Book - Outlines of Chordate Development 2
Outlines of Chordate Development by William E. Kellicott (1913).
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- 1 Chapter 2 The Early Development Of The Frog
- 1.1 Outline Of The Life History Of The Frog
- 1.2 Embryonic Period - A. From Fertilization Through Gastrulation
- 1.3 Embryonic Period - B. The Formation Of The Early Embryo
Chapter 2 The Early Development Of The Frog
The frog (Rana, sp.) is important zoologically, not as a central type representative of any large group of Chordates, but as a transitional form connecting the lower and higher groups of Craniata. This relation is no less apparent embryologically than morphologically. For there are comparatively few groups Lampreys, Ganoids, Dipnoans whose development can be compared closely with that of the Amphibia, while the types of development seen in the Cephalochorda (Amphioxus) and among the Elasmobranchs, Teleosts, and all of the Amniota, are in various respects quite special. Many of these special conditions may be more easily understood and compared through common reference to the Amphibia so that as a form transitional between the lower and higher Craniates the frog is a type of great importance.
There are also practical and historical reasons for emphasizing the development of the frog. The size of the eggs and their abundance at a season convenient for their study, the hardiness of the embryos under laboratory conditions and during experimental manipulation, and the ease with which the eggs may be fertilized in the laboratory, all make the frog's egg a particularly valuable laboratory subject. For such reasons this egg has served as the basis for many of the great embryological classics; in this respect the egg of the frog is second only to that of the fowl. And much of the important modern experimental embryology has had this same object as its basis, so that a thorough knowledge of the development of the frog is essential to the student of biology.
Outline Of The Life History Of The Frog
It will prove advantageous to recall, at this point, the most striking facts relating to the life history and development of external characters of the frog. The later development of this animal is marked by several abrupt changes in habit, accompanied by pronounced external modifications, but the earlier development is not so obviously divided into periods, marked by striking changes in habit or structure. The whole period of development may be subdivided as follows:
I. THE FORMATION AND PRODUCTION OF THE GERM CELLS. This period terminates with spawning.
II. THE EMBRYONIC PERIOD. This is conveniently divided into:
A. From Spawning through Gastrulation and Notogenesis. This includes fertilization, cleavage, the formation of the germ layers, the formation of the neural tube and notochord, and the establishment of an early embryo.
B. From the Early Embryo to the Time of Hatching.
III. THE LARVAL PERIOD. From hatching to metamorphosis. The actual developmental processes of this and the latter part of the embryonic period (II, B.) are most conveniently described together.
IV. THE ADOLESCENT PERIOD. From the beginning of metamorphosis to sexual maturity.
The durations of these periods can be given only in the roughest way since they vary with the species and particularly with the temperature and food supply; to a lesser extent the same is true regarding size. The ages and lengths given below are to be regarded then as only approximations under favorable conditions of development.
The period of formation of the germ cells occupies the long interval between the annual spawning seasons. For the most part the germ cells are formed during the summer so that in the following spring only the final steps remain to be accomplished. Fertilization is external and maturation of the egg is not completed until the entrance of the sperm cell. Spawning occurs during the first warm days of spring in most species, although some, like the bull-frog (R. catesbiana) may not spawn until summer. The cleavage of the egg terminates with the formation of a fairly typical blastula, followed by gastrulation, which is complicated during its later phases by the precocious processes of notogenesis and formation of the middle germ layer. During these early phases of development, which usually occupy about thirty-six hours, the spherical form of the egg is retained (Fig. 22, B), though a slight enlargement may result from the formation of internal cavities and the absorption of water. As notogenesis is completed the embryo begins to form, bent around the curved surface of the gastrula (Fig. 22, C, D). Soon, however, the embryo becomes slightly elongated, and shortly this elongation becomes quite marked chiefly as the result of the enlargement of the head region and the growth of the posterior part of the body (Fig. 22, E). Fig. 22, F, shows an embryo of about two days (2.5 mm.), at the stage which we may arbitrarily assume to represent the end of the first division of the embryonic period.
The neural tube is entirely closed, the blastopore roofed over, and in the head region are visible the rudiments of the mandibular and hyoid arches and the optic vesicle.
Fig. 22. Representative stages in the development of the frog. C-F, after Keibel(Kopsch). G, H, from Ziegler's models. A. Unfertilized ovum. B. Fully formed gastrula; posterior view. C. Early stage in the formation of the central nervous system; dorsal view. D. Side view of young embryo showing the rudiments of the visceral arches. E. Side view of an embryo with central nervous system established, and optic vesicles indicated. F. Side view of early embryo," showing formation of head and tail regions. G. Side view of embryo just before hatching. H. Fully formed tadpole, showing rudiments of hind-limbs, a, Animal pole; b, blastopore containing yolk plug; ba, branchial arches; eg, rudiments of external gills; hi, hind-limb bud; m, mouth; mh, mandibular and hyoid arches; nf, neural folds; ng, neural groove; np, neural plate; o, olfactory and stomodseal pit; op, rudiment of optic vesicle ; opr, right opening of opercular cavity, just before its closure; p, proctodseum; pn, pronephric eminence; r, branchial ridge (plate); s, oral sucker; t, rudiment of tail; tnf, transverse neural fold; v, vegetal pole.
The influence of the yolk mass upon the form of the embryo now diminishes rapidly, and during the next few days external change consists largely in the appearance of a definite body region, the elongation of the tail, and the enlargement of the head upon which appear olfactory pits, stomodaBum, and sucking disc, and just back of the head the rudiments of external gills and the pronephric elevations (Fig. 22, G).
At about six days (5 mm.) the embryo begins to show muscular twitchings, and about one or two weeks after fertilization the embryo wriggles its way out of the jelly and becomes a free living larva or tadpole (Fig. 22, G). This marks the end of the embryonic period. (In the higher temperatures of the laboratory the larva? may hatch within five days after fertilization.) For some days after hatching the larvse remain comparatively inactive, sometimes attaching themselves by their U-shaped suckers to the outside of the jelly mass, or to other objects in the water and hanging thus, singly or in groups. Or they may fall to the bottom and lie passively on one side. During the days just after hatching the larva? are still dependent for food upon the yolk contained within the wall of the intestine, but about two to five days after hatching the mouth opening is formed, and the tadpoles begin to take in food from outside. As the tadpoles begin to feed they become active, the sucker becoming functionless and diminishing; and soon they are in almost constant motion searching for food over the bottom, or in the surface film of the water. The mouth becomes fringed with lips, covered with horny rasping papilla? and furnished with a pair of horny beaks. Their food consists of almost any kind of plant or animal debris and this is consumed in immense quantities. In captivity tadpoles thrive perfectly on a diet of any cereal, with the occasional sacrifice of one of their own number. As the alimentary tract becomes functional the digestive glands increase in size rapidly, and the long intestine, coiled like a watch spring, can easily be seen through the ventral body wall. This enlargement of the digestive tract gives the body a well-rounded form, sharply marked off from the narrow tail, upon which develop large dorsal and ventral fin-like folds of skin; this is the only locomotor organ in the tadpole. A period of rapid growth follows upon this voracious feeding; the rate of growth depending upon temperature and food supply.
Immediately after hatching external gills develop rapidly on the sides just back of the head, and for a time these are the only respiratory organs, but about the time the mouth opens four pairs of gill slits successively perforate the pharyngeal wall, and their borders become folded forming the true internal gills: thereupon the external gills gradually diminish and after a few days disappear completely. At this time the branchial region becomes covered over externally by a protecting opercular fold of integument, the opercular cavity thus formed finally remaining open on the surface only by a single excurrent pore or " spiracle" on the left side.
During the next few weeks, while the tadpole continues to feed almost incessantly, there are few external changes except the general increase in size. About four or five weeks, ordinarily, after hatching (much sooner at room temperature) the limb buds appear, first the anterior pair within the opercular cavity and therefore not visible externally, and soon after the posterior pair either side of the cloaca (Fig. 22, H). By the end of the second month these have enlarged and become jointed.
For some time previous to this the tadpoles have been coming to the surface occasionally to expel small bubbles of air from the slowly developing lungs, and to gulp down a fresh supply, and as this aerial respiration increases the internal gills retrogress and the gill slits diminish.
If developmental conditions have been favorable and food abundant, about the end of the third month the period of metamorphosis commences during which, in the space of a few days, the tadpole loses many of its characteristic structures adapted to aquatic life and rapidly, almost suddenly, assumes the characteristics of the amphibious frog. "The tadpole and in the head region are visible the rudiments of the mandibular and hyoid arches and the optic vesicle.
The influence of the yolk mass upon the form of the embryo now diminishes rapidly, and during the next few days external change consists largely in the appearance of a definite body region, the elongation of the tail, and the enlargement of the head upon which appear olfactory pits, stomodaeum, and sucking disc, and just back of the head the rudiments of external gills and the pronephric elevations (Fig. 22, G).
At about six days (5 mm.) the embryo begins to show muscular twitchings, and about one or two weeks after fertilization the embryo wriggles its way out of the jelly and becomes a free living larva or tadpole (Fig. 22, G). This marks the end of the embryonic period. (In the higher temperatures of the laboratory the larvae may hatch within five days after fertilization.) For some days after hatching the larvae remain comparatively inactive, sometimes attaching themselves by their U-shaped suckers to the outside of the jelly mass, or to other objects in the water and hanging thus, singly or in groups. Or they may fall to the bottom and lie passively on one side. During the days just after hatching the Iarva3 are still dependent for food upon the yolk contained within the wall of the intestine, but about two to five days after hatching the mouth opening is formed, and the tadpoles begin to take in food from outside. As the tadpoles begin to feed they become active, the sucker becoming functionless and diminishing; and soon they are in almost constant motion searching for food over the bottom, or in the surface film of the water. The mouth becomes fringed with lips, covered with horny rasping papillae and furnished with a pair of horny beaks. Their food consists of almost any kind of plant or animal debris and this is consumed in immense quantities. In captivity tadpoles thrive perfectly on a diet of any cereal, with the occasional sacrifice of one of their own number. As the alimentary tract becomes functional the digestive glands increase in size rapidly, and the long intestine, coiled like a watch spring, can easily be seen through the ventral body wall. This enlargement of the digestive tract gives the body a well-rounded form, sharply marked off from the narrow tail, upon which develop large dorsal and ventral fin-like folds of skin; this is the only locomotor organ in the tadpole. A period of rapid growth follows upon this voracious feeding; the rate of growth depending upon temperature and food supply.
Immediately after hatching external gills develop rapidly on the sides just back of the head, and for a time these are the only respiratory organs, but about the time the mouth opens four pairs of gill slits successively perforate the pharyngeal wall, and their borders become folded forming the true internal gills: thereupon the external gills gradually diminish and after a few days disappear completely. At this time the branchial region becomes covered over externally by a protecting opercular fold of integument, the opercular cavity thus formed finally remaining open on the surface only by a single excurrent pore or "spiracle" on the left side.
During the next few weeks, while the tadpole continues to feed almost incessantly, there are few external changes except the general increase in size. About four or five weeks, ordinarily, after hatching (much sooner at room temperature) the limb buds appear, first the anterior pair within the opercular cavity and therefore not visible externally, and soon after the posterior pair either side of the cloaca (Fig. 22, H). By the end of the second month these have enlarged and become jointed.
For some time previous to this the tadpoles have been coming to the surface occasionally to expel small bubbles of air from the slowly developing lungs, and to gulp down a fresh supply, and as this aerial respiration increases the internal gills retrogress and the gill slits diminish.
If developmental conditions have been favorable and food abundant, about the end of the third month the period of metamorphosis commences during which, in the space of a few days, the tadpole loses many of its characteristic structures adapted to aquatic life and rapidly, almost suddenly, assumes the characteristics of the amphibious frog. "The tadpole variable sizes. The egg of the frog is therefore markedly telolecithal.
In the animal pole the nucleus is contained. At the time of egg laying this is in the metaphase of the second polar division (Fig. 26, /). The first polar body has already been extruded and though very small, may be found near the light spot in the flattened area at the upper pole.
Fig. 23. Egg and spermatozoa of frog. A. Spermatozoon of Rana fusca. After Broman. B. Spermatozoon of R. esculenta. After Broman. C. Section through the fully formed ovarian egg of Rana sp. From Morgan (Development of the Frog's Egg). The protoplasmic animal pole is covered with a thin layer of pigment; vegetal pole filled with yolk bodies; other deutoplasmic granules are distributed throughout the cell. The large nucleus, or germinal vesicle, surrounded by a definite nuclear membrane, lies eccentrically toward the animal pole, and contains the thread-like chromosomes and a group of nucleoli.
The specific gravity of the deutoplasm is slightly greater than that of the protoplasm, and this brings about the assumption of the definite position of the egg with the vegetal pole downward. But at this time the egg membranes are so closely adherent that its rotation to this position may be very slow.
At this time, i.e., preceding fertilization, the only symmetry of the egg is that expressed by its polarity. That is to say it has no single plane of symmetry, only an axis of symmetry (polar axis); this is the primary egg axis passing through the middles of the light and dark (vegetal and animal) poles.
The spermatozoa of the frog are quite typical in size and form (Fig. 23, A, B). They are about 0.1 mm. in length; the head is comparatively long, in some species tapered and curved at the apex with a laterally attached adhesive perforatorium or in others, quite blunt with a small rounded perforatorium. The anterior centrosome lies in the head, the posterior in the middle piece.
B. THE FORMATION OF THE GERM CELLS
1. The Reproductive Organs
Before describing the formation of the ova and spermatozoa it will be necessary to recall the essential arrangement of the gonads and their ducts in the mature organism. The development of the system will be described later.
The single parr of ovaries are proliferations of the cells forming the longitudinal genital ridges. These project from the body wall for some distance along either side of the dorsal attachment of the mesentery (Fig. 24). Each is surrounded by a peritoneal fold (mesovarium) , which also slings the organ from the dorsal body wall and transmits its nervous and vascular supplies. Each ovary is divided transversely into a series of compartments. In each of these is a small internal cavity the thick wall of which is formed by the germinal tissue proper. After spawning the ovaries are left as small rudiments, and during the following summer eggs are formed in large numbers and their growth is practically completed before the beginning of the period of hibernation. The ova are all formed from a few primitive ova which divide repeatedly, forming small groups or nests of cells, one of which enlarges becoming the ovum proper, while the others around it become the nutritive follicle cells (membrana granulosa). As the growth of the ova is completed, the ovaries are so enlarged that they occupy a large part of the body cavity and crowd upon the other viscera.
Loosely attached to the anterior ends of the ovaries are the fat bodies large masses of yellow streamers of lymphatic tissue, filled with drops of fatty substance chemically similar to the deutoplasm of the eggs. These are larger and more abundantly supplied with fat just prior to the breeding season. Their function seems, in part at least, to supply the material used in the final stages of the growth and maturing of the eggs, and also possibly, though doubtfully, for the nutrition of the animals themselves prior to and during the spawning season, for usually no food is taken after the end of their period of hibernation till after spawning is completed.
Fig. 24. Urinogenital system of the female frog. After Wiedersheim (Ecker). c, Cloaca; /c, kidney; o, ovary; od, oviduct; oo, opening of oviduct into cloaca; ou, opening of ureter into oviduct; ut, uterus.
Fig. 25. Urinogenital system of the male frog. After Wiedersheim (Ecker). ao, Dorsal aorta; c, cloaca; /, fat body; k, kidney; ou, opening of ureter into cloaca; t, testis; u, ureter; v, vena cava.
The remaining parts of the female reproductive system are the oviducts (Miillerian ducts). These are a pair of very long, much convoluted tubes, of small diameter but with rather thick walls, suspended from the dorsal body wall by folds of peritoneum attached just along the outer sides of the mesovaria (Fig. 24). Each oviduct opens directly out of the body cavity at its upper end by a ciliated ostium, and at its lower extremity it opens into the cloaca. The thickness of the oviducal wall is due chiefly to the presence of glands secreting the albuminous material which forms the outer egg membrane or jelly; the lumen of the duct is lined with ciliated epithelium. Both the length of the oviducts and the thickness of their walls are subject to seasonal variation, the glands being largest and the ducts most convoluted during the time of egg laying. The lower extremities of the oviducts are thin walled and easily dilatable forming the so-called "uteri," serving as storage spaces for eggs ready to be laid.
In the male there is a single pair of ovoid testes (Fig. 25), in a position corresponding to that of the ovaries, and similarly suspended 'from the dorsal body wall by a peritoneal fold (mesorchium). Each testis is drained by a variable number (ten to twelve usually) of vasa efferentia which, after penetrating the kidney, open into a longitudinal collecting duct the vas deferens (Wolffian duct) which serves also as a ureter. Just before the vasa deferentia open into the cloaca they are dilated into the seminal vesicles, where mature sperm are stored just previous to spawning. The testes are divided into lobes like the ovaries and each lobe is further subdivided into lobules, in the walls of which the sperm develop and mature. There are fat bodies in the male similar to those in the female (Fig. 25). The testes also show much the same seasonal variation in size as the ovaries, in some species in which the formation of spermatozoa is seasonal, while in others, which form the sperm continuously, there is less variation.
During the later ovarian history of the eggs the maturation processes are commenced and the deutoplasm or yolk material is accumulated (growth period). The nucleus of the early primary oocyte passes into synizesis on that side of the nucleus toward the attraction sphere (Fig. 26, A). After synizesis the chromosomes scatter through the nucleus as small feathery bodies (Fig. 26, C), which stain lightly and become vacuolated, finally losing their identity. Meanwhile small yolk particles of mitochondrial nature appear in the cytoplasm, in the region of the attraction sphere and apparently under its influence. During the growth period the mitochondrial particles and yolk bodies accumulate rapidly, especially around the attraction sphere giving it the appearance of a yolk nucleus, whence they extend to other parts of the cell except in the region immediately surrounding the nucleus. Finally the "yolk nucleus" breaks down and the deutoplasm around it scatters through the cytoplasm (Fig. 26, D, E).
Toward the close of this process the nucleus moves toward one side of the cell, marking the polarity of the ovum, and from the first the yolk accumulates in the side opposite the nucleus or vegetal region, while around the nucleus in the animal region the protoplasm contains much less deutoplasm; the superficial protoplasm of the animal pole also contains many pigment granules.
As the growth period of the primary oocyte is completed the nucleus moves up to the surface of the cell which becomes flattened or even depressed toward it; at the same time the pigment over the nucleus is partially displaced or withdrawn and the lighter fovea results. The nucleus is very large and clear; no chromatin network is visible and the only chromatic bodies in it are the nucleoli. The nucleoli are quite numerous and apparently of two kinds true nucleoli whose real nature is doubtful, and chromatin nucleoli. The nucleus becomes elongated parallel with the surface of the egg and pigment accumulates around it, while the nucleoli become vacuolated and much enlarged. Some of the nucleoli dissolve while others fuse into large masses and the chromatin nucleoli collect in a small group near the center of the nucleus.
While these events are occurring within the nucleus a pronounced cytoplasmic modification has appeared. Along the lower or inner side of the nucleus a small cytoplasmic area has become differentiated and from it radiations begin to pass downward into the cytoplasm. This area extends rapidly and the radiations pass around the nucleus and up toward the animal pole of the egg as well as centrally. Finally they extend even through the nuclear membrane and the nuclear meshwork takes on the same radiational arrangement which thus involves practically the entire animal pole. The nuclear membrane dissolves as the radiations become complete and the nucleoli are aggregated or dissolved, and just at this time the egg follicle is ruptured in some way, and the egg escapes freely into the body cavity surrounded only by its chorionic and vitelline membranes (Fig. 23, C).
Fig. 26. Oogenesis in the frog (R. temporaria). A-E, After Lams. F-I, After Lebrun. A. Primary oocyte in synizesis. B. Primary oocyte with vitelline substance 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. G. First polar spindle in primary position. From egg in body cavity. H. First polar spindle in metaphase. From egg in uterus. /. First polar body formed and second polar spindle forming. From egg in uterus, a, Attraction sphere; c, chromosomes; /, follicle cells; g, contents of germinal vesicle; n, chromatin nucleoli; fl, vitelline substance of mitochondria! (chromidial) origin; y, yolk plates; /, first polar spindle (polar body, in /); //, second polar spindle.
Certain areas of the peritoneum are covered with cilia which beat in the direction of the oviducal ostia. These too are abundantly ciliated, and as a result of the ciliary action in both regions the eggs are soon carried to and into -the upper ends of the oviducts. But by this time the first polar spindle is already formed.
As the egg leaves the ovary the small group of chromatin nucleoli becomes surrounded by a small spherical mass of fibrillar plasma; the nucleoli become more or less fused and vacuolated, and then give rise to the group of twelve small rod-like chromosomes which soon become rings or crosses (Fig. 26, F). The fibrillar plasma draws out into the elongated achromatic spindle, at first placed tangentially (Fig. 26, G), but soon rotating and coming to the surface of the cell in the radial position (Fig. 26, H). The spindle is quite blunt and no asters, centrospheres, or centrosomes have been seen. The chromosomes diverge as the egg is entering the oviduct, in the upper part of which the very small first polar body is cut off (Fig. 26,7).
The second polar spindle forms immediately, and by the time the egg reaches the lower end of the oviduct the second polar division has progressed as far as the mesophase or metaphase. In this condition the division is suspended, and proceeds considerably later and as a rule only after entrance of the spermatozoon.
Entrance of the eggs into the upper part of the oviduct stimulates the jelly-secreting glands of its walls, and as the eggs are carried along singly down the oviduct by the cilia of its own walls, each is smeared over the surface of the chorion with a thin coating of viscid albuminous material arranged in two or three layers. About two hours are occupied in the passage of an ovum down the duct. At the lower ends of the oviducts the eggs collect in the uteri, where they remain stored, usually for a day or two, pending the time of spawning.
The formation and maturation of the spermatozoa is completed within the testes. Each lobule of the testis is composed of a collection of tubules, in the walls of which the spermatogonia develop, surrounded by nutritive follicles the elements of which become in part the basal cells or Sertoli cells. So far as is known the formation of the spermatocytes and spermatids is fairly typical. The spermatid contains a large nucleus and two peripheral centrioles. During the metamorphosis of the spermatid into the spermatozoon, the inner centriole is taken into the nucleus while opposite the other the flagellum grows out. The sphere of idioplasm remains on one side of the anterior tip of the head, when this forms from the nucleus, and a part of the cytoplasm flows down around the base of the flagellum forming the middle piece; the remainder of the cytoplasm appears to be thrown off.
In some species of Rana the sperm form continuously, in others only seasonally, apparently just before hibernation begins. As the breeding season approaches they are produced more abundantly and collect in the dilated lower ends of the vasa deferentia or seminal vesicles, ready for extrusion.
In the more common species of Rana, spawning occurs during the first warm days of early spring; some forms spawn later in the spring, and in a few (e.g., R. catesbiana), breeding occurs during early summer. In the first mentioned, spawning follows immediately upon emergence from the period of hibernation, when the frogs collect in small ponds or streams, or about the margins of larger bodies of water. There is no true copulation, the male merely seizing the female firmly around the body dorsally, with the forelegs just behind those of the female. This embrace or amplexus usually begins some hours, even days, before the actual extrusion of the reproductive products begins, and quite likely this affords the normal, though not essential, stimulus to their discharge from the ovaries and testes respectively. This amplexus continues throughout the entire period of spawning of a single pair, which may occupy several days or even weeks; the duration depends upon the species and upon the temperature colder weather prolonging the period greatly.
Expulsion of the eggs usually occurs during the early morning hours and is an intermittent process. Apparently all the eggs contained in the uteri are spawned at one time, and then an interval of rest follows during which the uteri are again slowly filled. As each mass of eggs is forced out of the cloaca the male, at the same instant, expels quantities of seminal fluid containing enormous numbers of spermatozoa which mingle with the egg masses, insuring the fertilization of practically every egg. Fertilization is therefore strictly external.
In the common frogs there are no nursing habits so frequent among other Anura (e.g., Alytes, Nototrema, Rhinoderma, etc.) and the eggs are left to develop without further relation to the parent organisms, which, upon the conclusion of spawning, immediately leave the pools and scatter widely. The eggs surrounded by the jellies remain in large masses which sink to the bottow of the shallow water and there become loosely attached to sticks or debris.
The total number of eggs laid by a single individual during one season varies widely in different species, and seems to vary conversely with the size of the eggs. The European grassfrog (Rana temporaria) lays from one to two thousand large eggs (2-3 mm.), while the European water-frog (Rana escuknta) in which the eggs are small (1.5 mm.) lays from five to ten thousand during each season.
Embryonic Period - A. From Fertilization Through Gastrulation
Fertilization and the Development of the Symmetry of the Egg
During the first hour or two after the entrance of the sperm several changes of great importance occur within the egg. Although these are going on together and overlap to a certain extent we shall have to describe them separately.
Entrance of the Spermatozoon. Within a few moments after ensemination, a sperm cell bores its way through the thin jelly and the chorion, and enters the egg substance; in most cases the entire spermatozoon enters. Although there is no micropyle the sperm does not enter the egg at random, but normally only in the pigmented hemisphere, more frequently about forty degrees from the animal pole, and in any meridian (Brachet). The meridian passing through both poles of the egg and the point of entrance of the sperm is known as the fertilization meridian.
Of course many sperm tend to enter the egg but the entrance of the first seems to alter the chemical structure of the egg in such a way that additional sperm are deterred from entering; frequently many such sperm may be seen in the egg jelly. Polyspermy, although not rare, is never normal in any of the frogs, and should more than one sperm succeed in entering, the development of the egg becomes abnormal.
Once within the egg substance the sperm head and middle piece move rapidly inward, following approximately a radius of the egg. The path of the sperm is marked by a distinct trail of pigment, indicating unusual metabolic activity of the region, which remains visible for some time, occasionally even to the blastula stage. The first part of the sperm path iscalled the penetration path (Fig. 27, A, B). After the sperm has travelled along this path for a short distance it rotates, in the usual way, putting the middle piece with its centrosome in advance of the head, which begins to dissolve and to form a typical vesicular and enlarged nucleus. Then the sperm changes its course, often abruptly, and moves toward the region where the male and female nuclei will unite, unless, indeed, the penetration path may have led in that direction (Fig. 27,
Fig. 27. Sections through the egg of R. fusca, showing penetration and copulation paths, and the symmetry of the first cleavage plane. After O. Schultze.
A. Sagittal section through the egg before the appearance of the first cleavage;
B. Frontal section of the same stage as A, showing the symmetrical distribution of the egg materials. C. Frontal section through egg in two-cell stage, showing the symmetry of the egg; the penetration path is not shown, a, Anterior; cp, copulation path; I, left; p, posterior; pp, penetration path; r, right; s, remains of first cleavage spindle; sp, superficial pigment; 1, first cleavage furrow.
A). This second part of the sperm path is known as the copulation path and like the penetration path, it is marked by a trail of pigment left in the cytoplasm.
Swelling of the Egg Membranes. One result of the entrance of the sperm is the withdrawal of fluid from the egg substance. This fluid accumulates between the surface of the egg and the chorion forming the perivitelline space. This leaves the egg free to rotate within its membranes, and in a few moments after fertilization all the eggs are found with the pigmented pole uppermost. In unfertilized eggs these membranes are more adherent and while rotation occurs, it is very slow.
Probably some of the fluid in the perivitelline space is taken in from the outside, for the egg membranes, particularly the jelly, are extremely hydroscopic. The egg has been in the water only one minute when the thin jelly has visibly commenced its absorption of water. When the eggs are extruded
Fig. 28. Egg of frog a short time after laying and fertilization, showing the swollen egg membranes. From Zieglec (Lehrbuch, etc.), after O. Schultze. mb, The so-called vitelline membrane; p, pigmented penetration path of the spermatozoon; r, polar bodies; 1, 2, 3, inner, middle and outer albumenous membranes or layers of "jelly."
the thickness of the jelly is only about one-sixth the diameter of the egg; after three minutes contact with the water this is increased to one-half the diameter; and after ten to fifteen minutes its thickness equals the diameter of the egg; The swelling then becomes slower and unless fertilization has occurred it may almost cease. Usually however the absorption of water continues for several hours and the thickness of the jelly may equal twice the diameter of the egg proper.
As the jelly thickens it is seen to be arranged in definite strata a thin denser layer closely applied to the chorion, outside this a thick layer somewhat more fluid, and on the surface a thick layer, rather firmer than the middle layer. The two thick outer layers may, in some forms, be separate.d by a narrow dense layer which is distinctly fibrous in structure (Fig. 28).
The functions of the jelly are various. It serves to some extent to attach the egg masses, but chiefly to protect the eggs from pressure or mechanical injury, from being eaten by other organisms, from infection of various kinds. It seems likely, too, that the jelly assists in the elevation of the temperature of the egg, for as a transparent sphere it condenses the heat rays of sunlight which it allows to enter freely and at the same time checks their radiation from the egg. The black pigment of the upper pole seems to function toward the same end by absorbing readily the heat rays, so that altogether the temperature of the egg may be considerably higher than that of the surrounding water. While the eggs, and the spermatozoa also, are very resistant to cold, they are at the same time very sensitive to warmth, so that this slight elevation of temperature has the effect of hastening development an effect that may be quite important since the temperature of the water is often quite low at the time the eggs are laid, and the ponds in which the frogs spawn are quite likely to dry up during the summer, so that each day gained in development toward metamorphosis may mean much as regards survival.
Maturation. Another effect of the entrance of the sperm is the completion of the maturation process in the egg nucleus. As the sperm enters, this is in the mesophase or metaphase of the second polar division (Fig. 26, 7). This division is then rapidly completed and the second polar body cut off; this usually occurs about thirty minutes after entrance of the sperm. The second polar body is of the same size as the first, or smaller. The egg nucleus then reforms in the usual manner. The polar bodies are only loosely attached to the surface of the egg and frequently may be found floating in the perivitelline space.
By the time the egg nucleus has reformed the sperm nucleus Jias also become typical in form, and the two nuclei move toward the center of the egg, approach and meet in the usual manner. The path of the egg nucleus is not marked by any pigment, nor is it accompanied by any radiations such as were connected with it during its maturation. The sperm centrosome and centrosphere divide and form the poles of a small but typical cleavage figure which is not located near the center of the egg, but always toward the animal pole. The position of the first cleavage spindle is not entirely undirected, but before we can discuss this point we must consider some facts regarding the structure of the egg itself after fertilization.
The Symmetry of the Egg
Before fertilization the egg has a well-marked polarity and is radially symmetrical about its chief axis (Fig. 29, A). This form of radial symmetry (not spherical) has been termed 11 rotatory," i.e., radially symmetrical in any plane at right angles to the chief axis. The vegetal pole contains a large proportion of yolk, while the animal pole is relatively free from yolk and is covered externally by a thin but dense coating of brown or black pigment; moreover, the nuclear structures are hi the animal pole (Fig. 23, C). The specific gravity of the lower pole is the greater, on account of the heavy yolk contained in it, and therefore the pigmented animal pole is turned upward when the egg is free to rotate. This rotation, however, is not usually completed for some minutes after the spermatozoon has entered and the egg membranes are somewhat freed from its surface.
But this radial or rotatory symmetry is not retained after the entrance of the sperm, for this affords the stimulus which leads to a rearrangement of the substance of the egg, accompanied or followed by the rapid development of a bilateral symmetry in the egg, with which that of the embryo tends strongly to coincide. The factors determining the position of this new plane of bilateral symmetry are really three-fold, one primary and two secondary. The primary factor is the polar and rotatory symmetry of the unimpregnated egg; the plane of bilateral symmetry always passes through the chief egg axis. The secondary factors then determine through what meridian the plane will pass. One of the secondary factors is internal and one external; the former is the point of entrance of the spermatozoon together with the direction of its penetration path, the latter is the direction of the action of gravity with respect to the egg axis. These secondary factors alone cannot direct the position of this plane, but each acts only in connection with the primary factor which is, in reality, the essential structure or organization of the egg as expressed by its polarity and rotatory symmetry.
We should recall that the sperm enters the upper pole in any meridian (fertilization meridian) and that its penetration path is first approximately radial, while the latter part of its path, copulation path along which it passes after the sperm head has dissolved and become vesicular, may be at an angle with the penetration path.
Immediately upon the entrance of the spermatozoon the substance of the egg becomes more labile, and a sharper differentiation and more pronounced segregation of the various egg substances result. It is supposed that the influence of the sperm is first exerted in the cytoplasm in its own immediate neighborhood, and that the effects of its presence then spread gradually to the more remote parts of the egg; and further, that the influence of the sperm extends in a symmetrical wave like those from a vibrating body. The result of this would be that the rearrangement of the substances of the egg would be symmetrical with reference to the point of origin of the disturbance, namely, the sperm entrance point. Therefore the plane of symmetry of the egg would be that plane containing the three points: animal pole, vegetal pole, sperm entrance point (Fig. 27, B). This would be at the same time the plane containing the penetration path, and it would be marked superficially as the fertilization meridian.
Whether or not this is a true description of the effects of the sperm, the facts are that following impregnation there is a streaming of the protoplasm upward and of the deutoplasm downward so that the animal pole is largely freed from yolk, the vegetal pole composed more largely of it, and the polar differentiation thus more marked than heretofore. The pigment granules, whose specific gravity is really intermediate between that of the yolk and of the protoplasm, show little disturbance and redistribution except in one certain region. For some reason which is not clear, the pigment granules located in a definite area at the lower margin of the pigmented pole, on the side opposite that where the sperm has entered, and therefore in the region presumably the last to be affected by the sperm, are carried away from their original position leaving this region lighter in color. This area is crescentic in outline, the crescent extending one-half to two-thirds around the egg; it is known as the gray crescent (Fig. 29) .
Fig. 29. Frog's egg before and after fertilization, showing the formation of the gray crescent. A. Unfertilized egg, from side. B. Unfertilized egg, from vegetal pole. C. Fertilized egg before first cleavage, from side. D. Same from vegetal pole, c, Gray crescent; p, pigmented animal pole; w, unpigmented vegetal pole.
The rearrangement of substance which involves the formation of the gray crescent, is such that the center of the specifically lighter substance of the egg is not located precisely in the egg axis, toward the animal pole, but is displaced toward that side on which the gray crescent appears, i.e., on the side of the animal hemisphere farthest from the fertilization point. Normally this arrangement is sufficiently marked before the rotation of the egg is completed, so that when the egg comes to a position of rest the animal pole is not turned exactly upward. In most species of frogs the egg axis is inclined out of the vertical about thirty degrees; and of course at the same time the margin of the pigmented area is similarly tilted out of the horizontal and the gray crescent lies on that side which is the higher (Fig. 29) . In the egg at rest, therefore, we may describe a definite plane which is vertical and includes both the gravitational and the polar axes; from the mode of determination of the position of the gravitational axis this plane also includes the fertilization point and meridian, and the penetration path.
While the egg is in this position the streaming and rearrangement of its materials is completed, and since the specific gravity of the different materials is concerned in. the rearrangement, it takes place finally with reference to the direction of gravity in the egg at rest. The final steps in the determination of the structure of the unsegmented egg, therefore, take place with reference to this gravitational plane, which thus becomes the plane of bilateral symmetry of the egg structure. The symmetry of the egg is expressed externally at this time only by the gray crescent which is equally divided by the plane of symmetry, but this is merely an indication of the really important symmetry that of the arrangement of the materials within the egg, which is no longer rotatory about the egg axis, but bilateral with reference to the gravitational plane.
The final development of the internal structure or organization of the egg is completed (in Ranafusca) only shortly before the first cleavage, or about an hour and a half after the entrance of the sperm (Brachet). Before the end of the first hour, the structure of the egg is gradually becoming fixed and disturbances or artificial lesions are compensated, or regulated, so that the final structure is not affected and later development is not abnormal. But after this, by the time the egg and sperm nuclei have fused, the egg structure becomes fixed and the egg is incapable of perfect regulation under abnormal conditions imposed upon it, so that artificial lesions or other disturbances result in abnormalities of its structure which lead to abnormalities in cleavage or embryonic development.
To summarize, the bilateral symmetry of the egg is determined primarily by the polarity of the egg which has gradually developed during its formation and maturation. How extensive this essential bipolar structure is, we do not know, but it is expressed visibly, and probably only in part, by the polar arrangement of at least three different substances having different specific densities protoplasm, pigment, deutoplasm. Probably the arrangement of these is itself determined by some fundamental structure of the egg, but this we cannot observe directly. The bilateral symmetry is here only potential. It becomes actual only after ensemination when these substances are rearranged, first under the influence of the entering spermatozoon, which brings about the non-correspondence of the egg axis and gravitational axis, and then through the influence of gravity according to the plane fixed by these two crossing axes. In other words the bilateral symmetry is first determined by the egg structure as expressed through its polar differentiation and then through that as the result of the action of the entering spermatozoon and gravity, which latter is able to act finally only after the entrance of the sperm.
The penetration path of the sperm is usually in the direction of a radius of the egg from the entrance point, so that this portion of the sperm path tends to lie in the plane of symmetry to the same extent as, or even to a greater extent than the entrance point itself, and it may be that we should express the relation more truthfully by saying that the plane of symmetry tends to be directed first by the location of the penetration path rather than the fertilization point, since the influence of the sperm is exerted as it passes all the way along this portion of its course.
By placing the eggs under artificial conditions it has been found that the action of both these secondary factors is not essential, for normal development proceeds even when the directive effect of gravity is removed; it is not known, however, what the relation of the plane of symmetry to the fertilization point is under such conditions. And further, by placing the eggs in positions of constraint such that the sperm entrance point cannot lie in the median gravitational plane, it is found that the plane of symmetry is then that of the median gravitational plane. The secondary factors may thus be independent in their action, but in nature they usually tend to produce the same effect. However, in nature, and this also indicates their fundamental independence, there is some deviation between the plane of symmetry and either the fertilization meridian or the gravitational plane. The eggs in the interior of the mass are subject to some constraint due to pressure, and unknown factors may cause some variability in the effects of the factors named in the determination of symmetry. It is possible that the direction of the incident light (heat) rays plays some small part in the determination of the position of this plane. So that while the normal relation is that of coincidence of all these, all other relations are possible (the plane symmetry of course is always polar) and do occur with some frequency, and all that can be said is, that on the whole the median plane of the egg tends to coincide with the gravitational plane and with the fertilization meridian.
One of the reasons why the position of the plane of symmetry of the egg is of the greatest importance is that the plane of bilateral symmetry of the embryo and adult is directly related to it. Any factor which aids in the determination of the egg symmetry is at the same time influencing the symmetry of the developing embryo. By observing,, in a large number of specimens, the relation between the symmetry of the egg and of the embryo it is found that the tendency for the two to correspond is very marked. And yet variations of any extent may and do occur, showing that other factors may influence and development of the embryo (Jenkinson). The symmetry of the embryo and adult can be traced directly back into the gastrula or blastula, and it seems, therefore, that whatever causes the non-correspondence between these symmetries must operate during cleavage and not later, although there seems to be no definite causal relation between the direction of the first cleavage plane itself and the symmetry of the embryo, although there may be certain constancies in this relation.
We may finally mention briefly the relation between the symmetry of the egg (and therefore in general of the embryo) and the symmetry of cleavage, particularly the plane of the first cleavage furrow. The position of the cleavage plane is of course the direct result of the position of the cleavage spindle; it is therefore the position of this latter which is essential. The spindle always lies at right angles to the egg axis, in agreement with the law of Hertwig. In such a plane the position of the spindle is readily influenced by at least one external factor, namely pressure, in such a way that it tends to lie at right angles to the direction of the pressure, and the resulting cleavage would therefore occur in the direction of the pressure. In a large mass of eggs this factor is probably one of considerable importance, especially in affecting the direction of the first cleavage in those eggs in the interior of the mass. The relation between the direction of pressure and the symmetry of the egg is purely accidental and consequently we find much variation in the relation of these two planes.
When the egg is not subject to pressure there is a fairly marked tendency for the spindle to lie either transversely to the plane of symmetry or in that plane (Fig. 27, C). The symmetrical structure of the egg is fairly well established by the time the spindle forms, and there are only these two positions which the spindle can occupy and yet retain symmetrical relations to the internal structure of the egg. The former relation, in which the plane of the resulting cleavage would coincide with the plane of egg symmetry, is the more frequent and in approximately 25 per cent, of eggs the first furrow deviates less than five degrees, plus or minus, from this plane. The second relation, placing the first furrow at right angles to the median plane (within five degrees, plus or minus) is found in something like 10 per cent, of eggs (Jenkinson).
But the position of the spindle seems to be influenced quite considerably by the direction of the copulation path of the sperm nucleus, i.e., the direction of the plane passing through the contact surface of the copulating egg and sperm nuclei, and since this is subject to much variation with respect to the median plane, and also since the postion of the spindle is easily modified by external factors, we find that the relation between the first cleavage and the median plane of the egg is not at all regular, and deviations from the two relations mentioned are very frequent and all relations occur. There is no direct relation between the plane of the first furrow and the fertilization meridian; whatever relation there is results from their common relation to the plane of egg symmetry.
We may summarize briefly the relations of the plane of symmetry of the egg, the plane of symmetry of the embryo, and the plane of the first cleavage furrow, in normally developing eggs.
The position of the plane of bilateral symmetry of the egg is determined primarily by the polarity and rotatory symmetry of the unfertilized egg, in conjunction with the point of entrance of the sperm or the direction of the penetration path, and the direction of the action of gravity, in such a way that the median plane tends to lie in the gravitation plane, which also tends to coincide with the fertilization meridian. This determination, however, is not complete and variations may and do occur.
The median plane of the bilaterally symmetrical embryo tends to a marked degree to coincide with the plane of symmetry of the fertilized egg, but all other relations in the same axis are possible and actually occur.
The plane of the first cleavage furrow tends to lie either in or at right angles to the plane of symmetry of the egg, primarily on account of the tendency of the first cleavage spindle to assume some symmetrical position with reference to the egg structure.
There is, therefore, a tendency for the gravitational plane, the point of entrance of the spermatozoon, the penetration path of the spermatozoon, the median plane of the egg, the median plane of the embryo, and the plane of the first cleavage furrow, all to coincide, but all relations in the same axial plane are possible among these and are actually found.
Cleavage of the frog's egg is total and unequal. The first cleavage spindle lies in the direction of the greatest protoplasmic extent, i.e., transversely to the egg axis, and in a position determined by several different factors as described above (Fig. 27, C) . The first cleavage furrow becomes visible on the surface first at the animal pole, and gradually extends thence as a narrow groove around a meridian of the egg to the vegetal pole; it is completed about two and one-half hours after ensemination, or much sooner if the temperature is raised slightly. While this furrow is meridional, we have seen that it may or may not divide the gray crescent symmetrically. Throughout cleavage the blastomeres remain in close contact so that they are separated superficially by only shallow narrow grooves (Fig. 30), and do not become distinctly rounded and separate elements as in Amphioxus or in other eggs containing less yolk.
The second cleavage appears about one hour after the first; this is also meridional, at right angles to the first, dividing the egg into four adequal blastomeres. Succeeding divisions appear about an hour apart. The third cleavage is the first to divide the egg unequally; in the typical form of cleavage this appears similarly in all four quadrants, and is latitudinal or horizontal, i.e., at right angles to the first two (Fig. 30, A). Although this cleavage plane divides the protoplasmic material of the egg about equally, the accumulation of yolk in the lower pole actually displaces this middle plane above the equator of the egg, so that the cleavage furrow appears about sixty degrees from the animal pole, and the egg as a whole is divided unequally. Of the eight resulting cells, the four upper are small and richer in protoplasm, while the four lower are large and richer in yolk. This typical relation of the third cleavage is by no means invariable. A considerable proportion, in some lots nearly one-half, of the eggs show some departure from this arrangement, and the third cleavage may be horizontal in only one, two, or three blastomeres, and vertical in the remainder or, rarely, vertical in all four. These and also the later vertical planes frequently do not pass actually to the upper pole of the egg, and are therefore not strictly meridional,
Fig. 30. Cleavage of the frog's egg. After Morgan. Animal pole upward in all figures. (For earlier stages see Fig. 27.) A. Eight cells. B. Twelve cells becoming sixteen. C. Thirty-two cells. D. Forty-eight cells, more regular than usual. E, F. Posterior and anterior views of about 128 cell stage. G. Late cleavage or early blastula. H. Commencement of gastrulation (cell outlines indicated only in the region below the invaginating groove), i, Beginning of invagination.
although there is a decided tendency for them to lie in meridians. The location of the succeeding cleavages varies with that of the third. Typically the fourth cleavages (Fig. 30, B) are meridional forming eight small upper, and eight large lower cells, and the fifth again latitudinal, forming thirty-two cells arranged in four horizontal rows of eight cells each (Fig. 30, C). But the atypical appearance of some of the third cleavages may very early disturb this schema. With the appearance of the fifth cleavage the early, and brief, synchronism of the cleavages has become lost, the small upper cells dividing more rapidly than the large lower cells. Comparatively few eggs remain regular up to this stage, for eggs which were regular at eight cells usually become irregular at sixteen or thirty-two cells, and after that all regularity is lost (Fig. 30).
Turning back now to notice some of the internal arrangements of the cells during cleavage, we find that when four cells are cut into eight these all round off somewhat internally, as in Amphioxus, forming a small space among them, which is the beginning of the segmentation cavity or Uastoccel, and which from the position of the third cleavage has from the first an eccentric position toward the animal pole. During subsequent cleavages the blastoccel enlarges but always retains this eccentric location (Fig. 31, A).
After about thirty-two cells are formed not all of the subsequent cleavages are visible on the surface, for these early divisions, all passing through the axis of the egg, have formed cells elongated in a radial direction, and in some of these the cleavage spindles tend to take up a similar position* and the resulting division occurs parallel with the surface of the mass, forming a central cell bordering the segmentation cavity and a superficial cell visible externally (Fig. 31, B). There is no period at which such a delaminating cleavage occurs throughout the cell group, but scattered cells show this arrangement, first among the cells of the upper hemisphere and then later in the lower cells, which are divided quite unequally in this way. Many of these interior cells are formed by cleavages that are not exactly tangential but considerably oblique to the surface. By the time there are sixty-four or one hundred and twenty-eight cells, approximately one-fourth of the cells are interior, and line the blastocoel, so that at this stage the wall of the blastocoel is two or more cells thick.
We may assume that the arrangement of the cells forming the wall of the blastocoel as a more or less definite epithelium, marks the end of the cleavage period and the formation of the blastula. This arrangement is really definitely established by the time thirty-two to sixty-four cells are formed, i.e., before many interior cells are present. The immediately sub
Fio. 31. Sections through the blastula of the frog. A, B, from Morgan (Development of the Frog's Egg). C. After O. Schultze. A. Early blastula showing wall of segmentation cavity only one cell in thickness. B. Later stage showing multiplication of cells in wall of segmentation cavity. C. Late blastula showing the thinning of the roof of the segmentation cavity and the beginning of the germ ring, a, Animal pole; gr, germ ring; p, pigment; s, segmentation cavity or blastocoel; SG, Same as s; v., vegetal pole.
sequent cleavages do not modify their essential relations; the interior cells multiply rapidly and some cells migrate inward from the surface. The early blastula is spherical and about the diameter of the egg (Fig. 31, J3, C). The thinner roof and lateral walls of the eccentric segmentation cavity, are formed of cells derived from the animal region of the egg; these vary considerably in size and form, are irregularly and loosely arranged, and are roughly disposed in two sheets, one lining the blastocoel and one covering the surface. The thicker floor of the blastocoel is formed of the larger and less numerous vegetative cells. As the number of cells continues to increase several processes go on together. The small cells of the animal region divide the more rapidly and as they multiply they move gradually from the pole toward the equator, causing a thinning of the roof and a thickening of the walls of the blastocoel. This thickening toward the equator of the blastula is augmented by the rapid multiplication of cells there so that sections soon show a thicker ring, not very definitely delimited, of actively dividing cells forming what we may call the germ ring or growth zone, such as that described in the blastula and gastrula of Amphioxus (Fig. 31, C). This thinning of the animal pole increases the eccentricity of the blastoccel, which has meanwhile increased considerably in size. The later blastula increases somewhat in diameter, and accompanying this is the absorption or infiltration of water into the blastoccel, a part of the fluid content of which is, however, the secretion of its walls.
The germ ring is obviously formed of material from the animal pole of the egg, and apparently the substance contained in it can be distinguished at least as early as the eight cell stage, where it forms the upper quartet (micromeres) and the upper parts only of the lower quartet (macromeres) (Fig. 34). As the germ ring approaches the equator, one side (that of the gray crescent) commences to extend downward faster than the remainder; subsequent development proves this to be the posterior side. Soon the entire germ ring passes the equator, and by the time the blastula period is ended, it reaches, on the posterior side, a point about half way between the equator and the lower pole. These later phases in the downward movement of the animal cells can be observed externally, for these cells are easily distinguishable from the true lower pole cells on account of their dense pigmentation. Since the blastula retains its spherical form it is evident that the downward extension of the germ ring must displace the yolk cells in some direction, and as a matter of fact this displacement is evidenced by the elevation of the floor of the blastocoel. As the animal cells push downward the internal yolk cells rise till the floor of the blastocoel becomes first flat, and then convexly arched; at the same time the cavity widens somewhat so that in section it appears broadly crescentic (Fig. 32, A, B).
The first evidences of gastrulation now appear so that this stage must be taken to mark the completion of the blastula. We may state the characteristics of the fully developed blastula as follows. The completed blastula is spherical, in volume about one-fifth larger than the ovum, and bilaterally symmetrical; this bilaterality is accompanied by antero-posterior differentiation, and is indicated by the greater thickness of the anterior wall of the segmentation cavity, and by the more ventral extension of the pigmented cells on the posterior side, i.e., the side marked by the gray crescent in the egg. The small cells of the upper pole form the thin roof and thicker sides of the eccentric blastocoel; they are in two quite distinct sheets an outer layer of compactly arranged cells forming a distinct epithelium known as the superficial or epidermal layer, and, lining the blastocoel, a deeper or "nervous" layer of irregularly and loosely arranged cells, gradually increasing in thickness from the pole toward the base of the blastoccelic wall, about at the level of the equator of the blastula. Just at and below the equator actively dividing cells have accumulated from the whole upper pole region. Since the fate of these cells is to form the chief axial parts of the embryo this region is called the germ ring, although it lacks the distinctness of the germ ring as it is finally found in some other forms (e.g., Teleosts). This region seems comparable with a crescentic group of actively dividing cells having a corresponding position and function in the blastula of Amphioxus (Fig. 6). The floor of the blastocoel is formed of the larger vegetative cells which form practically the lower half of the blastula; these are compacted and show no definite arrangement, except that toward the lower pole they gradually increase in size. Although some yolk is contained in all the cells, the larger lower pole cells are particularly rich in deutoplasm and are known as the yolk cells. Immediately below the germ ring the cells are intermediate in character and form what is known as the transitional zone. The position of the polar axis in the blastula, with respect to gravity, remains the same as in the egg or the cleavage period.
The pigment, while chiefly superficial and in the cells derived from the animal pole, externally extends further toward the lower pole than in the egg; it is also found to a limited extent among the animal cells below the surface, and even in the smaller vegetal cells lining the blastoccel, which resemble closely the proper cells of the animal pole. This internal pigment is not derived from that more superficially located in the earlier stages, but it is deposited in situ as a by- or endproduct of metabolism. Pigment granules are laid down wherever developmental processes, including cell division, are in rapid progress. And since the smaller cells represent regions where cell multiplication has been more active, such cells contain relatively more pigment. This relation between metabolic activity and the accumulation of pigment may explain the pigmentation of the animal pole of the egg itself, although it is customary to refer this to the adaptational relation mentioned previously, a relation which need not be negatived by this method of its formation. The dense pigmentation of the path of the sperm is also referable to an unusual degree of metabolic activity.
The blastula of the frog differs from that of Amphioxus chiefly in that the blastocoel of the former is so decidedly eccentric, relatively smaller, and its wall several cells thick, the cells differing greatly in size, and already differentiated into superficial and deeper layers, at least in the animal region. The germ ring (growth zone in Amphioxus) extends completely around the blastula of the frog. These differences for the most part seem to be the direct results of the abundance of yolk in the frog egg, and its accumulation chiefly in one region. The effect of the yolk in modifying the course of development becomes more marked in the immediately succeeding phases of development, namely gastrulation and notogenesis; these processes are not at all as simple and diagrammatic as in Amphioxus.
Fig. 32. Median sagittal sections through a series of gastrulas of the frog (R. temporaria). After Brachet. The figures illustrate the change in position
Gastrulation and Notogenesis
The processes of gastrulation proper and of notogenesis overlap to a more considerable extent in the frog, than in Amphioxus, and are conveniently described together. Gastrulation results directly from a continuation of the downward extension of the germ ring, together with the consequent elevation of the yolk cells, which were such important features in the development of the late blastula.
The first external indication of gastrulation is the appearance of a slight irregular groove, approximately horizontal, lying across the sagittal plane on the posterior side of the egg, just at the lower margin of the germ ring, i.e., just below the equator (Figs. 32, 35, A). Thus located, the groove lies just between the animal cells and the yolk cells, and therefore comes to be lined by both kinds of cells on its opposite faces. From subsequent development we know that the formation of this groove is the beginning of invagination, the groove itself the beginning of the archenteron, its upper margin the rim of the blastopore, and the cells lining it above and below, ectoderm and endoderm respectively. Hemisection of this very early gastrula (Fig. 32, A), shows that the elevation of the floor of the blastoccel is very rapid at this time, and one of the first results of the arching up of the yolk cells (endoderm) is the formation of a narrow groove all around the margin of the blastocoel, between the base of its wall and its rising floor. As the yolk continues to rise, this groove soon becomes quite marked and compressed into a narrow slit which, though originating in the manner just described, seems to be continued ventrally all around the central cells as a definite splitting or delamination (Fig. 33, A). In effect this narrow space separates definitely the ectoderm and endoderm in the region within (above) the restricted invaginating region, which of course also gives rise to an ectodermal and an endodermal layer. This original groove is called the gastrular groove, and the delamination which extends it is the gastrular cleavage; the formation of these is not limited to the region of the dorsal lip of the blastopore, but extends entirely around the gastrula, even to the side opposite that of invagination. It remains a question whether the invagination process is the result of an active inturning of the cells forming the lower margin of the germ ring, or whether these cells are rather pulled inward by the elevation of the yolk cells, which results from the compression produced by the thickening and downgrowing germ ring; perhaps both factors are involved. However this may be, the invagination once begun continues rapidly, so that an elongating tongue of inturned cells continually pushes up under the superficial ectoderm which lies just above the invaginating region. This tongue is the invaginated endoderm derived from some of the cells of the animal pole and their descendants; at its inner limit it becomes directly continuous with the endoderm formed directly from the yolk cells, or cells of the transitional zone which have become entitled to the name endoderm, while still practically in situ, by the appearance of the gastrular groove and cleavage.
of the whole gastrula, as well as the phenomena of gastrulation proper. A. Commencement of gastrulation; earliest appearance of the dorsal lip of the blastopore. Internally the gastrular cleavage is indicated. B. Invagination more pronounced; beginning of epiboly. C. Invagination, epiboly and involution in progress. The gastrular cleavage is now indicated on the side opposite the blastopore. Rotation of the gastrula. D. Just before the ventral lip of the 'blastopore reaches the median line. The indentation of the wall of the segmentation cavity is an artifact. E. Blastopore circular and filled with yolk plug. Gastrula beginning to rotate back to its original position. Peristomial mesoderm differentiating. F. Segmentation cavity nearly obliterated. Neural plate established. G. Gastrulation completed, a, Archenteron; 6, blastopore; c, rudiment of notochord; ec, ectoderm; en, endoderm; gc, gastrular cleavage; ge, gut endoderm; m, peristomial mesoderm; np, neural plate; nt, transverse neural ridge; s, segmentation cavity or blastoccel.
Turning for a moment to the consideration of the external modifications during gastrulation we see that the germ ring, once having passed the equator begins to* rlarrow : as a 'whole (i.e., diametrically), and this is chiefly accomplished by the drawing in of its lateral regions toward the mid-line posteriorly. This, together with the very active multiplication of its constituent cells, causes this portion to push down more rapidly than the remainder, carrying along the layer of invaginating endoderm, and increasing considerably the vertical extent of the archenteron. Surface views show that at the same time the archenteric groove extends laterally, becoming first crescentic, then semicircular and finally circular (Fig. 35). That is to say, the first invagination of the pigmented cells forms the dorsal lip of the blast opore; then the invagination gradually extends laterally in each direction forming the lateral lips of the blast opore; and finally the process of invagination is carried around to the side of the gastrula, almost diametrically opposite to that where it began, and forms there the ventral lip of the blastopore, and the circular blastoporal margin is thus completed. During the completion of the blastoporal rim the germ ring has continued to extend downward over the yolk on all sides, so that by the time the rim is completed by the formation of the ventral lip, this is found at a level quite below that at which invagination began on the dorsal side (Fig. 35). The invagination involves the mturning of the cells transitional between the animal and vegetal poles, so that the cells of the pigmented and white areas are brought into sharp contrast. The circle of white yolk cells left within the blastoporal rim is called the yolk plug (Figs. 22, B; 32, E, F; 33, C). As the rim draws together, i.e., as the blastopore closes, the yolk plug appears gradually to diminish in size, while it really draws within, or some would say that it is pushed within, by the overgrowing lips of the blastopore, until finally it is no longer visible upon the surface (Fig. 38, B). The blastoporal opening then remains as a narrow elongated slit leading directly into the archenteron.
The precise way in which the germ ring narrows, or as we might say, in which the blastopore closes, is a matter of some importance. It has already been stated that the dorsal lip grows downward more rapidly than the remainder, due in part to the inflowing of the lateral portions toward the mid-line. This process is termed concrescence, or confluence, as in Amphioxus. In this way the materials found in the lateral, as well as posterior, regions of the germ ring are drawn to the median region, and as the ring there extends backward or downward, a thick median strand of tissue is left, from which are developed later, when the embryo begins to form, the rudiments of many of the important axial organs. The downward progress of the lateral and ventral margins of the blastopore is quite slow comparatively, so that the closure seems to occur mainly toward the lower pole of the gastrula. That is, as the diameter of the circular blastopore diminishes, its center, which is the center of the yolk plug, moves toward the lower pole and may finally reach this or even pass beyond it a short distance up on the opposite side. The form of the blastopore changes markedly during the later phases of its closure. The final rapid approach of the lateral margins alters its outline from a circle, so that it becomes ovoid and finally quite elongated and slitlike, in the direction of the sagittal plane of the gastrula (Figs. 35, 38, A). But before this occurs the blastopore is carried back near its point of origin by the rotation of the whole gastrula to be described shortly.
Fig. 33. Frontal and transverse sections through gastrulas of the frog (R. temporarid) of various ages. After Brachet. A. Frontal section through gastrula of same age as Fig. 32, C. B. Frontal section through gastrula of same age as Fig. 32, D. C. Frontal section through gastrula slightly older than Fig. 32, F. D. Frontal section through gastrula of same age as Fig. 32, G. E. Transverse section through gastrula slightly older than Fig. 32, D. F. Transverse section through gastrula slightly older than Fig. 32, G. a, Archenteron; b, blastopore; c, notochord; ge, gut endoderm; w, peristomial mesoderm; np, neural plate; s, segmentation cavity or blastoccel.
Sections through the gastrula during this period of closure of the blastopore show that many important processes are going on internally. Continuing from the stage described, where the archenteron had become a curved crevasse putting the invaginated and delaminated endoderm of its outer wall into connection, we find that the essential process of gastrulation is continued chiefly by the rising of the yolk cells from the floor of the blastocoel in advance of the archenteron, so that the inner limit of this cavity is carried upward under the animal pole and then beyond, toward the side of the gastrula opposite that of the first appearance of the archenteron (Fig. 32, C-F). The yolk cells at the same time are carried around the front of the advancing archenteron and form the outer wall of the primary gut cavity. This rearrangement of the yolk cells, involving their elevation in the dorsal or postero-dorsal region, draws away the yolk cells from beneath the original segmentation cavity so that as the archenteron advances the segmentation cavity recedes. Meanwhile the archenteron enlarges over the whole animal region and actually encroaches upon the blastocoel, so that as the latter moves toward the anterior side of the gastrula it diminishes in size and soon disappears (Figs. 32, 33). In some cases the wall of yolk cells separating the archenteron and blastoccel becomes thin and breaks through, before the blastoccel has completely disappeared; in this case the remnant of the blastocoel is added to the archenteron and the endodermal wall is completed by growth of the portion already formed.
Fig. 34. Diagrams of median sagittal sections through the frog's cleavage and gastrula stages, showing the changes in position during gastrulation. From Ziegler (Lehrbuch, etc.), after Kopsch. The arrow marks the vertical. (According to Morgan and others the figures of the first two stages should be rotated about 15 in the counter-clockwise direction.)
The formation of the archenteric cavity in a region formerly occupied by yolk cells, and the gradual enlargement and shifting of this cavity as well as of the blastocoel, obviously alter the position of the center of gravity of the gastrula as a whole, and the extensive changes in the relative positions of yolk and protoplasmic cells, whose specific gravities are unlike, contribute to the same alteration. This all results in a rotation of the gastrula about a horizontal transverse axis (Fig. 34). During the early phases of gastrulation, as just described, the more rapid growth of the dorsal (posterior) lip of the blastopore carries this to or even past the lower pole of the gastrula, even past the lower gravitational pole. Then as the blastopore continues to narrow, the whole gastrula rotates slowly in the opposite direction, carrying the blastopore back to the region where the dorsal lip first appeared and then on dorsally somewhat above the equator, into a postero-dorsal position, where it remains stationary for a time. Thus the shifting of the blastopore is the combined result of changes due to growth and to rotation. On account of the localization of the growth processes in the gastrula, which will be described presently, there is not a complete correspondence between the chief axis of the blastula or early gastrula and any single straight axis of the later gastrula.
Fig. 35. Diagrams of the frog's gastrula showing the position of the blastopore at various ages. A. Posterior view. B. Lateral view. 1-5 indicate the successive positions and forms of the blastopore. The change in position is due both to the actual growth movements of the blastopore, and to the rotation of the entire gastrula. Compare Figs. 32, 34.
The description of gastrulation from observation of median sagittal sections or hemisections does not give a complete idea of this process. We may return now to a consideration of some of the processes going on in the lateral parts of the gastrula. As the dorsal lip of the blast opore becomes crescentic, the deepening archenteron pushes laterally around through the mass of yolk cells (Fig. 33, B, C, D). But these have already become separated from the ectoderm by the gastrular groove and cleavage, except only in the region of the lower pole just anterior to the place where the anterior (ventral) lip of the blastopore will form. So that by the time the blastoporal lip becomes circular, i.e., by the time the ventral lip forms, this region has already been divided into ectoderm and endoderm, and therefore the extent of invagination at the ventral lip is greatly limited. The important result of this is that the actually invaginated endoderm is confined to a broad tongue of cells on the dorsal side (roof of the archenteron) and a ring-like strip extending around within the blastopore lip from the base of this tongue, narrowing rapidly toward the ventral side. The archenteron itself is at first a narrow slit, crescentic in cross section, but as it grows up to the animal pole and enlarges, it grows farther laterally so as to extend in a wide crescent (in transverse section) about to the level of the equator of the gastrula (Fig. 33, E, F). This leaves the yolk cells as a convex mass projecting into the archenteron from its floor.
The frog illustrates very well the way in which the process of gastrulation proper is complicated, in the Chordata, by the early formation of certain of the important axial organs which are the chief characteristics of the Chordate group; the formation of the rudiments of these structures is termed notogenesis. Gastrulation proper includes only those processes by which the singlelayered (monodermic) blastula is converted into the two-layered (didermic) organism, with definitely established ectoderm and endoderm the gastrula. The method by which this is accomplished may vary in different groups of Chordates; in Amphioxus, we have seen, invagination is the chief process, while in the frog this is less important, and the endoderm is more largely the result of delamination or of a simple rearrangement of cells forming different parts of the wall of the blastula. Among the higher Chordates invagination may be entirely lacking and gastrulation may be entirely accomplished by other methods (involution, epiboly, delamination). As a matter of fact, even in the frog, invagination is concerned less with the formation of the inner layer than with the establishment of the notochord and the formation of the rudiment which gives rise hi part to the mesoderm. It becomes necessary, therefore, to distinguish sharply between gastrulation and notogenesis. In the frog the strictly two-layered condition exists for a very brief period only, for the earliest phases of notogenesis, namely the formation of the mesoderm and chorda, occur quite precociously. The early formation of these structures may more conveniently be described together, and for the first stages we must return to the early gastrula.
In order to understand the origin of the mesoderm we must examine the early gastrula at the time the gastrular groove and cleavage extend down toward the incipient blastopore. Here the inner region of the germ ring and the yolk cells lining the blastocoel are continuous, and it is here that we find those cells which are later to form the mesoderm, and although distinguishable at this time, they are not definitely delimited within this zone which is transitional between the ectoderm and endoderm. On one side these cells are continuous with ectoderm cells, on the other with endoderm or yolk cells (Figs. 32, A; 33, A). As the lips of the blastopore extend laterally this mesoderm rudiment forms pari passu, and when the blastopore rim becomes circular the mesoderm rudiment can be distinguished in the ventral lip (Figs. 32, F; 33, C) . We may say then that the rudiment of the mesoderm appears first as a ring of cells just within the margin of the blastopore. But by the time the ring is completed ventrally, its dorsal region, in the more rapidly developing dorsal lip, has broadened considerably so that its general form might be compared with a signet ring.
As the blastopore is completed and begins to close, the confluence of its lateral margins transports masses of the cells toward the mid-line, and leaves them as a broad median band extending upward from the dorsal lip of the blastopore. As a result of the multiplication of these cells, and of the downward extension of this lip, a considerable axial thickening is formed. And at the same time the extension of the archenteron carries the yolk cells, with which the mesoderm is closely related on one side, upward toward the animal pole, so that altogether, even in these comparatively early stages, the extent of the mesoderm is nearly as great as that of the endoderm. And soon the mesoderm is definitely delimited from the endoderm by a rearrangement of cells giving the appearance of an irregular delamination (Fig. 33, D). This delamination commences in the dorsolateral regions either side of the thickened axial mass and gradually extends thence anteriorly and laterally around the sides of the archenteron separating a thin layer of definitive endoderm walling the gut cavity, and a much thicker mass of mesoderm between this and the superficial ectoderm (Fig. 44). In the region of the lower pole of the gastrula, under the thickest mass of yolk, the delamination comes to the surface of the yolk mass forming a free circular margin of mesoderm. From this free margin cells and groups of cells break or bud off passing farther ventrally, and finally reaching the lower pole, completing thus a fairly continuous layer between ectoderm and endoderm (yolk) (Figs. 44, 61, 63). In the region of the dorsal axial mass, particularly in the region of the blastopore, the delamination is delayed and its course somewhat modified. In these regions the cells concerned in mesoderm formation have very different relations from the remainder, since throughout they are the derivatives of cells which have been invaginated from the outer layer. And here too the development of the notochord complicates the matter somewhat.
Sections cut transversely through the blastopore and the region just in front of it, show that the rudiments of the chorda, mesoderm, and dorsal endoderm are for a time not distinguishable (Fig. 33, E). Sections through the narrowed blastopore, while it is still filled with the yolk plug, show that the rim of the blastopore is composed of a thick undifferentiated mass of cells, representing a part of the contracted germ ring. Farther laterally the ectoderm is separated by a line or narrow space which is formed in gastrulation ; the thin endoderm is separated from the middle layer by a line or space which results from delamination as described above. In front of the blastopore, in the region formed by confluence, the arrangement of the cells and germ layers is much the same, except that they are not interrupted in the mid-line. The pigmentation of the inner surface of the mass is an indication of the derivation of these cells from the outer layer through invagination.
At a later stage, when the yolk plug has withdrawn from the surface and the blastopore has become slit-like, transverse sections show several important changes in this axial mass. In front of the blastopore the separation between ectoderm and mesoderm has extended, by delamination, toward the midline, and just before reaching this, turns sharply downward toward the line of delamination between the endoderm and mesoderm, not, however, reaching quite to this, thus leaving in the mid-line a narrow vertical ridge of cells. In the regions where the endoderm and mesoderm remain continuous, a pair of slight depressions appear as shallow grooves out of the archenteron; these are continued into the cell mass a short distance as virtual grooves, indicated only by the arrangement of the pigmented cells. Further forward (Fig. 36) the lower margins of these grooves become better marked, as low lip-like structures approaching the mid-line, and the mesoderm in these regions is more extensively separated from the lining of the archenteron. Finally, still farther forward, the grooves disappear and the extension vertically of the spaces separating the mesoderm from the endoderm and ectoderm completely delimits the pair of mesoderm masses. The cells left in the mid-line between the proximal ends of the mesoderm sheets form a wedge-shaped elevation continuous with the endoderm; this is the rudiment of the notochord. In a still later stage the chorda begins to be cut off from the endoderm by a narrow split leaving the enteron roofed dorsally by a layer only one cell thick (Fig. 44). Passing posteriorly from the first section described above, into the region of the blastopore, we find the grooves out of the archenteron better marked and the ventral lip, as well as the dorsal, quite pronounced. These grooves are apparently indications of the enterocoelic evaginations; this relation will be mentioned more fully at the close of the description of notogenesis.
Fig. 36. Part of a transverse section through the young embryo of R. fusca, showing traces of enteroccel formation. After O. Hertwig. a, Archenteron; c, enteroccels; ec, ectoderm; en, endoderm; m, mesoderm; n, notochord; p, neural plate; y, yolk cells.
The Medullary Plate
The rudiment of another axial structure is developing at the same time as the chorda and mesoderm; this is the medullary plate. The medullary plate is formed in part from the median band of cells extending from the region of the dorsal lip of the blastopore nearly to the animal pole, and in part from the axial thickening due to the confluence of the lateral portions of the germ ring. In the former region the inner or nervous layer of ectoderm begins to thicken and by the time the blastopore has become circular and commenced to close, a thickened medullary plate has formed over the whole dorsal surface of the gastrula (Fig. 32, F). This is in the form of a broad plate, narrow just in front of the blast opore and widening gradually as it extends up toward and beyond the upper pole. By the time the yolk plug is withdrawn the margins of the medullary plate are considerably thickened so that its outline is visible externally; the median region has meanwhile become thinner and a shallow groove results (Fig. 22, C). The thickened margins are elevated slightly above the general surface of the ectoderm and form the two lateral neural ridges, extending from the sides of the blastopore along the dorso-lateral regions of the embryo, widely separated, to a level about opposite the blastopore, where they turn sharply and pass to the mid-line, meeting and forming thus the transverse neural fold, which marks the anterior limit of the medullary plate. The median groove soon becomes quite pronounced and is known as the neural groove.
Sections through the region just in front of the blastopore (Figs. 32, G; 33, F) show that the neural plate early begins to separate from the remainder of the ectoderm. Fig. 44 shows how the medullary plate is cut away laterally and superficially from the ectoderm by a narrow split resulting from cell rearrangements; we have already seen that a similar space separates the medullary plate from the underlying notochord.
Summary and Comparisons with Other Forms
Before continuing our account of the development of the rudiments whose formation has just been described, we should summarize the events of notogenesis and then 1 relation to gastrulation. It is evident that the distinction between gastrulation and notogenesis is real, and essential to an understanding of this period in development. Gastrulation involves only those processes which convert the monodermic embryo into a didermic embryo; notogenesis includes those processes involved in the formation of the medullary plate, notochord, and mesoderm. Gastrulation is accomplished primarily by delamination and the rearrangement of the yolk cells, and only secondarily, and to a very slight extent, by invagination. In the frog the process of invagination is chiefly concerned in the formation of the rudiments of the chorda and the mesoderm of the dorsal and dorso-lateral regions, although these structures are not formed wholly by invagination but also by the transport of the materials of the germ ring to an axial position, and then by delamination. In the development of the frog, therefore, invagination is a process of minor importance.
The actual materials out of which the axial structures of the embryo are formed are to be found in the animal half of the blastula, that is, in the walls and floor of the blastocoel, and these in turn are distinguishable in the eight cell stage, where they are contained in the upper quartet and the upper parts of the lower quartet; finally they can be traced back, approximately to the animal half of the egg. The later localization of the greater part of these materials in a true germ ring is an important characteristic in the development of the frog, and makes possible a close comparison of the gastrula and early embryo of this form, with the conditions in the lower as well as higher forms. It is also important that accompanying the downgrowth and closure of the germ ring there is a true confluence of its lateral margins, forming a thickened axial mass of cells, gradually elongating posteriorly through continued confluence. In this axial region the chief organs characteristic of the frog as a Chordate animal, have their origin. These organs gradually differentiate as the mass elongates posteriorly, and their rudiments, though not individually differentiated as such, are thus seen to form gradually from the cells contained laterally in the roots of the axial thickening, that is, in the germ ring itself. This germ ring is not so clearly indicated in Amphioxus, although we have seen in that form a rapidly growing ring of cells around the blastopore, wider on its dorsal side, from which are differentiated gradually the rudiments of the chorda and mesoderm, and a considerable part of the medullary plate. In Amphioxus, however, gastrulation is more nearly completed before notogenesis commences, so that while there is some overlapping it is not so extensive as to cause confusion of the two processes. In the frog not only does notogenesis commence so early as to obscure certain features of gastrulation, but these processes are both highly modified by the presence of a large mass of inert yolk cells. The presence of this rather immobile mass results in the formation of the embryo as on the surface of a sphere instead of as a simple elongated embryo, and such processes as the formation of enterocoelic or notochordal evaginations of the gut seem more or less abbreviated when compared with such a form as Amphioxus, where the embryonic layers are not impeded in their foldings by any such restraining influence as the yolk mass in the frog. Were we to assume that the development of Amphioxus represents truly primitive processes of development among the Chordates, we might describe the course of early development in the frog by saying that it is directed toward the accomplishment of the final result, rather than the carrying through of a definite program, so that the formation of the chief axial structures is effected more or less independently of the formal processes of development, as illustrated by Amphioxus.
However, there is considerable doubt as to whether Amphioxus does really represent, in these respects, conditions which may be considered primitive for the Chordates, Opinion remains quite divided upon the subject of the relation between developmental processes in Amphioxus and in such forms as the Amphibia. Some would point out that excepting only the Protochordates, all of the lower vertebrates have large eggs, containing a considerable quantity of yolk, definitely localized in one pole; that, indeed, the Mammals are the only Craniates which have small eggs, with little yolk, comparable with those of the Tunicates and Amphioxus. These would maintain that, while we may say that the presence of the yolk in the egg has modified the form of early development, we cannot call this modified development typical for the Chordata. It is quite likely that while the development of Amphixous is simpler and more diagrammatic than that of any Craniate, we. must after all regard this as a secondary or acquired simplicity, and not the simplicity of primitiveness. From the point of view of purely comparative embryology, Amphioxus should be the first to be considered; from the phyletic standpoint it should be considered after the more typically Chordate frog not that the frog is embryologically typical of all Chordates, merely that it represents that condition more truthfully than Amphioxus. On the other side, some would hold that the embryological simplicity of Amphioxus is that of true primitiveness, that Amphibian development is secondarily modified, and phyletically a modification of that of the Protochordates, interpretable only through the latter and not vice versa, and that many of the differences are the result of the accumulation of yolk in the Amphibia. We may mention from these two points of view only the development of the mesoderm as one of the central points.
Except in Amphioxus the Chordate embryo remains two layered or didermic only a very short time, on account of the very early development of the mesoderm in all other forms. In the frog the mesoderm cells are found, soon after the endoderm begins to be formed, first all around the margin of the blastopore forming an important part of the germ ring; that is, the mesoderm is first all blast oporal or peristomial. Then confluence begins and the lateral portions of the germ ring are carried to the mid-dorsal region and poured into the posteriorly elongating embryo, where they form the mesoderm bands; the mesoderm of the germ ring thus becomes axial in position and is known then as gastral mesoderm. The gastral mesoderm is here derived from the peristomial through a change in position due to confluence, and no essential distinction between the two is to be drawn. Only in the posterior region of the frog embryo immediately in front of the blastopore, are there traces of evagination in connection with the formation of the mesoderm in the form of a pair of slight grooves or slits. These may be considered as due merely to the rapid, unilateral and localized proliferation of the cells around the blastopore, such as often leads to a grooving of the surface, and as having nothing to do with the mesoderm folds and enterocoelic evaginations of Amphioxus. Or, on the other hand, these grooves may be regarded as vestiges of the enterocoelic grooves of a primitive Amphioxus-like embryo, which has been modified by the accumulation of yolk and the precocious formation of the mesoderm before confluence. It might easily be supposed that the formation of the mesoderm in advance of the definite establishment of the gut cavity would result in the loss of function and disappearance of the enteroccels. In Amphioxus the gastral and peristomial mesoderms have unlike origins, because the mesoderm is not formed until after gastrulation, and consequently, that formed from invaginated endoderm (gastral or axial) is unlike that (peristomial or blastoporal) formed from the "germ ring" or growth zone, around the posterior end or blastoporal region of the embryo.
While decisive evidence is perhaps lacking, and much may be said on both sides, on the whole the evidence seems to favor the first view; that the method of mesoderm formation in Amphioxus is not wholly primitive, that primarily there is no distinction between gastral and peristomial mesoderm, for all is first peristomial or blastoporal, and that the mesoderm grooves of the frog are not vestiges of enterocoelic evaginations, like those of Amphioxus, but represent the primary mode of origin of the mesoderm as a proliferation from the margin of the blastopore. This would, of course, involve the conclusion that the coelomic cavity is not phylogenetically derived from the gut cavity among the Chordata. In the frog the ccelom has no relation with the mesoderm folds, as it would have if these are vestigial enterocoelic grooves, and it will be recalled that in Amphioxus only the cavities of the more anterior somites are ever connected, even as grooves, with the gut cavity. And yet in some of the tailed Amphibia the ccelom is apparently sometimes directly connected with the grooves in the mesoderm folds.
Most of the differences in the arrangement of the mesoderm rudiments in Amphioxus and the frog can be traced to this difference: in the frog the mesoderm differentiates before gastrulation and confluence, in Amphioxus the mesoderm differentiates after gastrulation and after confluence has begun. Doubtless these unlike relations are largely the result of the absence of the yolk mass in Amphioxus and the consequent mobility of the blastomeres and the epithelia which they compose.
We may assume that the stage in which the rudiments of the central nervous system, notochord, mesoderm, and gut are all definitely established, marks the end of notogenesis and the beginning of the formation of a definite embryo in a restricted sense.
Embryonic Period - B. The Formation Of The Early Embryo
We may close our account of the early embryonic period by tracing briefly the further development of the rudiments formed at the close of notogenesis, up to the time the neural tube is closed. We have assumed arbitrarily to let this stage (about two days after fertilization) represent an "early embryo" (Fig. 22, E, F). At this time the embryo has elongated so that its length is about one and one-half times its depth or the diameter of the gastrula. The postero-dorsal region is narrowed and drawn out slightly into the rudiment of the tail. The dorsal surface is straight or even slightly concave and narrowed in cross section, the ventral surface remains broadly convex. The rudiments of several organs are visible externally as elevations or depressions; these will be described in connection with the internal structure. Externally the ectoderm alone still forms the covering epithelium, for as yet no part of the mesoderm has contributed to the formation of an integument. About the only change in the character of the ectoderm is the development, on some of its cells, o'f numerous short cilia. Just before the fusion of the neural folds the cilia begin to appear first along their margins. They extend rapidly more widely over the surface, and by the time this early embryonic stage is reached they are absent from only the ventral surface. A little later the ciliation is completed. The cilia beat in the posterior direction and give the embryo a slow rotary motion within the egg membranes.
The Nervous System
It is more convenient to describe the development of the nervous system first, partly because many of the chief external characteristics of this period are associated with the development of this system. The transverse neural fold, which marks the anterior limit of the central nervous system (Figs. 22, C; 32, G), forms from materials located in about the middle of the roof of the blastoccel, while the posterior limit of the nervous system is just above or in front of the dorsal lip of the blastopore. The downward extension of the latter, on account of the confluence, increases the length of the rudiment of the nervous system, so that before the blastoporal margin or germ ring has fully contracted, it extends nearly from pole to pole, around the posterior side of the gastrula. The rotation of the gastrula then changes the apparent, though not the true morphological, position of the anterior margin of the medullary plate, so that when the transverse neural fold actually appears, it is on the anterior side of the gastrula, and the medullary plate itself occupies nearly the whole dorsal surface of the early embryo (Fig. 32, G). We have already described the formation of the neural or medullaryplate, the neural groove, and the lateral and transverse neural or medullary ridges.
The elevation of the neural ridges, particularly their anterior portions, soon becomes very marked, and as it continues the middle of the plate sinks downward and soon the ridges bend over toward each other and meet along the mid-line, where they fuse, transforming the neural plate into the neural tube containing the neural canal or neurocoel (Figs. 22, D, E; 38) . For a long time after fusion a deep median groove marks the region where the folds have come together. The fusion of the neural ridges does not occur simultaneously throughout their extent, but first in about their middle, then extending posteriorly and anteriorly from this region (Fig. 38). This is approximately the location of the future medulla (myelencephalon) ; from this time, therefore, the spinal cord and brain are distinguishable (Fig. 37). The narrower cord region closes before the much wider brain. In the closure of the brain region the transverse fold plays an important part; this extends backward, roofing the expanded cavity of the brain, and meets the slowly fusing lateral folds in the region between the future fore- and midbrain. This is, therefore, the last region of the neural tube to close, and may consequently be termed the neuropore; in the later embryo this is the region just posterior to the epiphysis or pineal body (see next chapter). The neuropore has a very transitory existence.
Fig. 37. Diagrams of median sagittal sections of frog embryos. After Marshall. A. Just before the closure of the blastopore. B. Just after the closure of the blastopore. (See Fig. 38, D, E.~) a, Anal or cloacal aperture; b, blastopore; e, epiphysis; ec, ectoderm; en, endoderm;/, fore-brain; g, mid-gut; h, hind-brain; ht, rudiment of heart; hy, hypophysis; I, liver diverticulum ; m, midbrain; ms, mesoderm; w, notochord; nc, neurenteric canal; o, oral evagination; p, proctodseum; ph, pharyngeal region of gut cavity; r, rectum; s, spinal cord; y, yolk cells.
Those cells in the nervous layer of the ectoderm forming the lateral margins of the neural plate, that is, the neural ridges proper, do not themselves form an integral part of the neural tube. When the margins of the neural plate fold together they are left dorso-laterally, between the neural tube and the definitive ectoderm. These ridges of cells become broken into cell groups, lying along the lateral regions of the neural tube, forming the neural crests. These are concerned later in the development of the nerves, which, together with some additional details of this period, will be described in the next chapter.
At the posterior end of the embryo, the relations of the blastopore (germ ring) and neural folds are of considerable importance from a comparative point of view. We left the blastopore in the form of an elongated slit on the postero-dorsal surface of the embryo. The lateral walls then approach, about the middle of the slit, and finally it is there constricted completely, so that the blastopore is divided into two small openings, an upper and a lower (Fig. 38, A, B). The medullary groove extends forward from the upper opening, which remains open, leading directly into the archenteron (Fig. 37). The lower opening is soon closed by the fusion of its lips. The fusion involves only the ectoderm and endoderm of the region, and occurs some distance below the surface, so that a pit-like depression is left on the surface, lined with ectoderm; this is the proctodceum. When the posterior ends of the neural folds form, they extend into, or rather out from, the middle regions which have fused, and as they become elevated and form the neural tube they cover over the upper blastoporal opening (Fig. 38, C) which thus becomes the neurenteric canal, like that of Amphioxus or other Chordates, and similarly formed, putting the neurocoel and gut into communication. The lateral margins of the blastopore are formed of the remains of the germ ring, and when they meet, dividing the blastopore, they form a median cell mass in which ectoderm, endoderm, and mesoderm are fused in a more or less undifferentiated mass. This mass can no longer be called the germ ring, although it is really equivalent to the lateral parts of this; it is known as the primitive streak, and the groove that remains for a time on its surface, indicating its origin from originally separate lateral portions, is the primitive groove. The primitive streak and groove of the frog are homologous with the similarly named structures in Amphioxus and in the Amniota. The cells of the primitive streak continue to multiply rapidly, and bud forth strands of ectoderm into the neural folds and upon the surface of the body, mesoderm into the lateral bands, and endoderm into the wall of the gut. This leads to the formation of a postero-dorsal protuberance which is the rudiment of the tail, but in the stage we are describing this is only just indicated (Fig. 38, D, E).
Fig. 38. 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. After F. Ziegler. A. Blastopore nearly closed; neural folds just indicated. B. Blastopore becoming divided into neurenteric and proctodaeal portions; neural folds becoming elevated. C. Neurenteric canal forming; neural folds closing together. D. Neural folds in contact throughout. E. Neural folds completely fused; tail commencing to grow out. b, Blastopore, containing yolk plug; 61, rudiment of neurenteric canal (dorsal part of blastopore) ; 62, rudiment of proctodaeal pit (ventral part of blastopore) ; 60, branchial arches; g, neural groove; nf, neural folds; np, neural plate; p, proctodaeal pit; s, rudiment of oral sucker; t, rudiment of tail; x, neural folds roofing the blastopore and establishing the neurenteric canal.
At the opposite end of the body the enlarged brain protrudes, forming a head region faintly indicated externally. A sagittal section (Fig. 37, A) shows that the future regions of the brain are but faintly marked out. The chief characteristic of the brain is its abrupt bending or flexure around the anterior tip of the notochord; the region immediately in front of the chorda is that of the future mid-brain, while the large fore-brain lies entirely below the level of the chorda and the remainder of the neural tube. In the mid-line, just beneath the end of the forebrain, a tongue-like proliferation of ectoderm cells extends inward a short distance. This is the rudiment of the hypophysis.
The simple rudiments of the chief sense organs are also indicated at this early stage. The eyes are distinguishable, even before the brain closes, as small patches of deeply pigmented ectodermal epithelium in the antero-lateral regions of the medullary plate. When the neural tube is completed, they form a pair of hollow ventro-lateral outgrowths from the fore-brain to the superficial ectoderm. Frequently they can be seen externally, marked by a pair of slight darkened elevations, either side of the fore-brain region (Fig. 22, E, F). The ears are indicated as a pair of thickened patches of the inner or nervous layer of the ectoderm opposite the hind-brain region. They are scarcely visible externally at this time. The olfactory organs develop as thickened circular patches of ectoderm below and in front of the optic rudiments. At this stage a pair of slight depressions may sometimes be detected on the surface, marking the future olfactory pits.
By the time the neural tube is completed the chorda has become completely delaminated from the outer surface of the endoderm, except only in the region of the primitive streak where its formation is still progressing posteriorly. The separation of the chorda from the endoderm, or rather the enteroderm (see below), occurs in the posterior direction, beginning near but not quite at the anterior tip.
At the close of gastrulation and notogenesis the archenteron is a nearly hemispherical cavity on the dorsal side of the embryo, open posteriorly through the blastoporal opening. Its roof and sides are left as a thin layer of endoderm the enteroderm after the chorda and mesoderm have been split off; its floor is formed of the thick mass of yolk cells. By the time the neural tube is completed and the embryo has elongated slightly, the enteric cavity or mesenteron has enlarged considerably, chiefly in front of the yolk mass, which retains a postero- ventral position in the wall of the gut (Fig. 37). This anterior enlarged region of the mesenteron is known as the fore-gut, the entire wall of which is but one cell thick; this is the region of the embryonic pharynx and later of the oesophagus and stomach as well. At the posterior end of the mesenteron there is a slight enlargement, just in front of the neurenteric canal, which is the hind-gut or rectal portion of the intestine. Connecting these two regions the narrow mid-gut or intestinal region proper, is that containing the yolk cells, which are also to be regarded as enteroderm.
In the fore-gut of this stage there is an antero-ventral outpocketing toward the ectoderm just below the fore-brain, indicating the region where the mouth will form later. A posteroventral outgrowth beneath the anterior end of the yolk mass is the rudiment of the liver. Sections passing through the sides of the fore-gut show that even in this early stage the rudiments of the first two or three visceral pouches are present in the form of vertical outgrowths from the side walls of the pharynx to the ectoderm, with which they fuse. Along the region of the fusion the ectoderm is depressed so that these are externally visible as vertical depressions just back of the head (Fig. 22, E, F). Externally two of these are marked at this time as the external branchial grooves, and the ridges left between and in front of them are the rudiments of the second or hyoid, and first or mandibular arches respectively. The hyoid arch is sometimes known here as the "gill plate"; it extends dorsally nearly to the margin of the nervous system. The mandibular arches are less marked; these appeared even before the medullary plate became folded together, as a pair of low ridges diverging from the antero-lateral regions of the plate and sometimes called the " sense plates." They lie between the olfactory and optic rudiments and form the antero-lateral regions of the embryonic head.
The delamination of the mesoderm from the surface of the endoderm commenced in the dorso-lateral regions anteriorly, and spread thence posteriorly and ventrally. We have seen that posteriorly the mesoderm finally passes into the region of the germ ring, or what now represents a portion of that, the primitive streak, where it continues to be formed and budded off anteriorly as the primitive streak extends posteriorly. And ventrally the delamination ceased in the ventro-lateral regions of the endoderm, and the mesoderm then extended gradually toward the ventral side through the multiplication of its own cells and their downward extension, and through the splitting off of scattered groups of cells from the endoderm toward the ventral side. In this early embryo the mesoderm forms a distinct layer separating ectoderm and endoderm throughout (Fig. 44), except in the primitive streak and in the head region, where the mesoderm is never in the form of a definite sheet, but is represented by scattered cells filling the spaces between the wall of the mesenteron and the nervous system and ectoderm (Fig. 45). In the pharyngeal region the mesoderm becomes interrupted by the extension of the gill pouches out to the ectoderm; between successive pouches groups of mesoderm cells are enclosed which become the rudiments of the visceral arches mandibular, hyoid, and branchial.
Through the trunk region the typical mesodermal structures develop rapidly. The axial region toward the chorda thickens and becomes solid; this is the segmental plate or myotomal region (Fig. 39) . Farther laterally the thinner layer is the lateral plate; this splits into two more or less distinct sheets, the somatic and splanchnic layers, in contact with ectoderm and endoderm respectively. The space between these is the splanchnoccel, the rudiment of the body cavity or coelom. In the stage we are describing the coelom does not extend' completely through the lateral plate as it does finally. The coelom does however extend into the segmental plate, as the rudiment of themyocoels,
Fig. 39. Part of a section through the anterior body region of an embryo of R. sylvatica, just beginning to elongate, illustrating the differentiation of the mesoderm. After Field, c, Coelom; ec, ectoderm; en, endoderm; g, gut cavity; mp, medullary plate; my, myotome; n, notochord; nc, rudiment of neural crest; so, somatic layer of mesoderm; sp, splanchnic layer of mesoderm.
appearing toward the surface of this thicker mass. As the neural folds approach, the cells of this axial mesoderm are rearranged in such a way that the longitudinal bands are cut transversely into segments or somites. This process begins just back of the pharynx and extends rapidly toward the posterior ends of the bands (Figs. 44, E; 53). It is important that the mesoblast of the head region is not clearly divided into segments. By the time the neural tube is completed three or four pairs of somites have been formed. At first these are continuous with the lateral plate but shortly after they are marked out, the two regions become separate and the lateral plate itself is never segmented but remains uninterrupted. The coelomic spaces of the somites, the myocoels, then disappear without leaving any trace. These processes are progressive posteriorly, the formation of the coelom, somites, etc., continuing as the mesoderm forms from the primitive streak.
In the embryo of this period indications of two important structures are present in connection with the mesoderm but not as readily recognizable and definite rudiments; these are the pronephros and the heart. The connections between the second, third and fourth somites and the lateral plate remain as small masses of cells, in close relation with the somatic layer of the lateral plate. These are the cells forming the rudiments of the pronephric tubules. The definite formation of these rudiments and that of the pronephric duct must be left until a later stage is described. Just below the pharynx and in front of the liver, the mesoderm cells are arranged in somatic and splanchnic layers, and between these the coelomic space is well marked on either side, while elsewhere in the ventral region it has not appeared (Fig. 37, B). Between the mesoderm and the ventral side of the pharynx a few loose cells are scattered along. This is the region where the heart is soon to appear but this, too, must be described later. (References to the literature will be found at the end of Chapter III.)
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Outlines of Chordate Development: 1. Amphioxus | 2. Early Frog | 3. Later Frog Organogeny | 4. Early Chick - Embryonic Membranes and Appendages | 5. Later Chick - Organogeny | 6. Early Mammal - Embryonic Membranes and Appendages | Figures
Reference: Kellicott, W. E., (1913) Outlines of chordate development. New York: H. Holt and Company.