to the present author that in many, if not in all, cases at the '10 mm. stage the undilierentiated mesonephric primordium (nephrotomal tissue) extends across the


Fig. 113.——The development of the posterior part of the venous system in the Frog. From Kellicott (Chordate Development). After Shore. A. Portion of a transverse section through the posterior mesonephric region of an 18 mm. tadpole. B. Diagram of the veins of a 25—3O mm. tadpole. C. Diagram of the veins of the adult Frog.

2. Dorsal aorta. c. Vcna cava. e. Nuclei of the endothelial lining of the

mesonephric sinus, continuous with the vascular endothelium. f. Femoral vein. 1'. Iliac vein. lc. Lateral mesonephric channel of the posterior cardinal vein. in. Mesentery. mn. Mesone-phros. n. Mesonephric tubules. p. Posterior cardinal veins. (in C showing their original location). pv. Pelvic vein. rp. Renal-portal vein. rr. Revehent renal veins. sc. Sciatic vein. st. Nephrostome. u. Caudal vein. ucm. Median mesonephric channel of the posterior cardinal vein. W Wolffian duct. x. Connection between caudal vein and the lateral mesonephric channels. 1—1. Part of the renal-portal vein formed from the lateral channel of the posterior cardinal. 2-2. The posterior part of the vena cava formed from the median channel of the posterior cardinal vein. 224»! THE FROG: LATER OR LARVAL DEVELOPMENT

median line in many places as a single mass just above the fusing cardinals. This mass then seems actually to be divided by the dorsally pushing median cardinal vein instead of the reverse process as usually described.,Perhaps the real procedure is one of mutual interpenetration of mesonephric substance and veins as suggested in Figure

‘ 112, A. The writer regards this as most probable on

servations. Be this as it may the ultimate result is that the lateral vessels develop to become the renal portal veins; and the channels connecting them with the median posterior vena cava are then the renal veins. Later with the appearance of the legs each renal portal vein is joined by an iliac vein which. as these appendages develop. divides at its distal end into the femoral and sciatic veins. Finally with the loss of the tail the

Fig. 114..—-Ventral, lateral and dorsal views of P3” of the Poslerio" Vena the lymphatics in a 26 mm. tadpole of R. tempo- -cava caudal to [he kidneys

raria. From Hoyer. For description see text. vanishes so that most of the blood from the posterior region of the body must pass through the renal portal vessels and the abdominal (see below) (Figs. 112; 113).

The Pulmonary Veins. — These begin to develop very early (6 mm.) as a dorsal offshoot from the sinus venosus. Later this ofl"shoot opens

into the left atrium, while at the lungs the single pulmonary vein divides so as to receive blood from each.

bladder, making lateral connections with the femoral veins. Just anterior to the bladder the two vessels then fuse; while still further forward the right one later disappears entirely. The remaining single vessel is the abdominal vein, which finally loses its connection with the sinus

the basis of his own ob-' THE PRONEPHROS AND SEGMENTAL DUCT 225

venosus; it then acquires a connection with the hepatic portal vein, and also develops two branches opening into the capillaries of the liver (Fig. 112, B). ‘

The Lymphatic System. ~— Just before" hatching, the anterior lymph hearts appear to arise from a superficial plexus of veins between the third and fourth somites. They lie between the peritoneum and the integument, and soon become incased in muscle fibers. In connection with each “ heart ” there develop from other parts of the above venous plexuses two vessels just beneath the skin. One proceeds anteriorly, and the other posteriorly, while into these vessels drain numerous anastomos ing capillaries; the latter eventually form the characteristic subcutane- ‘

ous lymph sacs of the Frog. Sometime after hatching (26 mm.), the anterior vessels open downwards into large lymph sinuses in the branchial region (Fig. 114«) . The lateral posterior trunks unite at the root of the tail, and divide into a dorsal and a ventral vessel, which pass into it. The thoracic ducts seem to be outgrowths of the anterior lymph hearts, which extend posteriorly between the dorsal aorta and the posterior cardinal veins. When the hind legs appear, posterior lymph hearts develop from the segmental veins of that region also.

All the lymph hearts are guarded by valves between themselves and the lymph channels on the one hand, and between the hearts and blood vessels on the other. Thus the lymph always passes into the blood, never in the reverse direction.

The Spleen. —— At about 10 mm. there appears in the mesentery, on the anterior mesenteric artery, just dorsal and posterior to the stomach, a collection of lymph cells. They multiply, and later (25 mm.) the cell mass becomes very vascular. The body thus formed is the spleen.

INTERNAL DEVELOPMENT: THE LARVAL EXCRETORY SYSTEM

Although both the ‘larval and adult systems are paired, we shall re fer only to the development upon one side. This is done with theunderstanding that the processes on the opposite side are identical.

THE PRONEPHROS OR HEAD KIDNEY, AND THE SEGMENTAL DUCT

The Pronephros.——When last described, the somatic wall of the nephrotomal region had thickened until it slightly overhung the side 226 THE FROG: LATER on LARVAL DEVELOPMENT

Fig. 115.—-Sections through Frog larvae illustrating the later development of the pronephros. From Kellicott (Chordate Development). A. A section through the first nephrostome of a larva of Rana sylvatica of about 8 mm., with prominent external gills. After Field. B. A section through the region of the second nephrostome of a 12 mm. larva of Rana temporaria. After bringer.

c. Coelom. ‘cu. Sinuses of posterior cardinal vein. g. Gut cavity. gl. Glomus. gX. Ganglion nodosum (part of the ganglion of the vagus nerve). l. Lung. m. Mesencliyme. myz. Second myotome. p. Peritoneum. s1, 52. First and second pronephric neph rostomes. tr.‘Common trunk. X. Root of vagus nerve.

of the lateral plate between it and the ectoderm; in the region of the second, third and fourth somites, cavities were beginning to appear within the thickening, especially in its lateral portion (Fig. 84) . These laterally placed cavities now tend to run together so as to form in this region a continuous longitudinal lumen, the common trunk. At the same time, other spaces between this lumen and the coelomic cavity enlarge and unite with one another to form three separate tubules connecting the trunk with the coelom. These are the pronephric tubules, and each of them is opposite one of the three somites referred

‘to. The opening of each

tubule into the coelom is in the form of a funnel named the nephrostome (Fig. 115), which presently becomes lined with long cilia. The tubules, together with the common trunk, now become somewhat convoluted, and these convolutions begin to become imbedded in the sinus-like cardinal vein which partially surrounds them (Figs. 115, ,,,.,...,,4«-«a<.—«‘.,.er..‘«,»....-.,.r~«~,.. .. . .. .

THE PRONEPHROS AND SEGMENTAL DUCT 227

116). At the same time the mass which is thus formed becomes enclosed on its dorsal and outer sides by connective tissue derived from the myotomes of this region and from the somatic mesoderm. This covering is termed the pronephric capsule.

Although not directly connected with the pronephric tubules, there develops with them another organ which because of its position and structure is probably concerned with their function. It arises as an outpushing or fold of splanchnic mesoderm at the extreme dorsal limit of the coelom in the region just opposite the nephrostomes. In this way a


Fig. 116.—Total views of the pronephros of the Frog (R. sylvatica). From Kellicott (Chordate Development). After Field. A. Right pronephros oi an embryo of about 3.5 mm. The crosses mark the location of the nephrostomes. B. Right pronephros of a larva of about 6 mm. First tubule dotted; second white; third obliquely ruled; pronephric (segmental) duct shaded with lines.

small mass of tissue becomes suspended directly opposite these openings. Presently numerous capillaries form within it and become connected with the nearby dorsal aorta. This vascular body is then called the glomus, and it has been shown by transplants in Amblystoma that the stimulus to its development depends upon the presence of the pronephric tubules (Fales, ’35), even though the latter have no direct connection with it. The pronephric tubules, together with the glomus, may henceforth be referred to as the pronephros or head kidney (Figs. 116, 117).

The Segmental Duct. —-— So far as has yet been indicated, the larval kidney has no external outlet. While the above changes are going on, however, the lumen of the common trunk has extended backward through the lateral border of the nephrotome until it has established a connection with the cloaca. The outer ‘portion of the nephrotome containing this lumen is then called the pronephric or segmental duct. Rosterior to the fourth sornite it gradually becomes more or less separated from the more median portion of the undifferentiated nephrotomal tissue which occurs in this region. 228 THE FROG: LATER OR LARVAL DEVELOPMENT.

Changes Subsequent to Hatching. - This is approximately the condition reached at the time of hatching, when the tadpole is from 6-7 mm. long. The pronephros does not attain its maximum development, however, until. the animal is about 12 mm. in length. During this particular period the pronephric tubules increase their convolutions to a considerable extent, and the coelomic space into which the nephrostomes open and in which the glomus is suspended becomes cut off ventrally from the main coelomic cavity. This is accomplished by the development of the lungs in this region (see Fig. 115). These organs are covered by a fold of the splanchnic mesoderm, and, as they grow, this covering fold is eventually brought into contact with the somatic mesoderm, with which it fuses for a short distance. The cavity thus formed, though it is separated from the coelom beneath, remains open to it both anteriorly

Fig. 117.-—Transverse section of an advanced Frog embryo. From J enkinson (Vertebrate Embryology) .

m.!. Medullary tube. rz. Notochord. s.n. Subnotochordal rod. my. Myotome. a. Aorta. p.c.v. Posterior cardinal vein. prn. Pronephric tubule. prn.f. Pronephric funnel (i.e., nephrostome). gl. Glomus. C. Coelom. so. Sornatopleure. spl. Splanchnopleure. g. Gut. l. Liver. v.v. Vitelline vein. ec. Ectoderm.

and posteriorly. It is termed the pronephric chamber.

By the time the larva reaches a length of 20 mm., the head kidney begins to degenerate. Thus the pronephric region of the segmental duct becomes cut off from the part posterior to it. The former portion of the duct, together with the pro nephric tubules and their nephrostomes, then gradually disappears; ° the glomus at the same time shrivels up, though remnants are visible even after metamorphosis. As the larval kidney is thus eliminated, its

place is taken functionally by the mesonephros whose development is now to be described.

5 Hall states that during the degeneration of the pronephros the three nephro stomal openings, at least in R. sylvatica, always become fused into one, the common nephrostome (Fig. 118 C ‘ THE MESONEPHRIC OR WOLFFIAN BODY 229


‘ - .g—..-ma"


Fig. 118.—Sections through the developing mesonephros and the degenerating pronephros of R. sylvatzca. From Kellicott (Chordate Development). After Hall. A.

. Section through the eighth somite of an 8.5 mm. larva. B. Section through the meso nephric rudiment of a 25 mm. larva. C. Section through the pronephric chamber and the common nephrostome of the pronephros of a 25 mm. larva.

(I. Dorsal aorta. c. Coelom. en. Common nephrostome. g. Germ cell. 1'. Inner tubule. m. Mesonephric rudiment. my. Myotome. 0. Outer tubule. p. Remains of pronephros. pc. Posterior cardinal vein. s. Shelf cutting off the pronephric chamber from the remainder of the coelom. so. Somatic rnesoderm. sp. Splanchnic mesoderm. W. Wolffian duct. I. Primary mesonephric unit. II. Secondary mesonephric unit.

THE MESONEPHRIC OR WOLFFIAN BODY

Posterior to the pronephros the outer margin of the nephrotome went to form the segmental duct. The inner portion medial to the duct appears meantime to have fused to some extent with that from the opposite side, thus forming a continuous mass ventral to the dorsal .aorta,

' and above the fusing, or fused, posterior cardinal veins. This inner por tion now starts to form the adult kidney in the following manner. The Mesonephric Vesic1e.——As indicated above, the inner part is for a brief time divided into segmental nephrotomes. These, however, 230 THE FROG: LATER on LARVAL DEVELOPMENT

Fig. 119.—Series of diagrams illustrating the development of the primary ymesonephric tubules in R. sylvatica. From Kellicott (Chordate Dewelopment). After Hall. The Wolflian duct is drawn in outline simply. The mesonephric vesicles are shaded; the somatic part of the tubule is shaded by continuous lines, the splanchnic part by dotted lines. A. Wolflian duct and simple mesonephric vesicle. B. Mesonephric vesicle dividing into the large primary mesonephric unit and the small dorsal chamber. The latter elongates anteroposteriorly and represents the rudiment of the secondary and later mesonephric units. C. Formation of the rudiment of the inner tubule. D. Inner tubule extending upward and toward the mesonephric duct; formation of rudiment of outer tubule. E. Outer tubule fused with peritoneum and rudiment of nephrostome thus established. Bowman’s capsule forming. Commencement of differentiation of secondary mesonephric unit. F. Separation of nephrostomal rudiment from remainder of tubule. G. Connection of nephrostome with branch of posterior cardinal vein; separation of secondary unit.

a. Bowman’s capsule. 13. Inner tubule. n. Nephrostome. 0. Outer tubule. p. Peritoneum. 1;. Branch ‘of posterior cardinal vein. 1. Primary mesonephric unit. II. Secondary mesonephric unit. Tertiary mesonephric unit not yet developed. M... 4.1;-.5

THE MESONEPHRIC OR WQLFFIAN BODY 231

disappear almost at once so that a single nephrotomal band extends from the seventh to the twelfth somites. Within either side of this hand there then arise a series of thickenings somewhat more numerous than the somites, and in each thickening there soon appears a cavity (Figs. 118, 119). This cavity, which is called the mesonephric vesicle, eventually becomes divided into two parts, the second and smaller part still later giving rise to a third. These parts are called primary, secondary, and tertiary units, in the order of their appearance, and their further development, though not simultaneous, is identical in character. lt will be necessary, therefore, to describe the process in only one of the primary units.

The Development of a Primary Vesicular Unit.—Upon the dorsal side of the unit a small hollow outgrowth appears (Fig. 119, B). This, as later events prove, represents the rudiment of the secondary unit, but for the present does not develop further. Next (Fig. 119, C), an evagination pushes out from the ventro-lateral side of the primary unit in the direction of the segmental duct. This is the inner tubule, which presently becomes connected with the segmental duct, the latter being henceforth known as the mesonephric or Wolfiian duct. It is to be noted, moreover, that, by virtue of the partial rotation of the primary unit, this connection occurs dorsally rather than ventrally (Fig. 119, D, E). A part of the inner tubule later becomes greatly convoluted and the coils press down into the median cardinal vein (15 mm.), perhaps helping to divide the latter, as indicated above. Meanwhile there has grown out from what is now the ventral side of the unit, another evagination which presently become connected with the peritoneal (coelomic) cavity. This is the outer tubule, whose subsequent history in the

Frog is very peculiar." It soon (20 mm.) breaks away from the main

portion of the unit and acquires an opening into the lateral division of the median cardinal vein, i.e., the future renal portal vein. At the same time its opening into the coelomic cavity becomes ciliated as a typical nephrostome, this curious connection between body cavity and blood vessel persisting throughout life (Fig. 119, F, G).

The growth of these tubules has meanwhile been accompanied by a loss of the round or ‘vesicular character of the region of the original primary unit. Thus between the point of origin of the secondary unit and that of the inner tubule, this region has become stretched out, and at the same time invaginated in a ventro-medial direction (Fig. 119,

7 Some authorities assert that the outer tubule probably never actually opens into the cavity of the primary unit from which it arises (Marshall Hall). 232 THE FROG: LATER OR LARVAL DEVELOPMENT

E, F, C). In this manner a cavity is produced which is later filled by a mass of capillaries connected with the dorsal aorta and also with the posterior vena cava. Such a capillary mass is called a glomerulus. The occurrence of the venous connection and the location of the structure within the kidney rather than in the coelom are two essential features in which a glomerulus diifers from a glomus. The surrounding walls of the


Fig. 120.-——-Parts of sections through young R. temporaria, showing the origin of the adrenal bodies. From Kellicott (Chordate Develop m.en.r). After Srdinko. A. Through 30 mm. tadpole. B. Through 11 mm. Frog after metamorphosis.

a. Dorsal aorta. ac. Corticle cells of adrenal body. am. Medullary

cells of adrenal body. ct. Connective tissue. g. Gonad. gs. Sympa thetic ganglion. m. Mesentery. n. Mesonephros. rv. Revehent renal

vein. v. Vena cava. x. Point where ganglion cells enter mesonephros and adrenal bod_y.

invaginated unit in which the glomerulus thus lies embedded then constitute Bowman’s capsule, the capsule and capillaries together being termed a renal corpuscle or Malpighian body. 7

The occurrence of similar processes in the other units finally results in a mass of tubules, glomeruli, and nephrostomes, which constitute the

adult mesonephric organ or kidney. This organ is virtually complete by the time metamorphosis is ended.

THE ADRENALS

Though in no sense a part of theexcretory system, ways occur in such close connection w

to describe them at this point. Indeed, ship of the adrenals and kidneys is Vertebrates, so much so that it is difii animal, the former organs appear 111

these organs alith the kidneys that it seems best

in the mature Frog the relationmore intimate than in the higher cult to separate them. Thus in this erely as an area of thin yellowish ADRENALS AND GONODUCTS 233

tissue attached to the ventral side of the mesonephros. They are com posed, however, of two kinds of cells, the so-called medullary su bszance, and the cortical substance, which originate as follows:

The cortical substance is so named from the fact that in higher forms it occurs on the outside of the organ, though this is not true of the Frog. Here it consists of anastomosing cells apparently derived (at about 12 mm.) from the rnesonephric blastema cells (Segal, ’53) near the cardinal veins. These cells form a meshwork into which branches from the veins soon penetrate. The medullary substance consists of pigmented cells which appear later. They are derived originally from sympathoblasts in the sympathetic ganglia of the mesonephric region, and become scattered throughout the cortical tissue (Fig. 120).

INTERNAL DEVELOPMENT: THE GENITAL SYSTEM

THE GONODUCTS

In the Male. ———The vas deferens of the Frog is simply the meso-nephric or Wrolliian duct, which serves as both ureter and sperm duct. Posteriorly, in the region of the cloaca, each duct develops a glandular seminal vesicle. Anteriorly each vas deferens becomes connected with the respective testis as follows: From the latter certain strands of tissue known as rete cords (see below) develop into fine ducts which grow into each rnesonephros along its median edge. Within the kidney these fine ducts become connected with the Bowman’s capsules of some of the kidney tubules. The fine ducts together with the tubules of the kidney with which they thus connect then constitute the vase eflerentia, opening into each mesonephric duct (vas deferens) .

At about 20 mm., there appears on each side of the coelomic wall just beneath‘ the pronephric region, a longitudinal thickening of the peritoneum. Along the dorsal border of this thickening ‘there is then proliferated a ridge of cells, whose edge grows downward and presently fuses with the ventral border of the thickening. In this manner a tube is formed, which, when completed, is held close to the body wall by a thin covering of the general peritoneum (Fig. 121). This process continues anteriorly to a point opposite the base of the lungs and posteriorly to the cloaca, which it reaches subsequent to metamorphosis. In the male this tube develops no further, and is very inconspicuous and without function, but is the rudiment of a Miillerian duct ( see below) .

In the Female.——The mesonephric duct is of course present in the female, but in this case acts only as a ureter. It possesses, nevertheless, , ,.:.;..=.- . . ;_- .. .

234 THE FROG: LATER OR LARVAL DEVELOPMENT

extremely slight enlargements, representing rudimentary seminal vesicles.

Each Miillerian duct or oviduct, on the other hand, develops as described in the male, but does not stop at the point there indicated. Instead, the rudimentary duct moves away from the body wall somewhat, though it still remains attached to that wall by its peritoneal covering. Between the duct and the wall the two layers of the covering then fuse


Fig. 121.—Sections through the developing Miillerian duct of a 34 mm. tadpole of R. syluatica, From Kellicott (Chordate Development). After Hall. A. Section passing through the beginning of the Miillerian evagination. B. Section posterior to A. Duct established but still connected with peritoneum. C. Section still farther

posterior, showing the separation of the duct from the peritoneum with which, however, it is covered.

M. Miillerian duct. p. Peritoneum. 2:. Third pronephric tubule.

to form the mesentery-like sheet supporting the oviduct. Anteriorly the duct turns down slightly, and its end becomes dilated as the infunclibulum, while posteriorly it acquires an opening into the cloaca; between these points it gradually becomes greatly convoluted and thickened.

THE GONADS

The Indifferent Period. ——-As the early stages of these organs are identical in the male and female, a single account will suflice for both. At about the time of hatching, a slight median dorsal ridge appears on the outside of the enteron (Fig. 122, A). It is composed of primordial germ cells, which, as in other cases, have apparently arisen from

among the cells of the gut. Indeed, at this time it is difficult to distinguish the cells of the ridge from the enter'

as noted above, the lateral plates of In

esoderm press in toward each other in this region, and as they meet, t

hey separate the ridge of cells THE GONADS V 235

Fig. 122.—Sections showing the origin of the sex-cells (germ cells) in R. sylvatica. From Kellicott (Chardate Development). After Allen. A, B. Sections of a 7.5 mm. larva showing (Al sexcell ridge of endoderm and (B) its separation as the sex-cell cord. C. Part of a section of an 8.3 mm. larva showing the beginning of the migration of the sex-cells, resulting shortly in the division of the sex-cell cord into two parts.

a. Dorsal aorta. ch. Notochord. cv. Posterior cardinal vein. e. Endoderm cells. g. Gut cavity. l. Lateral plate of mesoderm. m. Mesentery. my. Myotome. n. Nerve cord. sc. Sex-cell cord (not to be confused with sexual cords). sch. Subchordal rod (hypochorda). sr. Sex-cell ridge. W. Wolifian duct.

(sex-cell ridge) from the enteron, so that the former lies just dorsal to the newly formed mesentery (Fig. 122, B). This ridge, now the sex-cell card (not to be confused with the sexual cords), soon divides in two longitudinally andgeach part moves a short distance ventro-laterally, taking up its position just beneath one of the cardinal veins. The two parts covered by coelomic.epithelium (peritoneum) project slightly into the coelom in these regions and are known as the genital ridges. As each ridge increases in size it projects further into the body cavity in 236 THE FROG: EATER OR LARVAL DEVELOPMENT

which it is suspended by the peritoneal epithelium which covers it. This epithelium gradually presses in above the organ, and thus forms a double sheet of tissue similar to that which supports the oviduct. As noted in the description of the adult organ, this sheet in thetcase of the ovary is termed the mesovarium and in the case of the testis the mesorchium. At this stage sex is still indistinguishable, and the gonad


A


Fig. 122. ——./1. Section through the gonad of a 30 mm. tadpole of R. catesbeiana. B. Section through a young ovary from a tadpole of the same species. The secondary genital cavity lined with rete cord cells is small, but the germ cell nests of

which the rest of the gonad ‘is composed are already beginning to break up. After Swingle.

gc. Germ cell. gcn. Germ cell nest. pc. Primary genital cavity. rc. Rete cord cells. sgc. Secondary genital cavity or ovarial sac.

whether male or female consists simply of an elongated sac in which the germ cells are coming to be arranged about the periphery. Throughout the interior there exists a space which is filled by a jelly-like substance containing a few nuclei, and though thus occupied by jelly this region is termed the primary genital cavity (Fig. 123, A). The develop strands, the ret'e cards, which grow ventrally into the primary genital cavity, and dorsally into the mesonephros (Witschi, ’52) .3 At this point in most Frogs the sexes begin to be differentiated as follows:

3 These strands are sometimes designated as the sexual cards, or sea; cords

(Swingle), but it seems preferable to reserve these terms for the strings of germ

cells coming from the germinal epithelium, and found in many of the higher vertebrates (see Chick). THE GON ADS 237

The Period of Sexual DiFferentiation.——In the case of a gonad destined to become an ovary the germ cells about the periphery begin to multiply. Simultaneously the masses of rete cord material which at certain points have grown down into the primary genital cavity begin to develop spaces within themselves. These new spaces within the rete cord material are known as the secondary genital cavities, and though at first occurring at intervals along the length of the organ they presently become more or less confluent. The larger cavities formed by this confiuence are called ovarial sacs, whose walls composed of rete cord cells, are everywhere in contact with the innermost layer of germ cells. These germ cells soon become arranged in groups or

nests’ each nest being sup Fig. 124.-—A. Section through a gonad of R.

catesbeiana showing the first signs of a begin


rounded by a layer of follicular cells apparently derived from the peritoneum. Later the nests break up, and each growing oiicyte has its own follicle (Fig. 123, B). As this growth of the oocytes and their follicles proceeds,

ning testis. Note the rete cord material extending out among the germ cells, and the absence of any extensive secondary genital cavity. B. A developing testis from the same species showing nests of germ cells, the forerunners of ampullae, and eventually tubules. Near the hilus or base of the organ note the rete cords forming the distal parts of vasa efierentia, which lower down branch out to connect with the ampullae. After Swingle.

a. Ampullae. gc. Germ cell. pc. Primary genital cavity. rc. Rete cords.

their pressure upon the walls

of the ovarial sacs causes these walls to approximate one another until the cavities of the sacs are virtually obliterated. According to most accounts there always remain in the Frog a few nests of oiigonia close against the periphery of the ovary, and from these are derived the new oiicytes for each breeding season.

In each gonad which is to form a testis on the other hand a different procedure occurs. The multiplication of the germ cells is less at first, while the proliferation of the rete cord material is greater. The latter also does not develop extensive secondary genital cavities as in the case it i 3%

238 THE FROG: LATER OR LARVAL DEVELOPMENT

of the ovary, but instead remains relatively condensed. Into this, germ cells from the periphery seem to migrate (R. sylvatica, ~Witschi, ’29), or in some cases cords of rete material grow out and surround groups of the germ cells (R. catesbeiana, Swingle, ’26, Fig. 124, A). In either event cysts are thus formed lined partly by rete material, and partly by connective tissue or stroma. These may at first be described as ampullae, but eventually they lengthen out to form the seminiferous tubules of the adult. Within a given tubule most of the germ cells are usually at the same stage of development, except that a few residual spermatogonia apparently always remain to furnish sperm for the next season. As indicated above the seminiferous tubules are connected with the vas deferens through the vasa efferentia, the latter being formed partly from the rete cords and partly from mesonephric tubules (Fig. 124, B).

In both sexes the anterior third or half of each genital ridge fails to develop as indicated above. Instead, some time previous to metamorphosis this portion of the organ starts to become converted into the fat bodies.

It may also be noted that while this is the normal situation in Frogs, in Toads an interesting modification occurs. In most species of the latter animal the male possesses a small ovary-like body lying between the testis and fat body. It is called Bidder’s organ, and has long been an object of interest. It is now believed by some (Witschi, ’33) to represent an incipient ovary. According to this view it is held that the undifferentiated gonad in this region is deficient in medullary substance, thus allowing the cortex here to develop to a limited degree. Though as indicated, it is most common in males, it also occurs in some female Toads where it again appears, according to this interpretation, as an undeveloped piece of the ovary (Witschi, ’33) .

SEX REVERSAL IN AMPHIBIA

The occurrence of hermaphroditism and of sex reversal is always of interest in any animal which normally has two distinct sexes. Hence since considerable experimental work in this connection has been done upon the Amphibia it is appropriate to say a few words about it at this point.

From the foregoing account of normal gonad development in the Frog, it is obvious that the gonads of the two sexes start out from common primordia. What then causes their differentiation? It will be recalled from statements in the chapter on the germ cells that the initial determination of sex in general is believed to depend on a balance beSEX REVERSAL IN AMPHIBIA 239

tween male and female determining genes. The female determining genes in most animals occur in the X-chromosome and the male determining genes in the autosomes. It was also noted, however, that these gene effects, like others, can be modified by the environment, and that the Amphibia afford good examples of this fact. The complete story here is not yet entirely clear, but experiments on both Frogs and Urodeles by Burns, Humphrey, Witschi and others seem to have elucidated the more essential factors. These experiments involve transplanting gonad primordia of various stages between animals of opposite sex, uniting at random many individuals to form pairs (parabiosis, Humphrey, ’36), injecting sex hormones, and altering the temperature at critical stages. For example in the Frog the cortex of the partially differentiated gonads is apparently inhibited by excessive warmth (32 degrees C.) , causing prospective ovaries to become testes (Witschi, ’29) . Or in various species of Amhlystoma it was shown that the implantation of a gonad preprimordium of the opposite sex in a larval host of another stage shifts the sex of the implant or the host (Humphrey, ’53). Also injection of male hormone, testosterone, during differentiation of prospective female gonads in Amhlystoma, produces partial reversal to males, while injection of oestrone in prospective males causes reversal toward the female (Burns, ’38, ’39). Chang, ’53, however, thinks substances other than these hormones are involved. Lastly Bruner and Witschi, ’54, showed that early use of testosterone actually causes the pre-medullary component of the prospective male gonad to form mesonephric tubules instead of medulla, without which the cortex partly differentiates into ovary. _

Without going into detail the conclusions suggested by the results of these procedures may be summarized as follows: The chromosome com plex gives the first impetus to sex determination, apparently by affect- ing the character of the mesoderm at the gonad site (gonad preprimordium) . The character of this preprimordium having been thus initially influenced then determines whether, in the seemingly indifferent gonad rudiment arising from it, the cortex or the medulla shall acquire the ascendancy. As soon as one or the other of these tissues does gain a start it begins to produce a substance with two effects. One effect is to stimulate still further the development of the favored tissue, cortex or medulla, and the other effect is to inhibit the development of the opposite tissue. Thus when once initiated the general result is cumulative. Finally when the mature gonad has formed, it produces the usual sex hormones, testosterone‘ or oesterone, and these tend, to control such secondary sex characters as may be characteristic of the species. With this 240 THE FROG: LATER OR LARVAL DEVELOPMENT

history in mind we may better understand various types of sex anomalies in ‘animals possessing a perfectly normal chromosome complex. Thus it is possible to have complete sex reversal in both gonads and gonoducts, or there may be partial reversal in these organs giving a sort of nondescript neuter. Also there may be complete reversal on one side only, resulting in real hermaphrodites. Lastly there may be reversal in parts of both gonads producing a kind of sex mosaic.

INTERNAL DEVELOPMENT: THE SKELETON

Only a brief outline of the development of the specific parts of this system as it occurs in the Frog, Chick and Pig will be given in this text. For further details the reader is referred to more extended accounts cited in the bibliography. However, since the general histogenesis of the different types of bone is essentially similar in all true Vertebrates, it seems desirable to give some details concerning the basicvprocesses involved‘. This will therefore be done at this point, with the understanding that though the fundamental pattern is similar in all the forms studied there are some variations in detail. The more important of the latter will be indicated in connection with the forms concerned.

THE HISTOGENESIS OF BONE

Dermal or Membrane Bone.—This type of bone is peculiar in that ossification (deposition of calcium salts) occurs directly within membranous connective tissue without the intervention of a cartilaginous stage. It is a method of bone formation which occurs extensively, though not exclusively, as we shall see, in certain bones of the skull, and may be described as follows: .

Within a connective tissue layer where the bone is to form, certain undifferentiated mesenchymal cells become arranged in isolated strands, each strand being several cells in thickness. These cells then lose their fine processes characteristic of the cells of mesenchyme, and begin to secrete in their midst a delicate fiber, for which reason they are termed fibroblasts. The fiber they secrete is called an ossein fiber, but is not essentially different from other nonelastic or white connective tissue fibers consisting of collagen. Soon numerous fibers thus formed in a particular region come to constitute a thickened strand. In ‘the next step the fibroblast cells which deposited the fibers become modified chemically, and about each fiber they begin to deposit calcium salts. ‘When this stage has been reached the cells involved are called osteoblasts. THE HISTOGENESIS or BONE 24.1

The fibroblasts and osteoblasts, continuing to form respectively both ossein fibers and calcium salts about each original strand, add to its

thickness and length. As a consequence of the latter type of growth, .

these thickened and ossified strands, now termed trabeculae, are brought into contact with each other, and thus a bony network is produced (Fig. 125) . Since, moreover, the deposition of fibers and calcium (matrix) is more or less‘periodic we find any given trabecula consisting of layers of bone somewhat like the growth rings of a tree. It should


,1


bone trabecula connective tissue ,3 (membrane)

csteoblasu

Fig. 125.—Trabecu1ae of a piece of membranous jaw bone from a Mammal in the process of being thickened by fibroblasts and osteoblasts. Drawing from Turtox preparation.

also be obvious that as the osteoblasts deposit their matrix they must keep moving away from the original center of deposition or else be‘ imprisoned in their own products. As a matter of fact different ones do both these things. Those which move, and thus remain at the surface continue to function as osteoblasts. Those which are trapped, so to speak, cease deposition, but do not die. They remain as permanent bone cells with delicate processes extending out into the matrix. These processes. Qonvey nutriment from the spaces containing blood vessels to the cell bodies, which furnish it to the organic ossein fibers. When these cells and the fibers deteriorate and finally disappear with senescence only the calcium salts are left, and the bone becomes brittle.

The bone formed as described evidently contains many irregular spaces, and so long as these exist it has a spongy texture. In the central parts of membrane bones which are under discussion this condition is permanent, and the bone is known as cancellous. The spaces in such bone, however, are not empty. They are filled with blood vessels and large thin walled sinusoids, surrounded and supported by reticulate 242 THE F ROG: LATER OR LARVAL DEVELOPMENT

connective tissue (stroma). The stroma contains all types of mature blood corpuscles which are being constantly produced by its undifferentiated cells, and passed as needed into the sinusoids, which communicate with the blood vessels. This conglomeration of loose connective tissue, blood spaces, vessels and cells is termed marrow. Sometimes it is permeated with fat containing cells, and is then known as yellow marrow as compared with the corpuscle producing red marrow. The spaces thus occupied by marrow of one sort or the other are lined by a more dense flat connective tissue layer, now containing fibroblasts and osteoblasts, and known as the endosteum. It is not to be assumed of course that marrow exists only in ‘dermal bones. It occurs as much or more in the other type of bone as will presently be pointed out.

As so far described it might be supposed that dermal bone is entirely cancellous, but this is not the case. Surrounding the first formed cancellous material is a layer of connective tissue similar to the endosteum which comes to line the marrow spaces. This being outside, how I ever, is called periosteum, and it also contains fibroblasts and osteo blasts. ‘These fibroblasts and osteoblasts, like those of the endosteum covering the trabeculae, deposit fibers and bone, in this case in continuous layers completely surrounding the cancellous bone -and marrow. Thus is formed one type of compact bone, between whose layers entrapped bone cells occur at intervals, just as in the case of the layers deposited on the trabeculae. As implied, however, this is not the only type of compact bone that may be formed. In some cases, as will be described more in detail below, some of the more outer marrow spaces are filled with concentric bone layers which thus make the region so involved compact. More will presently be said of this method of forming compact bone. Also curiously enough some of the first continuous peripheral layers deposited may prove not to be permanent. Another type of connective tissue cells, known as osteoclasts, may invade this peripheral bone and eat out cavities in it so that it in turn becomes cancellous. Later, however, such secondary cavities will be filled in again in the manner noted in the case of the other cancellous bone, thus making it again compact. In any case a few of the continuous peripheral layers are always finally Ieft surrounding the entire bone. The end result of all these processes is that the completed dermal bone consists of a cancellous and marrow filled central region surrounded by varying thicknesses of compact layers of onersort or another. Bones of this type it should be added are more or less flat in shape, occurring for the most part, as noted, as covering bones of the skull. ’ THE HISTOGENESIS OF BONE 243

Cartilage or Endochondral Bone.— In the case of bone of this type, which comprises the larger part of the skeleton, ossification does not occur directly from membrane, but from an intervening cartilaginous stage. The process is as follows:

epiphysial cartilage

cartilage being

replaced by bone

trabeculae covered by fibroblasts

and osceoblasts

dyiaphysls

Fig. 1Z6.—-—The epiphysis and a portion of the diaphysis of a developing mammalian long bone. The epiphysis is still entirely cartilaginous. At the boundary between the two regions, however, the cartilage is being ‘reduced to fine strands by means of chondroclasts. Further down in the diaphysis these strands are being built up into bony trabeculae by the fibroblasts and osteoblasts which cover their surfaces. Photo of a Turtox preparation by the author.

As before the initial condition is that of a mass or layer of mesenchyme. The mesenchymal cells then lose their processes much as in the preceding case. Now, however, instead of becoming aggregated in strands they form a densely packed mass of multiplying cells which gradually assumes the shape of the future bone. These cells, however, do not form bone. Instead each cell begins to secrete a gelatinous matrix of a substance called chondrin. This is at first quite elastic, and thus the cells are able to move away from each other as they secrete. 244 THE FROG: LATER OR LARVAL DEVELOPMENT

Later the chondrin condenses to form the mature cartilage matrix. When this stage is reached, the cells can no longer push each other apart, or multiply much. Each cell may divide once or twice, and the small group secretes just enough to cause the cartilage immediately around it to become especially dense. Thus we have formed a mass of cartilage the shape of the future bone. It consists of a dense chondrin matrix containing numerous small groups of cells. Finally this mass of cartilage has surrounding it a firm connective tissue layer called perichondrium, whose cells, like those of the periosteum, continue for a time to add to the cartilage peripherally. The next step is the destruction of the cartilage and its replacement by bone.

The destruction of the cartilage is brought about by the same cells which previously deposited it. Now, however, these cells behave like the osteoclasts noted above, only in this case they act as chondroclasts, and erode cartilage instead of bone. They proceed in such a way that soon they have reduced the cartilage to delicate strands whose surfaces they cover. Meanwhile certain cells of the perichondrium become active and, along with blood vessels, start to invade the disappearing cartilage. These cells turn out to be fibroblasts and osteoblasts which soon replace the cartilage eroding cells surrounding the cartilaginous strands. These cartilaginous strands thus take the place of the fibrous strands of cancellous membrane bone, and around them the new fibroblasts and osteoblasts deposit fibers and calcium salts to form cancellous endochondral bone ( Fig. 126). The resulting bony trabeculae surrounding marrow filled spaces are the same as before, only in this instance the bone was preceded by cartilage. In View of its behavior the surrounding perichondrium is from now on termed periosteum. This endochondral cancellous bone may now become compact in the same way that the cancellous bone ofmembranous origin does so. The details of that process, which were merely suggested previously, are as follows:

The bone forming cells, fibroblasts and osteoblasts, covering the trabeculae gradually so arrange themselves while depositing bone that the marrow spaces become tube shaped. Then as the osteoblasts and fibroblasts continue to deposit layers of calcium salts and fibers, part of the cells withdraw toward the center of the constantly decreasing marrow space. Others, as previously described in another connection, are trapped between the layers to form permanent bone cells. In this manner concentric layers of bone are produced surrounding a marrow space which finally is reduced to a small canal containing only a couple of blood vessels and a few cells. This is called an Haversian canal, and toTHE HISTOGENESIS OF BONE 245

gether with the concentric arrangement of the bone layers about it constitutes an Haversian system (Fig. 127). Compact bone so formed therefore would consist of many such systems filling completely the spaces between the original trabeculae. The canals of the numerous systems are, moreover, interconnecting, so that the blood vessels in them ultimately reach the periosteum on the one hand or the central marrow on the other.

It should again be emphasized that the actual process of bone depo


location of V a bone cell


Haverslan canal P"l°“°3l bone

Fig. 127. -——I-Iaversian systems from a section of adult ‘bone.

sition just indicated as occurring in compact endochondral bone is exactly the same as that referred to in the case of one type of compact membrane bone. The difference is entirely in the preceding processes. In the former case the compact bone was preceded simply by cancellous bone. In the present case the cancellous bone was itself preceded by cartilage. In addition to this difference in the method of development between membrane bones and the part of all endochondral bones thus far described, there is one other feature characteristic of the final structure of most of the latter. A good deal, or all, of the central cancellous material in mature endochondral bones is usually removed entirely by osteoclasts, and the relatively large single space so produced occupied by the marrow. Any other marrow in such bones will, as in membrane bones, occupy the spaces of any cancellous bone which remains (Figs. 126, 128). l

' It must now be added that even so called endochondral bones are not entirely so. This is because the endochondral compact bone formed in 246 THE FROG: LATER OR LARVAL DEVELOPMENT

the manner we have indicated is always ultimately surrounded by bone formed directly from the periosteum, and hence entirely membranous in origin. This may involve simply the laying down of the final circumferential layers. Usually, however, as in the case of completely membranous bone, some of the early surrounding layers are rendered can


marrow

’ bony

bone)



  • 'ln,llu Hui | s\‘/

‘lu,'"lu.',"'l mmnum u$““I\}‘,‘ \‘gnuf,':g1_n_p-u:uuI_I_I_l_L‘}§“u-{‘:/

L


Fig. 128.— A semi-diagrammatic representation of a cross section of a mammalian

long bone (endochondral) , showing periosteum, periosteal bone lamellae, Haversian systems and marrow.

cellous by osteoclasts, with the subsequent development of Haversian systems. And in this case the latter were obviously not immediately preceded by cartilage. Thus it is to be remembered that when, in later discussions, we refer to certain bones as being endochondral in origin, it is only a part of such bones which were really preformed in cartilage. Socalled “ membrane bones” are, however, entirely preformed in membrane. ’

Finally it should be noted -that in the case of any kind of bone the later stages in its formation involve a very intimate connection with the periosteum. This is because that, in; addition to blood vessels, innumerable white periosteal connective tissue fibers are surrounded by THE VERTEBRAL COLUMN 247

the final calciferous deposits. Thus these fibers, known as the fibers of Sharpey, are directly continuous from the periosteum right into the compact bone forming an extremely tight union between connective tissue and the bone itself. It may also be noted that at certain points these fibers are aggregated into bundles called tendons which are continuous in the opposite direction from the bone into the connective tissue sheaths of its muscles. We are now prepared to turn to a brief consideration of the formation of the various parts of the skeleton of the-Frog.

THE VERTEBRAL COLUMN

At or a little before the time of hatching, the skeletogenous sheath has already come to surround the notochord and nerve cord, as in-. dicated above. Some time after hatching (about 15 mm.) , cartilage develops within this sheath and presently becomes divided into sections corresponding in position and num-' ber to the future vertebrae. Within each such


Fig. 129. —-— Transverse section through the vertebral column in the body region of a larva of Xenopus capensis. From Kelli cott (Chordate Develop section, moreover, the cartilage about the chorda soon forms a ring which completely surrounds it (Fig. 129). Within these cartilaginous rings, ossification now starts and gradually spreads inward until the notochord at the core of every ring is entirely obliterated. Thus is formed the centrum of each vertebra. Meanwhile between these vertebral centra the notochord is also obliterated by the ingrowth of cartilage. Each intervertebral disc thus developed, later splits into an anterior and a

posterior part. Finally, during metamorphosis each of these parts he

ment) . After Schauinsland. c. Notochord. d. Dorsal vertebral cartilaginous arch. s. Sclerotomal (skel etogenous) sheath. n. Nerve cord. cs. Chorda sheath (primary and secondary). t. Perichondral connective tissue. 12. Ventral (hypochordal-) vertebral cartilage. The dorsal and ventral cartilaginous elements have not yet come to surround the noto chord.

\

comes ossified and fused with the end of the contiguous centrum. In a like way the neural arches ossify from cartilage which extends

dorsad from the centra around the nerve cord, while the transverse processes arise as bits of cartilage projecting laterally from each centrum, which also later ossify. Eventually minute cartilaginous ribs form at the ends of the processes, but are soon fused with the latter. -Vertebra formation is induced by nerve cord rather than notochord (Holtzer, ’52) . 1»

248 THE FROG: LATER OR LARVAL DEVELOPMENT

As already noted, the Frog possesses only nine real vertebrae, and the above description applies only to them. The skeletogenous elements of the last two somites, however, form a single tubular piece of cartilage which surrounds the end of the notochord. Later it also becomes mostly ossified, and is known as the urostyle.


Fig. 130. ——Dorsal views of the chondrocranium of the Frog larva. A. Cl1ondrocranium of a 7.5 mm. larva of R. temporaria. From Kellicott (Chardaze Development). After Gaupp, from Stiihr-Ziegler model. B. Chondrocranium of a 14 mm. larva of R. fusca. After Gaupp, from Ziegler model. .

a. Auditory capsule. bp. Basal plate. c. Notochord. ct. Trabecular cornu. f. Basicranial fontanelle. in. Internasal plate. ir. lnfrarostral cartilage. j. Jugular foramen (for IX and X cranial nerves). m. Muscular process. M. Mecke1’s cartilage. mo. Mesotic cartilage. o. Occipital process. pa. Anterior ascending process of palatequadrate cartilage. pl. Parachordal plate. pp. Posterior ascending process of palata quadrate cartilage. pq. Palato-quadrate cartilage. sr. Suprarostral cartilage. t. Trabecular cartilage.

THE SKULL

The F1oor.—-«The posterior portion of the skull floor, i.e., that part which lies beneath the hind brain, is formed medially by the notochord. On each side of the notochord a cartilaginous rod develops which fuses with the chorda or rather with the cartilage which soon takes its place, thus completing the floor in this region. These rods are called the parachordals, and the fused mass is the parachordal plate (Fig. 130, A).

In front of each parachordal is another rod. These rods are curved THE SKULL 249

I!’

Fig. 131.—/1. Anterior portion of chondrocranium of R. fusca during metamorphosis. Lateral view. From Kellicott (Chordate Development). After Gaupp, from Ziegler. B. Skull of a 2 cm. R. fusca, after metamorphosis. Dorsal view. Membrane bones removed from left side. After Gaupp, from Ziegler.

a. Auditory capsule. am. Anterior maxillary process. an. Annulus tympanicus. art. Articular process of palato-quadrarte cartilage. ea. Exoccipital bone f. Frontaparietal bone. fpo. Proiitic foramen. mx. Maxillary hone. n. Nasal bone. 0. Olfactory cartilages. on. Orbitomasal foramen. pa. Anterior ascending process of palatequadrate. pg. Pterygoid bone. pl. Plectrum. pm. Posterior maxillary process. pp. Posterior ascending process of palato~quadrate. pq. Palato-quadrate cartilage. pt. Pterygoid process of palate-quadrate. px. Premaxillary bone. qj. Quadratojugal

bone. 11. Foramen for optic nerve. III. Foramen for ‘III cranial nerve. IV. Foramen for IV cranial nerve. 250 THE FROG: LATER OR LARVAL DEVELOPMENT

somewhat, with their concave sides facing each other, and their posterior ends fused with the anterior ends of the parachordals. Their own anterior ends grow toward each other and fuse between the olfactorv organs; these rods are the trabeculae. The space between them in the anterior floor of the skull is the basicranial fontanelle, which temporarily lodges the infundibulum. Later, as the trabeculae grow together, this opening is closed. I

The Sides, End, and Roof.—The floor has reached the stage indicated only a short time after hatching. The other cartilaginous parts of the skull then develop as follows:

In the posterior region the cartilaginous auditory capsules appear at the sides of the head (Fig. 130, B). Ventrally they are presently united with the skull floor by the mesotic and occipital cartilages. The capsules thus form the sides of the posterior part Fig. 132.——Hyoid and branchial arches of a29 of the skull’ while the 0c.

mm. larva of R. fusca. Ventral view. From Kelli- cipital cartilages grow up

cott (Chordate Development). After Gaupp, to form the Posterior walls from Ziegler.

bb. Basibranchial (first), or copula. bh. Basi- and the l‘00f Of this region.

5‘32‘=F:f;. S:':;::::‘-.;:;,§::::%::;*:hfie‘ Between the eeeeveeele is e ‘ posterior opening, the fa ramen magnum, through which the spinal cord passes into the brain.

Anteriorly the trabeculae grow up to form the sides of the skull in the neighborhood of the orbits. Their more anterior portions then grow together dorsally forming the anterior roof. Between this anterior roof and the posterior one formed by the occipitals is the supra-cranial fontanelle. The extreme anterior ends of the trabeculae go to form the olfactory capsules, which are partly separated from the brain cavity by a septum. All of these changes, both anterior and posterior, are virtually completed in larvae of 3 cms.

Dermal Elements in the Skul1.——The cartilaginous skull thus formed later becomes ossified, in the usual way. Before this occurs, however (about 20 mm.), many of the parts begin to be covered by

51:

ch THE SKULL 251'

bony plates originating in the dermis (in the manner indicated above) and hence called dermal bones (Fig. 131). Some of these plates, such as the fronto-parietals, serve to cover open spaces left in the cartilage, e.g., the supra-cranial fontanelle. Most of the dermal bones as well as those formed in the cartilage have appeared before metamorphosis is com plete.


Fig. 133.—A. Hyobranchial apparatus of R. fusca, toward the end of metamorphosis. The left side is shown in a more advanced stage than the right, in that less cartilage is present. The original cartilage is indicated by fine stipples. The coarse stipples indicate the cartilage added during the early part of metamorphosis. From Kellicott (Chordate Development). After Gaupp, from Ziegler. B. Hyobranchial apparatus of a 2 cm. R. fusca, after metamorphosis. After Gaupp, from Ziegler.

a. Alar process. ac. Anterior process of hyoid cornu. 17. Body of hypobranchial cartilage. bb. Basibranchial (first), or copula. ch. Ceratohyal (hyoid cornu in B). ho.

Hypobranchial plate. 1. Postero-lateral process of hypobranchial cartilage. m.'Manu brium. 2. Remains of second ceratobranchial (postero-medial process of hypobrar.chial cartilage).

The Visceral Arches.—These arches at first consist merely of

concentrations of mesoderm, as indicated above. Shortly after the

mouth opens, however, all have developed skeletal elements of cartilage. The cartilage of the mandibular arch early becomes divided into a dorsal portion, the palato-quadrate, ‘and a ventral portion, Meckel’s cartilage. The ‘former becomes fused anteriorly and posteriorly with the trabeculae and at metamorphosis is considerably modified to form a part of the upper jaw. As noted above, furthermore, a small outgrowth becomes separated from the *posterior or quadrate portion of this cartilage and gives rise to the annulus tympanicus of the middle ear. Meek252 THE FROG: LATER OR LARVAL DEVELOPMENT permanent cartilaginous

'. epiphysls erlosteum cartilage blood vessel invading Eériiflng blood vessel forming marrow perlosteal bone Iamellae of diaphysls marrow


Fig. 134-.-—-Semi-diagrammatic representations of medial longitudinal sections of growing long bones of Bullfrog tadpoles. A. A young stage in which cartilage is still the dominant element in both diaphysis and epiphysis. In the diaphysis, however, the periosteum has already replaced some of the cartilage with circumferential bony lamellae. Also a blood vessel along with chondrioclasts has invaded the cartilage, and is beginning to form the marrow. B. A later stage in which the diaphysial cartilage has all been replaced by marrow and circumferential bone lamellae laid down by the periosteum. Note that in this case there are not, and never would have been, any. I-Iaversian systems, all the bone of the diaphysis being formed from periosteal lamellae. The epiphyseal cartilages, at this and the preceding stages, contain a lozenge-shaped growing zone characteristic of the Frog. The epiphyses remain permanently cartilaginous in this animal. After studies by Marvin.

9 THE APPENDICULAR SKELETON 253 epiphysis

 .. __ _

l ’ lozenge l shaped

«,3 region . 4,


diaphysis

circumferential membrane depositing bone lamellae

marrow

Fig. 135.—The epiphysis and part of the diaphysis of a developing Bullfrog femur in a condition similar to that diagramed in Fig. 134, A. Note the cap of epiphyseal cartilage extending down on either side of the diaphysis. Also in this cap note the lozenge-shaped region of dividing cells. On each side of the diaphysis the heavy lines represent. dense circumferential connective tissue within which the layers (lamellael of circumferential bone are about to form. A small region of marrow which occupies the middle portion of the bone shows at the

lower edge of the picture. (Author’s photograph of preparation by Marvin.)

el’s cartilage remains small throughout larval life, but constitutes the core of the lower jaw in the adult-.

The hyoid arch (Ceratohyal) and the second branchial arch, together with certain median elements, form the hypobranchial apparatus of the adult. In the latter the hyoid arch becomes the so-called hyoid (greater) cornu or horn, while the second branchial arch becomes the

lesser cornu. All of the other arches disappear entirely at metamorphosis (Figs. 132, 133). A \ 254 THE FROG: LATER OR LARVAL DEVELOPMENT

THE APPENDICULAR SKELETON

Both the pectoral and pelvic girdles are said to be endochonclral in origin, with the exception of the clavicle, which as in other animals is a membrane bone. The long bones of the limbs are also usually thought of as endochondral, but in the Frog, unpublished investigations by R. W. Marvin (’47) in the author’s laboratory would seem to show that in a strict sense they are not so at all. In the case of these bones in this animal what appears to occur is this:

A cartilaginous core as usual first replaces the condensed mesoderm or membrane, and around this the bone is later laid down exclusively by the periosteum in circumferential layers (Fig. 134»). The cartilage is then removed, as well as some of the first formed inner layers of bone. This removed material, however, is all replaced by marrow, none of it by bone. Hence if this account is correct there is no true endochondral bone involved, i.e., none which replaces cartilage or bone preceded by cartilage in the manner described above. The situation as so far indicated refers only to the bone shaft, i.e., the part defined in all such bones as the diaphysis. The condition at the ends, whichare known as the epiphyses, remains to be discussed. In the case of the Frog the ends of the cartilaginous cores of the shaft of a long bone never become ossified at all, even after all growth has ceased. Thus the ends or epiphysis in this case consist of permanent caps of cartilage whose borders extend down somewhat over the bony cylinders which constitute the diaphysis (Fig.4 135) . These procedures in both diaphysis and epiphyses are at variance, as we shall see, with what occurs in both the Bird and the Mammal, which also differ somewhat from each other.

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CHAPTERS IV, V, VI

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7

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ence of the Annular Tympanic Cartilage on the Formation of the Tympanic Membrane,” Physiol. Zob'l., I, 1928.-—“ V. The Atrophy of Anuran Tail Muscle during Metamorphosis,” Physiol. Zo6l., II, 1929.—-“VIII. The Role of the Urostyle in the Atrophy of the Tail,” Anat. Rec., XLVII, 1930.——“ VII. The Influence of the Columella on the Formation of the Lamina Propria of the Tympanic Membrane,” Jour. Exp. Zoo'l., LIX, 193l.—“ XII. Potential Influences of the Quadrate and Supra-Scapula on Tympanic Membrane Formation in the Anuran,” Jour. Exp. Zo6l., LXVII, 1934.

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Holtfretter, J., “Morphologische Beeinflussung von Urodelenektoderm bei xenoplasticher Transplantation,” Arch. f. Entw.-mech., CXXX, l935.—-“A Study of the Mechanics of Gastrulation: Part I," Iour. Exp. Zoiil., LXLIV, 1943.

Humphrey, R. R., “ The Early Position of the Primordial Germ Cells in Urodeles; Evidence from Experimental Studies,” Anat. Rec., XLII, 1929.—“ Studies on Sex Reversal in Amblystoma. VII. Reversal of Sex Type in Gonadic Preprimordia of A. punctatum males implanted in Females of More Rapidly Growing Species,” Anat. Rec., LXII, l935.—“ IX. Reversal of Ovaries to Testes in Parabiotic A. tigrinum,” Iour. Exp. Zo6l., LXXIII, 1936.

Janes, R. G., “ Studies on the Amphibian Digestive System. III. The Origin and Development of Pancreatic Islands in Certain Species of Anura," Jour. Morph., LXII, 1938.

Jenkinson, J. W., “On the Relation between the Symmetry of the Egg and the Symmetry of Segmentation and the Symmetry of the Embryo in the Frog,” Biometrika, VII, 1909.—Experimental Embryology, Oxford, 1909.

Kaan, H. W., “ Further Studies on the Auditory Vesicle and Cartilaginous Capsule of Amblystoma punctatum,” Jour. Exp. Zo6l., LXXVIII, 1938.

Knouff, R. A., “ The Origin of the Cranial Ganglia of Rana,” Jour. Comp. Neur.,'

XLIV, 1927. —“ The Developmental Pattern of Ectodermal Placodes in Rana pipiens,” LXII, 1935.

Lehmann, F. E., “ Further Studies on the Morphogenetic Role of the Somites in the Development of the Nervous System of the Amphibians. The Difierentiation and Arrangement of the Spinal Ganglia in Pleurodeles waltli,” Jaur. Exp. Zo¢'2'l., XLIX, 1927. _

Lewis, W. H., “ Experimental Studies on the Development of the Eye in Amphibians. I. On the Origin of the Lens," Am. Jour. Anal, III, 1904.

Liedke, K. B., “ Lens Competence in Rana pipiens,” four. Exp. Zob'l., LXL, 1942.

Lindeman, V. F., “Integumentary Pigmentation in the Frog (R. pipiens) during Metamorphosis, with Special Reference to Tail Skin Histolysis,” Physial. Zoo'l., II, 1929.

Mangold, 0., “ Transplantationsversuche zur Frage der Spezifitéit unter der Bildung der Keimbliitter,” Arch. Mikr. Anat., C, 1924.

Marx. A., “ Experimentelle Untersuchungen zur Frage der Determination der Medullarplatte,” Arch. Mikr. Amzt., CV, 1925.

Maximow and Bloom, Text-Book of Histology, Philadelphia, 1938.

McClendon, J. F., “ The Development of Isolated Blastomeres of the Frog’s Egg,” Am. Jour. Anat., X, 1910. 258 THE FROG: LATER OR LARVAL DEVELOPMENT

Morgan, T. H., “The Formation of the Embryo of the Frog,” Anat. Anz., IX, 1894-.——-“ Half-embryos and Whole-embryos from One of the First Two Blastemeres of the F1-og’s Egg,” Anat. Anz., X, 1895.—-The Development of the F rog’s Egg: An Introduction to Experimental Embryology, New York, 1897. “ The Relation between Normal and Abnormal Development of the Embryo of the Frog, as Determined by Injury to the Yolk-Portion Of the Egg.” Arch. Entw.-mech., XV, 1902.—“ The Relation between Normal and Abnormal Development of the Embryo of the Frog (III), as Determined by Some Abnormal Forms of Development,” Arch. Entw.-mech., XVIII, 1904. —“ The Relation between Normal and Abnormal Development of the Embryo of the Frog: X. A Re-examination of the Early Stages of Normal Development from the Point of View of the Results of Abnormal Development,” Arch. Entw.mech., XIX, 1905.—-“ Experiments with Frog's Eggs,” Biol. Bull., XI, 1906. ——“ The Origin of the‘Organ-forming Materials in the Frog’s Embryo,” Biol. Bull., XI, 1906.—Experimental Embryology, New Yorlc, 1928.

N eedham, Joseph, Biochemistry and Morphogenesis, Cambridge, 1942.

Pasteels, J., “ New Observations Concerning the Maps of Presumptive Areas of the Young Amphibian Gastrula (Amblystoma and Discoglossus),” Jour Exp. Zob'l., LXXXIX, 1942.—“ On the Formation of the Primary Entoderm of the Duck (Anas domestica) and on the Significance of the Bilaminar Embryo in Birds,” Anat. Rec., LXLIII, 1945.

Piatt, J., “ Nerve-Muscle Specificity in Amblystoma, Studies by Means of Heterotopic Cord Grafts,” Jour. Exp. Zo¢'il., LXXXV, 1940.

Porter, K. R., “ Androgenetic Development of the Egg of Rana pipiens,” Biol. Bull., LXXVII, 1939.

Raven, Chr. P., “ Zur Entwicklnng der Ganglienleiste. V. Uber die Diflerenzierung des Rumpfganglienleistenmaterials,” Arch. Entw.-mech., CXXXIV, 1936. Roux, W., “ Beitrage zur Entwickelungsmecbanik des Embryos, Nr. IV. Die Richtungsbestimmung der Medianebene des Froschembryo durch die Copulationsrichtung des Eikernes nnd des Spermalcernes,” Arch. Mikr. Anat., XXIX, 1887.—“Beitr§ge zur Entwickelungsmechanik des Embryo.” V. “Ueber die kiinstliche Hervorbringung halber Embryonen durch Zerstiirung einer der beiden ersten Furchungskugeln, sowie iiber die Nachentwickelung der fehlen'den kb'rperhiilfte,” Virchow’s Archiv., CXIV, 1888.——“Ueber die Lagerung des Materials des Medullarrohres im gefurchten Froschei (Verh. Anat. Ges. 2),” Anat. Anz., III, l888.—“ Ueber die ersten Teilungen des Froscheies und ihre Beziehungen zu der Organbildnng des'Embryo,” Anat. Anz., VIII, 1893.

Rugh, Roberts, “ Heterchromatic Radiations and Early Amphibian Development,” Coll. Net, VIII, 1933.—“A Quantitative Analysis of the Pituitary-Ovulation Relation in the Frog (Rana pipiens),” Physiol..Zoo'l., X, 1937.—“ Release of Spermatozoa by Anterior Pituitary Treatment of the Male Frog, Rana pipiens,” Proc. Soc. Exp. Biol. and Med., XXXVI, 1937.

Schectman, A. M., “Unipolar Ingression in Triturus: A Hitherto Undescribed Movement in the Pregastrular Stages of a Urodele,” Univ. Cal. Press, XXXIX, 1934.— “ Mechanism of Ingression in the Egg of Triturns torosus,” Proc. Soc. Exp. Biol. and Med., XXXII, 1935. —“ The Mechanism of Amphibian Gastrulation. I. Gastrulation-Promoting Interactions Between Various Regions of an Anuran Egg (Hyla regillal,” Univ. Cal. Press, LI, 1942.

Schleip, -W., Die Determination ,cler Primitioentwicklung, Leipzig, 1929.

—-, W. and Penners, A., “Weitere Untersuchungen iiber die Entstehung der Schultzeschen Doppelbildungen beim braunen Frosch,” Ver. Phys.-Med. Ges., Wiirzburg., LI, 1926.

Schotté, O. E. and Edds, Mac V., “Xenoplastic Induction of Rana pipiens AdREFERENCES TO LITERATURE 259

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Schultze, 0., “Die kuenstliche Erzeugung von Doppelbildungen bei Froschlarven mit Hilfe abnormer Gravitationswirkung," Arch. Entw.-mech., I, 1894.

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Shore, T. W., “ On the Development of the Renal-portals and Fate of the Posterior Cardinal Veins in the Frog,” Jour. Anat. Physiol., XXXVI, 1901.

Smith, P. E., “The Pigmentary Growth and Endocrine Disturbances Produced in the Anuran Tadpole by the Ablation of Pars Bucalis of the I-Iypophysis,” Am. Anat. Mem., II, 1920.

Spemann, H., “ Entwicklungsphysiologische Studien am Triton-Ei,” I, II, III, Arch. Entw.-mech., XII, XV, XVL, 1901—1903.——“Ueber experimentelle erzeugte Doppelbildungen mit cyclopischem Defekt,” Zool. Jahrb. Supplement, VII, 1904. —“ Ueber die Determination der ersten Organanlagen des Amphibienemb1-yo,” I—II, Arch. Entw.-mech., XLIII, 1918.—“Die Erzegung tierischer Chimiiren durch heteroplastische ernbryonale’ Transplantation :.urschen Triton criszatus and Triton taeniatus,” Arch. Entw.-mech., XLVIII, 1921.——“Ueber Organisatoren in der tierischen Entwicklung,” Nat.- W iss.,_ XII, 1924. —-“ Some Factors in Animal Development,” (Translation), Brit. Jour. Exp. Biol., II, 1925.

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Swett, F., “Experiments upon Delayed Determination of the Dorsoventral Limb Axis in Amblystoma punctatum (Linn.),” Jour. Exp. Zob'l., LXXV, 1937. “ Further Experiments upon the Establishment and the Reversal of Prospective Dorsoventral Limb-Axis Polarity,” four. Exp. Zob'l., LXXXII, 1939.——“ Establishment of Definitive Polarity in the Dorsoventral Axis of the Fore-limb Girdle in Amblystoma punctatum (L_inr_1.),” ./our. Exp. Zob'l., LXXXVI, 1941. '

Swingle, W. W., “ The Germ Cells of Anurans,” I. “ The Male Sexual Cycle of Rana catesbeiana.” II. “ An Embryological Study of Sex Differentiation in Rana catesbeiana,” Jour. Morph. and Physiol., XLI, 1926.

Taylor, A. C., “ Selectivity of Nerve Fibers from the Dorsal and Ventral Roots in the Development of the Frog Limb,” Jour. Exp. Zo¢'il., LXLVI, 1944.

—, and Kollros, J. .I., “Stages in the Development of Rana pipiens Larvae,” Anat. Rec., LXLIV, 1946. _

Vogt, W., “Operativ bewirkte ‘ Exogastrulation’ bei Triton und ihre Bedeutnng fiir die Theorie der Wirbeltiergastrulation,” Verh. d. Anat. Gesell., LV, 1922. —“ Gestaltungsanalyse am Amphibienkeim mit iirtlicher Vitalflairbung,” Arch. Entw.-mech., CVI, 1925.—“ Ueber Wachstun und Gestaltungsbewegungen am hinteren Kfirperemde der Amphibian,” Verh. J. Anat. Gesell., LXI, 1926.

Weber, A., “ Etude de la torsion de Pébauche cardiaque chez Rana esculenta,” Bibliographic Anatomique, XVIII, 1908 (1909).

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Witschi, E., “Studies on Sex Differentiation and Sex Determination in Amphibians,” I. “Development and Sexual Differentiation of the Gonads of Rana sylvatica.” Jour. Exp. Zoc'il., LII, 1929.——‘‘ Studies on Sex Differentiation and Sex Determination in Amphibians. II. Sex Reversal in Female Tadpoles of Rana sylvatica Following the Application of High Temperature,” Jour. Exp. Zob'l., LII, 1929.—-“VI. The Nature of Bidder’s Organ in» the Toad,” Am. Iour. Anat., LII, 1933.—“ VIII. Experiments on Inductive Inhibition of Sex Differentiation in Parabiotic Twins of a Salamander,” Anat. Rec., LXVI, 1936. -—“ IX. Quantitative Relationships in the Induction of Sex Differentiation, and » V.

260 THE FROG: LATER OR LARVAL DEVELOPMENT

the Problem of Sex Reversal in Parabiotic Sainruanders,” Iour. Exp. Zob'l., LXXV, 1937.——“ The bronchial columella of the ear of larval Ranidae,” Jour. Morph., XCVI, 1955. .

Yntema, C. L., “An Experimental Study of the Origin of the Cells which Con. stitute the Vllth and Vlllth Cranial Ganglia and Nerves in the Embryo of Amhlystoma punctatum,” Jour. Exp. Zo6l., LXXV, 1937.—“An Experimental Study on the Origin of the Sensory Neurones and Sheath Cells of the IXth and Xth Cranial Nerves in Amblystoma punctatum,’ Jour. Exp. Zo5l., LXLII, 1943.

Zwilling, E., “An Experimental Analysis of the Development of the Anuran Olfactory Organ,” Iour. Exp. Zo6l., LXXXIV, 194-0.~—-“ The Determination of the Otic Vesicle in Rana pipiens,” Jour. Exp. Zo6l., LXXXVI, 194-1.

APPENDIX To Face BIBLIOGRAPHY

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Brunet, J. A. and Witschi, E., “ Pluripotentiality of the mesonephric blastema and the mechanism of feminization of male salamanders by androgenic hormones,” Anat. Rcc., CXX, 1954.

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