Book - Introduction to Vertebrate Embryology 1935-2

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Shumway W. Introduction to Vertebrate Embryology. (1935) John Wiley & Sons, New York

Shumway (1935): Preface - Contents | Part I. Introduction | Part II. Early Embryology | Part III. Organogeny | Part IV. Anatomy of Vertebrate Embryos | Part V. Embryological Technique
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Introduction to Vertebrate Embryology (1935)

Part II. Early Embryology

Chapter III The Germ Cells

The germ with which the development of the vertebrate commences is the fertilized egg, or zygote. Before discussing the development of the zygote, it is advisable to examine the gametes, egg and sperm, whose union results in its existence. We shall proceed first to the description of the gametes, comparing them with each other and with a generalized cell. Next we shall consider the way in which the germ cells originate and become mature. Thereafter we shall turn to the study of fertilization.

A. The Gametes

Vertebrates are characterized by the bisexual method of reproduction, in which there are two distinct sexes: the female, or egg-producing individuals; and the male, or sperm-producing individuals. Among the protochordates (tunicates) we find groups in which the same individual produces both eggs and sperms. Such individuals are called hermaphrodites. This phenomenon is rare among the vertebrates and is not typical of any species.

The two kinds of gametes, eggs and sperms, differ from each other in appearance, size, and structure. These differences will be more apparent after a brief review of cell structure in general.

Table 3 Structure Of The Cell

A. Nucleus (composed of karyoplasm). 1. Reticulum (composed of chromatin). 2. Karyolymph (nuclear sap). 3. Nucleolus (plasmasome). 4. Nuclear membrane.

B. Cytosome (composed of cytoplasm). . Hyaloplasm (ground-protoplasm). . Centrosomes (centrioles). . Mitochondria (chondriogomes). . Golgi bodies (dictyosomes). . Plastids. . Metaplasm (relatively lifeless accumulations). Plasma membrane.


C. Envelopes or matrix (cell wall).

The cell. — The familiar definition of a cell (Fig. 5) is, “ a mass of protoplasm, containing a nucleus, both of which have arisen by the division of the corresponding elements of a preéxisting cell.” Protoplasm in this sense refers to the living substance of the cell, including both the material inside the nucleus and that in the cell body or cytosome. It is customary to use the term karyoplasm (nucleoplasm) for the nuclear protoplasm, and the word cytoplasm for the protoplasm of the cell body. Some writers employ only the words nucleus and cytoplasm to distinguish between nucleus and cell body.


Fig. 5. — Diagram of a composite cell. (After Wilson.)


The nucleus. — The cell nucleus is generally a rounded body separated from the eytosome by a delicate nuclear membrane. Within this is a transparent ground substance known as the karyolymph or nuclear sap. But the characteristic substance of the nucleus is its chromatin, a substance staining sharply with basic dyes, and arranged usually in a network of threads called the reticulum (Sharp). Sometimes swellings, chromomeres, are apparent at the nodes of the network. The nucleus usually contains a smaller body known as the nucleolus, a droplet of some material heavier than the nuclear sap, but staining with acid dyes. Its staining properties alter during cell division.

The nucleus may fragment to form polynuclear cells. It may also divide, often many times, while the cell body remains undivided, resulting in the formation of a syncytium. Sometimes the nucleus may be ejected to leave enucleate cells such as the red blood corpuscles of mammals. But in general every cell has one nucleus.

The type of nucleus here described is known as the vesicular type. There is distinguished also the massive or compact type of nucleus, in which the chromatin forms apparently a solid mass, as in the sperm cell. Then there is a diffuse type, in which the nuclear membrane is absent and the chromatin is scattered through the cell body in granules called chromidia.

The cytosome. — The cytoplasm of the cell body includes an outer delicate semipermeable membrane known as the plasma membrane. This is the surface at which the protoplasm of the cell is in contact with its environment. Within this is the liquid ground substance or hyaloplasm, in which are distributed a number of differentiated bodies. Of these cytoplasmic inclusions the more important seem to be the centrosomes, mitochondria, and Golgi bodies, all of which appear to have the properties of independent growth and division.

The centrosomes (centrioles), small spherical bodies, one or two in number, lie near the nucleus. They seem to be concerned in the process of cell division. In cells with locomotor organs, like the tail of the sperm, the centrosomes are connected with the contractile element of the cell.

The mitochondria (chondriosomes) are small rods, or granules, very numerous and scattered through the cytoplasm. They are dissolved by many common methods of preparing cells for observation, but can be demonstrated in the living cell by a stain called Janus Green B. They are preserved by special chemicals, e.g., osmium tetroxide.

The Golgi bodies (dictyosomes) are sometimes scattered through the cytoplasm but often aggregated into a network, the Golgi apparatus. Some authors deny that there is a real structure of Golgi bodies and speak therefore of the Golgi material or the Golgi zone. Other investigators have sought to identify these bodies with the plastids, cytoplasmic elements which are found in plant cells. Golgi bodies are hard to identify in living cells but can be demonstrated by special techniques involving the use of osmium tetroxide or silver nitrate. Their function is doubtful, but there is some reason to believe that they are concerned with the elaboration of substances within the cell such as enzymes.

Still another type of inclusion in the cytosome is represented by the plastids. These bodies are found more frequently in plant cells, e.g., chloroplasts, the chlorophyll bodies, which appear to have the capacity of independent growth and division.

Metaplasm is the name given to all those bodies in the cytoplasm which clearly do not possess the properties of independent growth and division. These may be aggregated in vacuoles or distributed in tiny droplets, granules, etc. Among these are such bodies as secretory granules, intermediate stages in the production of cell secretions (enzymes, etc.). Storage granules are end stages in the accumulation of reserve food materials such as yolk, oil, starch, etc. Here also we may include the minute pigment granules. Embryologists sometimes use the term deutoplasm for reserve food materials in the cell.

The cell wall. — In concluding this brief review of cell structures we must recall that the cell may secrete a wall around itself such as the vitelline membrane. In some tissues these cell walls unite to form a matrix such as the intercellular substance of cartilage or bone.

The sperm.— The male germ cell of vertebrates is a very minute flagellate cell ranging in size from 20 microns (crocodile) to more than 2 mm. (Discoglossus, an amphibian). The general shape is that of a tadpole with an excessively long tail, but there are sufficient differences among these tiny cells for them to be identified by specialists.

The sperm (spermatozoon) consists of a head and a tail (Fig. 6). The head contains the nucleus, which is compact and stains very deeply with basic dyes. Here also is the acrosome, usually at the apical end, originating from Golgi bodies, possibly connected with the production of some secretion involved in fertilization. The head is surrounded with a delicate plasma membrane.

The tail consists of three divisions: middle-piece, main-piece, and end-piece. The middle-piece contains two centrosomes.


Yolk. — The bulk of the egg is due to the presence of metaplasm in the form of yolk. This substance contains the principal foodstuffs for the developing embryo. Studies on the yolk of the hen’s egg indicate that it contains water (50 per cent), proteins, fats, carbohydrates, inorganic salts, vitamins, pigments, and enzymes (Needham).

The yolk is present in the form of spheres, ovoids, or discs, which stain usually with basic dyes. The yolk tends to accumulate in one hemisphere of the egg, forcing the nucleus into the other. Since the yolk is heavier than the other constituents of the egg, the yolk-laden hemisphere is the lower one when the egg is suspended in water. In large-yolked (macrolecithal, megalecithal) eggs, such as those of the frog and chick, the accumulation of the yolk in one region is so marked that they are known as telolecithal eggs. In small-yolked (microlecithal, oligolecithal) eggs, like those of the amphioxus and of man, the yolk is distributed more generally and they are called isolecithal (homolecithal).

Polarity. — Even in isolecithal eggs there is a visible distinction between the two hemispheres of the egg, so that an axis exists from the center of one hemisphere to that of the other. This, known as the polar axis, is the earliest indication of a differentiation in the egg. The two ends of the axis are known as the poles. The polar bodies, referred to in the preceding chapter, are formed at one of these which is known as the animal (apical) pole. It is sometimes called simply the pole. The other is called the vegetal (vegetative, abapical) pole, sometimes the antipole. The nucleus always lies in the polar axis, more or less towards the animal pole. The yolk shows a gradation from the animal towards the vegetal pole. We shall observe in later chapters that the animal pole marks the anterior end of the developing embryo and the vegetal pole marks the posterior end. There is also reason to believe that the polar axis, in addition to being the first expression of symmetry in the egg, marks a gradient of metabolism (Child). By this is meant that metabolic processes are accelerated at the animal pole and progressively retarded towards the vegetal pole.

A considerable body of evidence shows that the animal pole of the egg is the one which was most active in physiological exchange with its environment while still in the ovary. It is the pole of the egg which is attached to the ovary in the amphioxus (Conklin) and the chick (Conklin). It has been suggested that in the frog the animal pole of the egg is the one lying nearest the arterial blood supply (Bellamy).

Egg envelopes. — The ovum usually possesses, in addition to the plasma membrane, a variety of protective envelopes which are divided into three classes according to the mode of their formation. Primary envelopes are those formed by the egg itself, such as the delicate vitelline membrane. The secondary envelopes are those formed by the follicle cells which immediately surround the egg in the ovary. A good example is the so-called “chorion” of one of the cyclostomes, Myzine (Fig. 9). It is usually quite difficult to distinguish primary from secondary envelopes, and it is probable that many vitelline membranes are compound in origin. In those vertebrates in which fertilization is external, such as the cyclostomes and bony fish, the primary and secondary envelopes are often perforated by openings called micropyles through which the sperm may have access to the egg. The tertiary envelopes include all those formed by the walls of the oviduct during the passage of the egg. Examples are the egg albumen, shell membranes, and shells of such groups as the reptiles, birds, and the egg-laying mammals; the egg capsules of the elasmobranchs, and the egg jelly of the amphibia and many bony fish. These envelopes are not formed until after fertilization, except in the case of the egg jelly, and this does not attain its final thickness until after the entrance of the sperm, when it swells by the absorption of water.


Fig. 9. — Egg of Myxine, showing “chorion” and micropyle (after Dean).


THE EGG OF THE AMPHIOXUS. — The eggs (Fig. 10A) are 0.1 mm. in diameter. Before maturation the large nucleus is roughly 0.05 mm. in diameter displaced well towards the animal pole. The cytoplasm consists of a thin outer layer relatively free from yolk, and probably containing mitochondria. The rest of the cytoplasm contains yolk. There are no egg envelopes except perhaps a vitelline membrane. The egg is classed as isolecithal.


Fig. 10. — Typical eggs. A, amphioxus, approx. X70 (after Wilson in Willey). B, frog X8. C, hen X? (after Duval). D, human X250 (after Allen in Arey).


THE EGG OF THE FROG. — The diameter of the egg (Fig. 10B) is 1.7 mm. (R. pipiens, Wright), with a large nucleus before maturation. There is a thin outer layer of cytoplasm, containing granules of pigment in the animal hemisphere. Pigment is also found around the nucleus. The yolk is distributed with fewer and smaller platelets in the animal hemisphere grading down to more and larger platelets in the vegetal hemisphere. There are a vitelline membrane (primary), ‘‘ chorion ”’ (secondary), and one to three layers of egg jelly (tertiary). The eggs are discharged 40 THE GERM CELLS

in large masses which adhere to each other by means of this jelly. The eggs are classified as telolecithal.

THE EGG OF THE CHICK.— The hen’s egg (Fig. 10C) is extremely telolecithal. The cytoplasm, with the nucleus in its center, forms a small germinal disc upon the great mass of yolk.

This yolk is arranged in

wei e, Albumen or concentric layers of

Outer shell yellow and white mateshen Yial around a_ central



x membrane

Inner shell

membrane mass of white yolk, chamber called the latebra (Fig.

11). From this latebra a stalk of white yolk (the neck of the latebra) exFia. 11. — Diagram of hen’s egg sectioned tends up ward. The (after Lillie). germinal disc rests on this isthmus. The yolk and germinal disc are surrounded by a delicate vitelline membrane (primary). This in turn is surrounded by the albumen, a viscous tertiary membrane twisted spirally about the egg from left to right, starting from the broad end of the egg. The albumen next to the vitelline membrane is denser than the rest and is prolonged into two spirally twisted cords, the chalazae, one at cither end of the egg. The albumen is in turn surrounded by two parchment-like shell membranes, of which the inner one is the thinner. These two are separated at the blunt end of the egg, thus forming the air chamber. The egg shell is a calcareous deposit upon the outer shell membrane. Its color is due to bile pigments of the hen. The germinal dise is about 4 mm. in diameter, the yolk about 40 mm. The size of the egg as a whole varies largely, depending on the amount of albumen deposited around the yolk.

Giant and dwarf eggs are sometimes recorded. In the hen’s egg, double- and triple-yolked eggs are known, as well as those which have no yolk at all. A very strange abnormality is known as the “‘ ovum in ovo,” where one egg is formed around another. The eggs of birds are either male-producing or female-producing, a statement, based solely on the evidence of genetics as no visible differences have been observed.


THE EGG OF MAN. — The human egg (Fig. 10D) is extremely smal}. The yolk granules are concentrated about the nucleus, which is slightly excentric. It is not positively known whether a vitelline membrane is present. But the egg is enclosed in a thick capsule with radial striations (canals?), the zona pellucida. It is not clear whether this is a primary or secondary envelope. At the time the egg leaves the ovary it is still surrounded by a layer of follicle cells which make up the so-called A ge \4- Zona pellucida corona radiata (Fig. 12). sacs Rp ie The egg may be termed ee NE Saas isolecithal. Its diameter ae : is about 0.13 mm.

Eggs and their environment. — Needham has recently pointed out that eggs differ from one another in respect to the physico-chemical constitution of the unfertilized egg, and the possibility of obtaining necessary material from the environment. The marine egg, exemplified by the amphioxus, develops in a medium containing oxygen and inorganic salts. The egg is organized in such a manner as to facilitate the exchange of materials with the environment, and the yolk is small in amount and (to judge from analyses made on marine fish) relatively poor in fats and inorganic salts. Development is rapid up to the hatching stage, but thereafter the larva takes a long time to attain its full size and sexual maturity.

The egg which develops in fresh water, like that of the frog, does not have a medium so rich in salts as the marine egg. It is therefore originally equipped with a larger store of this material. But the aqueous medium still affords facilities for the exchange of carbon dioxide and oxygen and for the disposal of nitrogenous wastes. The jelly with which the frog’s egg is provided consists almost entirely of protein and water. Diffusion takes place through it readily, and it affords protection against mechanical injury and bacterial infection, as well as furnishing a source of nourishment immediately after hatching.


Via. 12.— Human egg (ovarian) X200 (after Waldeyer).


The terrestrial (cleidoic) egg, such as that of the hen, stands easily first in respect to the amount of yolk present. The ratio of fat to protein in the yolk is also the highest. It is obvious that the egg must contain all the material necessary for growth except free oxygen and water, for these are the only substances passing from the atmosphere through the protective envelopes of the egg. Hence, as pointed out by Milnes-Marshall, except in the earliest stages the chick develops more rapidly than the amphioxus and attains its adult form in a much shorter time. The egg albumen also a source of food is a watery solution of protein with some carbohydrates. As we shall see in later chapters, the relative isolation of the embryo in the cleidoic egg is correlated with the development of its extra-embryonic sacs, i.e., the amnion or water bath, and the allantois which serves in the first instance to store nitrogenous wastes. _ The uterine egg, typical of the mammals, is characterized by little yolk, for, from a very early period, its nourishment is derived exclusively from the body of the mother. Accordingly there is a precocious separation of a special layer, the trophoblast, concerned with implantation, and later the development of a special organ of interchange, the placenta.

Comparison of the egg and the sperm. — Both gametes are morphologically complete cells. Each has a nucleus and a cytosome containing representatives of the centrosomes, mitochondria, and Golgi bodies. Each hasa plasma membrane. Yet neither is capable of independent, continued existence, for physiologically they are unbalanced. The egg is large, inert, and contains a vast store of metaplasm, is protected by egg envelopes, and has lost the power of continued division. The sperm is small, highly motile, contains little cytoplasm and no metaplasm, is devoid of protective membranes, and in itself has lost the power of continued division. We shall now turn to the study of the development of the germ cells and see how the structural differences, at least, arise.


TABLE 4 CoMPARISON OF THE VERTEBRATE EGG AND SPERM


Ovum

Ceil structures

Sperm


Large amount One, disappears in maturation Diffuse


Cytoplasm Centrosomes Mitochondria


Small amount Two, retained in maturation Spiral coil



Diffuse Golgi bodies Acrosome Present Plasma membrane | Present Vesicular Nucleus Compact Present Nucleolus Indistinguishable Present Nuclear membrane | Present

Other differences Large Size Small Quiescent Movement Swims actively None Motile organs Flagellum Egg envelopes Protection None Spheroid Shape Tadpole Few to many Numbers produced | Very many


B. Gametogenesis

Gametogenesis is the term applied to the history of the gametes

— their origin and development (Fig. 13).

The special history

of the male gametes is called spermatogenesis, that of the female

gametes odgenesis.

The origin of the germ cells. — Weismann is responsible for a

theory that the germ cells separate completely from all the other cells of the body (soma cells) ata very early stage in development. There is some evidence for this-in the embryology of a few invertebrate animals such as Ascaris, a parasitic roundworm. In the very first cleavage of the fertilized egg, the two daughter cells show a-striking difference, for when one of the daughter cells divides it retains all the chromatin of its nucleus whereas the other gives up a portion of this material to the cytosome. This phenomenon has been called chromatin diminution, and the cell showing this characteristic becomes a soma cell. The other is THE GERM CELLS

Soma Cells

Meiosis <

st

\ 7

o———> o—-e0-+83-|= ow-~

a o

Zygote Fig. 13. — Diagram of gametogenesis, male on left, female on right (after Wilson). THE ORIGIN OF THE GERM CELLS 45

known as a stem cell (Fig. 14), and in its division it produces in turn one cell which will be a soma cell and one which will be a germ cell. Eventually a stem cell gives rise to two identical cells, both of which are germ cells. These are known as primor



cg Fia. 14. — Origin of stem cells in Ascarts. A, first cleavage. B, C and D, second cleavage. P; and Ps are stem cells. 8S, (which gives rise to A and B) and 8; are soma cells. (From Richards after Boveri.)

dial germ cells, and, from this time on, they and their descendants produce germ cells only.

This theory of the distinction between germ cells and soma cells has held an important place in the history of biology because 46 THE GERM CELLS

it seemed to deny the possibility of the inheritance of characteristics acquired after fertilization. In other words, the characteristics would be acquired by the soma cells whereas inheritance is transmitted ‘by the germ cells which are entirely distinct. Now that we know that the nuclei of all cells are identical, whether they are germinal or somatic, the theory of the continuity of the germ cells has less theoretical importance.

Primordial germ cells. — In all vertebrates, so far as is known, the germ cells are first recognizable in the lining of the gut at a very early stage of development. These primordial germ cells, as they are called, are distinguishable by their large size, clear cytoplasm, and heavily staining nucleus (Fig. 15). From the gut

Mesonephric duct



Postcaval vein




ee ee = - ¥ — “ P me a i * , q Else sé: \ \__ Primordial 8 5D a 4% germ cell

Dorsal mesentery

Fia. 15. — Primordial germ cells in the frog (Rana sylvatica). Part of transverse section through 10 mm. larva, showing coelomic roof, X375. (After Witschi, 1929.)

wall they migrate into the mesentery suspending the gut from the roof of the coelom, and thence to the wall of the coelom. Here they multiply rapidly and produce two longitudinal ridges, which are the primordia of the sex glands, or gonads.

The ’gonia. — There are two opinions concerning the fate of the primordial germ cells:i in vertebrates: one that they give rise to all the later generations of germ cells; the other that they degenerate and the later germ cells arise independently from the tissue of the gonads. In any case, the germ cells which continue to multiply actively in the gonads are known as ’gonia: spermatogonia i if they are to give rise to sperm, odgonia ifthey givé Tise

to eggs. THE MATURATION DIVISIONS 47

The ’cytes. — When the individual becomes sexually mature, individual ’gonia undergo a period of growth by means of which they become transformed into ’cytes (auxocytes, meiocytes): spermatocytes if male, odcytes if female. The ’cyte (Fig. 16) is a large cell with a vesicular nucleus, two centrosomes surrounded by a clear area sometimes known as the sphere substance, which is in turn surrounded by a layer A of Golgi bodies, and a cloud of mitochondria.

The maturation divisions. — Each ’cyte gives rise to four iS daughter cells or gones (Sharp) by __ diosome Golgi bodies means of two cell divisions. i , These divisions are unique because 4,, 1g — Diagram of an early cyte of certain internal phenomena and (auxocyte). (After Wilson.) are known as the maturation divisions. The nature of these divisions will be discussed in more detail in a later chapter (page 64). Meantime we note that the spermatocyte gives rise to four cells of equal size, the spermatids, each of which will be transformed into a sperm. The odcyte on the contrary gives rise, by the first maturation division, to two cells one of which is very minute, the first polar body (polocyte I). The larger cell undergoes a second unequal division, resulting in the production of a second polar body (polocyte IT) and the mature egg or ovum. It will be recalled that among the vertebrates the

TABLE 5 Stages IN GAMETOGENESIS

Nucleus

Mitochondria


Spermatogenesis General Odgenesis (Stem cells) ¢ Primordial germ cells * Period of migration ~ Spermatogonia ’Gonia O&gonia Period of multiplication Spermatocytes ’Cytes. Odcytes Period of maturation coe Spermatids Gones (Sharp) Ovum and polocytes (Odtids) Period of metamorphosis Sperms ® 48 THE GERM CELLS

sperm enters the egg before the production of the second polar body. Sometimes the first polar body also divides so that -four cells (odtids) may be produced by the odcyte. .

Spermatogenesis. — The male ’cyte (primary spermatocyte) is a large cell containing a large vesicular nucleus, more or less excentric. Near the nucleus, and in the center of the thicker layer of cytoplasm surrounding it, are to be seen one or two centrosomes, surrounded by a clear substance known as the sphere substance. This compound body is known as the idiosome, and with it are often associated the Golgi bodies, sometimes so closely connected as to form an investing reticulum or even a shell. Around the idiosome are also grouped the mitochondria forming a cap which sometimes includes the nucleus as well.

The primary spermatocyte divides, giving rise to two secondary spermatocytes, which divide again, often without intermission, each forming two spermatids. The four spermatids thus produced from the primary spermatocyte are later transformed into the sperms.

During the two divisions mentioned above, the chromatin of the spermatocyte nucleus is distributed to the spermatids in such a way that they will differ from each other in respect to the nuclear contents. The details will be discussed later (page 64). The centrosomes divide at each cell division so that each spermatid has a centrosome. The Golgi bodies, each with a small amount of sphere substance, are divided among the four spermatids, in each of which they aggregate to form an idiosome. The mitochondria are divided with almost exact evenness among the spermatids, in each of which they assemble to form a paranucleus (nebenkern). A plasma membrane is present.

In the transformation of the spermatid into the mature sperm (Fig. 17), the nucleus, having previously extruded a large amount of material, condenses into a deeply staining mass which elongates into its final shape. The centrosome divides, and the two centrosomes take up a position which marks the posterior end of the future sperm, one centrosome (proximal) lying against the nucleus, the other (distal) posterior to the first. The paranucleus also takes a posterior position while the idiosome moves around the nucleus to the anterior end. The greater part of the cyto plasm is sloughed off. SEMINATION 51

Ovulation. — Within the ovary the vertebrate egg is surrounded by nurse cells which make up a nest or follicle (Fig. 19). Within this it enlarges and may undergo its first maturation division. Periodically, varying from once a year in most vertebrates to once a month in the human species, or daily in the domestic fowl, eggs are discharged from the ovary. In numbers this discharge varies from a single egg as in man or the fowl to thousands in the frog or millions in many fish.

The factors bringing about ovulation are diverse. In the frog it has been shown by Rugh! that ovulation is brought about by the contraction of a thin Fia. 19. — Transverse section through part of muscular layer in each fol- frog ovary. X95. licle, plus the action of an enzyme which digests the outer wall of each follicle and thereby weakens it. In mammals a follicular fluid is secreted about the egg, enlarging the follicle until it protrudes from the surface. Finally the outer wall of the follicle, now very thin, ruptures, owing perhaps to factors similar to those acting on the frog’s egg. It has been shown in many vertebrates that ovulation can be induced at any time by means of a hormone secreted by the anterior lobe of the pituitary gland (page 332).

From the ovary the eggs are caught up in the open end of the oviduct, down which they pass to the exterior. In many aquatic forms they are discharged directly. In others they accumulate in an enlarged portion of the oviduct known as the uterus, awaiting discharge from the body; such animals are known as oviparous (amphioxus, frog, chick). In still others the egg remains in the uterus until development has reached an advanced stage; these are the viviparous animals (man, etc.).

Semination. — This term is applied to the discharge of the sperms. These cells remain in the testis (Fig. 20) until mature,

1 Jour. Exp. Zool. In press.



Wall of ovary

Follicle cells Odcyte Oégonium often attached to nurse cells. When discharged they pass through tubules of the testis which lead directly to a sperm duct. They become motile upon reaching the medium in which fertilization takes place. Enormous numbers are produced at a single discharge (over 200,000,000 in man).

In aquatic animals such as the amphioxus and fish the two sexes congregate together at the breeding season, and eggs and sperms are discharged together. In some cases even aquatic animals have copulatory organs which introduce the sperms into the oviduct, bringing about internal fertilization. In the frog, the males and females unite in pairs (amplexus), thus ensuring that the sperms are discharged simultaneously with the eggs so that fertilization, although external, is regulated. In all terrestrial vertebrates fertilization is internal.


Fig. 20. — Section through part of frog testis. 200.

C. Fertilization

Fertilization. — The actual fertilization of the egg (syngamy) has been observed in the amphioxus and the frog, but our detailed knowledge of the process is obtained from the study of such marine invertebrates as the sea urchin. The essential act in fertilization is the entrance of a single sperm into the egg and the coming together of the two nuclei (pronuclei) (Fig. 21). But this phenomenon is preceded by other events concerned in bringing the sperm to the egg.

Attraction. — One of the factors believed to bring the sperm towards the egg is an attraction (chemotaxis) caused by the emission of some chemical substance by ‘the egg or the female sex organs. It is known also that sperms swim in a spiral path, and it has been suggested that when they come in contact with a solid object they remain in contact with it (thigmotaxis). If the spiral brings a sperm obliquely towards the egg the contact flattens out the spiral, causing the sperm to remain in contact without penetration. But if the sperm arrives at the egg in a radial direction penetration is facilitated. Lillie has shown that the sea-urchin egg emits a secretion (fertilizin), which brings about a temporary and reversible adhesion of the sperm heads in clusters (agglutination). Fertilizin is produced only after the egg is mature and before it is fertilized.

Penetration. — The sperm bores its way through the egg envelope but then apparently comes to rest against the plasma membrane. Meantime there appears at the surface of the egg a cone or even a long filament of cytoplasm which comes in contact with the sperm head. It then retracts drawing the sperm to the egg and engulfing it (Fig. 21A, B). Thereafter, and commencing at the point where the sperm head was engulfed, a thin layer of surface protoplasm is elevated to form what is known as a fertilization membrane (vitelline membrane?). In older days, when it was thought that the sperm bored its way into the egg, it was believed also that the fertilization membrane acted as a bar to other sperms. Apparently the elevation of the membrane is due to the secretion of some fluid from the egg, which decreases in diameter at the same time. Okkelberg describes a loss of 14 per cent in the volume of the egg of the brook lamprey. The formation of this membrane with its perivitelline fluid underneath marks the successful fertilization of the egg. For example, the fertilized frog’s egg will rotate within this membrane.


Fig. 21. — Diagram to show fertilization of the egg. A, fertilization cone. B, penetration path. C, female copulation path, and rotation of sperm head. D, male copulation path. E, cleavage path. F, first cleavage.


The pronuclei. — After the second maturation division, which does not take place in vertebrates until after the entrance of the sperm, the nucleus of the ovum (female pronucleus) is near the periphery at the animal pole, while the nucleus of the sperm (male pronucleus) is at the periphery near the point of penetration. The sperm head rotates 180° so that the male pronucleus now lies distal to the middle-piece containing the centrosome (Fig. 21C). The two pronuclei come together (Fig. 21B, C) by a route which may be analyzed into the following components: (1) the sperm penetration path, which is usually the radius of the egg at which the sperm entered; (2) the sperm copulation path, which is directed towards the point at which the pronuclei will meet and is often at a considerable angle to the penetration path; (3) the egg copulation path, along which the female pronucleus moves towards the meeting point; and (4) the cleavage path (Fig. 21E), along which the two pronuclei move to their final position on the egg axis, often slightly nearer the animal pole. The two pronuclei may unite to form a common reticulum, or they may remain close together contributing independently to the first division of the zygote (Fig. 21F). See also page 156.

The centrosome of the egg disappears after the second maturation division. The centrosome of the zygote, therefore, is either the centrosome of the sperm or, as it is believed in some cases, a new one developed in the egg cytoplasm near the engulfed sperm head.

The mitochondrial material of the sperm fragments and is distributed throughout the cytoplasm of the zygote. The later history of the acrosome has not been followed.

There is much divergence among different kinds of animals with respect to those parts of the sperm which are actually engulfed in the egg.- In some, it is the entire sperm; in others, only the sperm head.

Presumptive organ regions. — In many different kinds of eggs, the student of cellular embryology has been able to recognize different regions by differences in the cytoplasm, such as the presence or absence of pigment, mitochondria, yolk, etc., and to trace the distributions of these materials into the different daughter cells as cleavage takes place. These presumptive organ regions, as they may be called, are usually more easily demonstrated after fertilization. For example, before fertilization the living egg of the tunicate Styela (Cynthia), according to Conklin (1905), has orange pigment 6) Ground granules uniformly distributed protoplasm in its outer layer of cytoplasm. ais During fertilization, following an intricate series of stream movements, the orange pigment (Fig. 22) is concentrated in a crescentic area at what will later be the posterior surface. Immediately above this is a similar area of clear protoplasm. On the opposite side of the egg at @) Grey yolk what will become the anterior ,,, 22, — Presumptive organ regions surface is a gray crescent. Be- (organ-forming substances) in egg of low these crescents the vegetal Styela after fertilization, viewed from hemisphere is marked by the im) approx. X250. (After Conk: presence of gray yolk. In later cleavage, the yellow crescent will be distributed to the cells which form the mesoderm, the gray crescent to the cells which form the notochord and neural plate, the gray yolk to the cells of the endoderm, and the remainder of the egg goes to the cells of the epidermis. The materials contained in the different presumptive organ regions are frequently called organ-forming substances. The regions themselves are called presumptive endoderm, presumptive mesoderm, etc.

Parthenogenesis. — This term is applied to the development of a new individual from an unfertilized egg. It does not occur naturally among the vertebrates but may be illustrated by the honey bee, in which the unfertilized egg develops into a male 56 THE GERM CELLS

(drone) and the fertilized egg becomes a female (either queen or worker according to the type of food supplied).

Artificial parthenogenesis has been produced in the frog’s egg by slight punctures with a finely pointed glass needle. Most of the parthenogenetic eggs do not go far in development, but Loeb was able to raise, out of many thousands of treated eggs, a few adult frogs (15 males, 3 females, 2 doubtful).


FERTILIZATION OF THE AMPHIOXUS EGG. — In the case of the amphioxus, fertilization is external. The males and females leave the sands to swarm in the shallow waters during late afternoons of spring and summer. Eggs and sperms are discharged, from the segmental gonads in which they develop, into the cavity of the atrium, and escape to the exterior through the atriopore. The first polocyte is given off before fertilization. Immediately after fertilization the vitelline, now the fertilization, membrane expands greatly, leaving the egg in a large perivitelline space (Figs. 23 and 10A). The second polocyte given off after the formation of the fertilization membrane remains attached to the egg while the first is usually lost to view.


Fig. 23. — Presumptive organ regions in egg of the amphioxus, one hour after fertilization. Sagittal section, approx. X220. (After Conklin 1932.)


The fertilized egg of the amphioxus (Conklin 1932) shows in sections (Fig. 23) a crescent of more deeply staining protoplasm on the side of the egg which will give rise to the posterior part of the body. This crescent will form the cells of the mesoderm (compare the orange crescent of Styela). Opposite this is a less clearly defined crescentic area from which the cells of the notochord and neural plate will be formed (compare the gray crescent FERTILIZATION OF THE FROG’S EGG 57

of Styela and of the frog). The material of the vegetal hemisphere, bounded above by these crescents, will form the endoderm; the material of the animal hemisphere above the crescents will form the epidermis.

FERTILIZATION OF THE FROG’S EGG. — The fertilization of the frog’s egg is external, but the sperm are brought into close proximity to the eggs during the sexual embrace or amplexus. During the breeding season the males embrace the females with the fore-legs, at which time the germ cells of each are extruded. The sperms make their way through the egg jelly before this envelope has attained its final thickness. The entire sperm enters the egg usually within 40° of the apical pole. The vitelline membrane is thrown off as the fertilization membrane, leaving a perivitelline space within which the egg may rotate. The second maturation division then occurs, followed by the conjugation of the pronuclei. The penetration and sperm copulation paths are marked by a trail of pigment dragged in with the sperm head. A single sperm enters the egg. Immediately upon fertilization the cortical cytoplasm of the egg rushes towards the point of penetration, carrying with it the black pigment (melanin) of the animal hemisphere. Upon the side of the egg opposite the point of penetration there appears a crescent-shaped area in which the pigment is less dense and which is therefore known as the gray crescent (Fig. 24). The region gives rise to the notochord and neural plate.


Fig. 24. — Diagram of frog’s egg after fertilization to show gray crescent. The line immediately external to the vitelline membrane and polocytes represents the “chorion.” X10.


FERTILIZATION OF THE HEN’S EGG. — In the fowl, fertilization is internal. The sperms, introduced into the cloaca of the female during copulation, make their way to the upper end of the oviduct, where fertilization takes place. Five or six sperms enter the germinal disc, where they remain inactive until after the second maturation division. One of them then moves inward until it comes in contact with the female pronucleus, which has itself moved downward from the surface of the germinal disc. The supernumerary sperms move outward to the border of the disc, where, after a few divisions, they degenerate. The fertilized egg moves slowly down the oviduct while the tertiary envelopes are forming about it.



Fig. 25. — Fertilization of the guinea pig egg. Three stages following that shown in Fig. 20A. (After Lams.)

FERTILIZATION OF THE HUMAN EGG. — Fertilization is internal and occurs at the upper end of the oviduct (Fallopian tube). It is probable that a single sperm enters the egg after the first maturation division. Further details are lacking, as no direct observations have been r recorded. Figure 25 illustrates fertilization in the egg of the guinea pig.

Summary

The gametes are atypical cells, the egg and sperm differing both from each other and from a composite cell. The egg most resembles a composite cell, from which it differs in the absence of a centrosome after it has become mature. It is large, quiescent, and protected by envelopes. The sperm, almost devoid of cytoplasm, is small, active, and naked.

The gametes are derived from primordial germ cells which are first recognizable in the wall of the gut. Thence they migrate into the roof of the coelom where they multiply rapidly giving rise to the gonad. In the gonad multiplication continues until, when the individual is attaining maturity, some of the ’gonia enlarge to become ’cytes, each of which will undergo two meiotic divisions. The spermatocyte gives rise to four spermatids each of which will be transformed into asperm. The odcyte, on the contrary, gives rise to an ovum and two or three polocytes.

The zygote, or fertilized egg, arises from the union of an egg and a sperm. This union is preceded by the discharge of eggs from the ovary (ovulation) and sperms from the testis (semination). The sperm, attracted to the egg, enters it due to the mutual action of the two gametes, and the nuclei of the two gametes come together, each to contribute to the first division of the fertilized egg.

After fertilization, and sometimes even before, it can be seen that the egg has a definite organization, manifest in its polarity (seen even in ovarian eggs) and, in especially favorable material, evident in presumptive organ regions.

References

Allen, E. (ed.) 1932. Sex and Internal Secretions, Chap. 14.

Brachet, A. 1921. Traité d’embryologie, Book 1.

Cowdry, E. V. (ed.) 1924. General Cytology, Sections I, V-VIII.

Hertwig, O. (ed.) 1906. Handbuch, etc., I, Chaps. 1 and 2.

Jenkinson, J. W. 1913. Vertebrate Embryology, Chaps. 3 and 4.

Kellicott, W. E. 1913. General Embryology, Chaps. 2 to 5.

Kerr, J. G. 1919. Textbook of Embryology, I, Chap. 1.

Aorschelt, E., and Heider, K. 1902. Lehrbuch, etc. Chaps. 4 to 6.

Lillie, F.R. 1919. Problems of Fertilization.

Loeb, J. 1916. The Organism as a Whole from a Physicochemical Viewpoint.

Meisenheimer, J. 1921. Geschlecht und Geschlechter im Tierreiche. organ, T. H. 1927. Experimental Embryology.

Wilson, E. B. 1925. The Cell, etc. Chaps. 1-6, 9-12.

Sharp, L. W. 1934. Introduction to Cytology, Chaps. 1-16.

Chapter IV The Chromosomes And The Genes

The germ cells are really cells detached from the bodies of the parents. When they unite in fertilization they bring together material from both parents. Herein lies the explanation of the inheritance of parental characteristics, of the fact that the fertilized egg develops in a way characteristic of the species and the fact that individuals differ from one another. In the following paragraphs we shall review the theory that the individual units of heredity are the genes, borne in the chromosomes, distributed in the maturation divisions, and brought together in fertilization.

A. The Chromosomes

It will be necessary first to describe the chromosomes as they behave in ordinary (somatic) cell division, then to point out the peculiar features of the maturation (meiotic) divisions and of fertilization, and finally to indicate how this behavior of the chromosomes fits the known laws of heredity.


The chromosomes in mitosis. — The division of most cells is accompanied by the formation and longitudinal division of threads of chromatin, called chromosomes, in the nucleus. This type of cell division is known as mitosis (Fig. 26). Some cells, however, divide without the formation of chromosomes (amitosis), and the daughter cells are thereafter incapable of mitotic division. For the sake of convenience we may use the terms karyokinesis for the division of the nucleus in mitosis and- cytokinesis for the division of the cytosome.


Karyokinesis. — Before cell division the metabolic (“‘ resting ’’) nucleus is a reticulum of chromatin lying in the fluid karyolymph with a nucleolus, the whole surrounded with a nuclear membrane . (Fig. 26A). In mitosis we distinguish four stages, prophase, metaphase, anaphase, and telophase.

In the prophase the reticulum separates into its constituent threads, chromonemata, by the breaking down of the smaller threads connecting them. Very early it can be seen that these threads are double or split longitudinally (Fig. 26B). Soon thereafter a matrix is visible about the two chromonemata. This compound structure, consisting of the two chromonemata and the surrounding matrix, is a chromosome (Fig. 26C). The number of chromosomes so formed is the same in every cell of every individual belonging to any particular species. (This statement is subject to exceptions. See pages 69 ff.) Towards the end of the prophase the chromonemata are usually invisible.


Fig. 26. — Diagrams of somatic mitosis. A, metabolic (‘‘resting”’) stage. B, early prophase showing chromonemata and attachment points. C, middle prophase, matrix appearing. D, late prophase, chromonemata obscured. E, metaphase. F, anaphase. G, early telophase, matrix disappearing. H, middle telophase, nuclear membrane forming. I, late telophase, reticulum developing. (Based on a diagram by Sharp.)


Finally the nuclear membrane disappears, and the karyolymph assumes the form of a double cone or spindle (Fig. 26D).

In the metaphase (Fig. 26E), the chromosomes line up in an equatorial plane through the spindle. Each has a definite attachment region lying in the equatorial plane even though the ends of the chromosomes may lie outside of the plane.

In the anaphase (Fig. 26F), the chromosomes separate into two longitudinal portions each containing one of the original chromonemata with surrounding matrix. Preceded always by its attachment region each daughter chromosome moves towards a pole of the spindle. Carothers (1934) describes the growth of a fiber from the attachment region of each daughter chromosome to the nearest pole of the spindle. Eventually two equivalent sets of chromosomes are formed, one in the vicinity of either pole, each set containing a daughter chromosome from each of the original chromosomes formed in the prophase.

In the telophase (Fig. 26G, H, I), each set of chromosomes assumes the metabolic condition. The matrix loses its staining capacity and the chromonemata reappear, often already split longitudinally. The nuclear membrane is formed about each group, the chromonemata are united by tiny cross-strands, the nucleolus reappears, and the nucleus is seen to be filled with karyolymph. The cell now contains two daughter nuclei each identical with the other and with the parent nucleus.

Cytokinesis. — Other striking events are taking place in the cytosome during mitosis. During the prophase the centrosome, if not already divided, separates into two daughter centrosomes which move apart. About each of them is a spherical mass of protoplasm, often containing radial striations, known as the aster. Between them is a central spindle apparently containing fibers. Cytologists distinguish three types of fibers: (1) primary or continuous fibers extending from centrosome to centrosome, (2) half spindle components extending from chromosome to centrosome, and (3) interzonal connections extending between the separating daughter chromosomes (Schrader). The centrosomes reach the opposite sides of the nucleus just as the nuclear membrane disappears. The karyolymph apparently unites with the material between the two centrosomes to form the mitotic spindle along which the chromosomes move in the anaphase. In the telophase, asters and spindle disappear and the centrosomes alone remain in the positions they occupied at the poles of the mitotic spindle. Sometimes they divide in anticipation of the next mitosis.

The mitochondria usually divide en masse (Fig. 27A). This division of the mitochondria is approximately an equal one, and there is some reason to believe that the individual mitochondria divide during mitosis or just prior to it.

The Golgi bodies, even when aggregated into a Golgi apparatus, separate during mitosis and are segregated into the daughter cells, usually associating themselves with the two centrosomes (Fig. 27B). It is uncertain whether each Golgi body divides individually at mitosis, but some evidence has been brought forward to support this contention.



Fig. 27.— The mitochondria and Golgi bodies in mitosis. A, mitochondria. B, Golgi bodies. (After Bowen.)


In animal cells the cytosome as a whole divides by construction. In this process the cell elongates in the direction of the spindle during the anaphase and telophase. Following the reconstruction of the daughter nuclei in the telophase, a furrow appears at the periphery of the cell, around the equatorial belt, and at right angles to the axis of elongation. This furrow advances towards the center of the cell until the cell is completely divided.

Distribution of the chromosomes. — Each daughter cell has approximately half of the cytoplasm proper, half of the mitochondria and Golgi bodies, a centrosome derived from that of the 64 THE CHROMOSOMES AND THE GENES

parent cell, and a nucleus built up from a set of chromosomes, each of which was produced by the division of a chromosome in the parent cell. It is apparent from the foregoing account that the key to the complexities of mitosis is the division of the chromosomes. The achromatic figure is the framework upon which this division takes place. The division of the mitochondria and Golgi bodies is still too little understood. But the chromosomes, appearing in the prophase, halved with such accuracy in metaphase and anaphase, and disappearing again in the telophase, are characterized by a constancy in number, an individuality evinced in form and behavior, and a persistence from generation to generation. In some favorable material it has even been possible to demonstrate that the chromonemata arise in the prophase exactly as they merged into a reticulum in the previous telophase. From the statements above, it is not unreasonable to draw the conclusion that the chromosomes are directly concerned with inheritance in cell reproduction.

The chromosomes in meiosis. — During the two maturation divisions by which the gametes are formed, the number of chromosomes is reduced to one-half the number characteristic of the species. Since in the ordinary somatic mitosis the number of chromosomes given to each daughter cell is exactly the same as that of the parent, it is evident that we are dealing with a peculiar type of mitosis (Fig. 28). The name meiosis is frequently applied to the maturation divisions.

First meiotic division. — The essential feature in which the first meiotic division differs from the ordinary (somatic) mitosis is that during the prophase the chromosomes unite in pairs (Fig. 29, 2). This is synapsis and occurs only in the first meiotic division. Since each of the chromosomes always divides during the prophase also (Fig. 29, 4), the net result is that at the end of the prophase there are only half the number of chromosomes seen in somatic mitosis, but each of these consists of four parts (chromatids) instead of two (Fig. 29, 5). These compound bodies consisting of four chromatids are called tetrads (Fig. 29, 6). The quadripartite nature of the tetrad may be expressed by the formula 42 Tn where A represents one of the synaptic mates and an the other.

DIAGRAM COMPARING SOMATIC MITOSIS WITH THE MEIOTIC MITOSES

First columns Equational fo division of a diploid somatic ° chromosome complement,

Second column: The meiotic be ABe divisions, changing the diplaid °

to the monoploid state.


Fig. 28. — Comparison of somatic and meiotic mitosis. (From Sharp.) (5) 66 THE CHROMOSOMES AND THE GENES


Fig. 29.— Diagram of meiosis. 1, first meiotic division, prophase, (leptonema stage). 2, do. showing synapsis (zygonema stage). 3, do. showing thickening of the chromosomes (pachynema stage). 4, do. showing forma tion of tetrads (diplonema stage). 6, do. showing condensation of matrix (diakinesis stage). 6, metaphase I. 7, anaphase I showing dyads. 8, telophase I. 9, second meiotic division, prophase showing dyads united at attachment points. 10, metaphase IT. 11, anaphase II showing the separation of the chromatids which composed the dyads. 12, telophase II. Each of the four germ cells now has the haploid number of chromatids (chromosomes). (From Sharp.) DISTRIBUTION OF THE CHROMOSOMES 67

In the anaphase (Fig. 29, 7), the daughter chromosomes each possess two chromatids and are known as dyads. But there are two different ways of dividing a tetrad. In one case the two chromatids derived from one of the synaptic mates (4) might be separated from those derived from the other mate (2) in a reduc tion (disjunction) division. In the other, each dyad might contain one chromatid from each of the synaptic mates (A : a) as the result of an equation division.

The telophase (and prophase of the second meiotic division) sometimes is omitted if the second division succeeds the first immediately.

Second meiotic division. — If these omissions take place, each of the daughter ’cytes divides immediately, the chromosomes, still in the dyad condition, lining up on the spindles for the metaphase of the second meiotic division. But even if the telophase of the first and prophase of the second meiotic divisions are not omitted (Fig. 29, 8), it is obvious that the chromosomes arising in the prophase (Fig. 29, 9) are dyads and that they undergo no other longitudinal split. The anaphase of the second meiotic division (Fig. 29, 11) merely separates the two chromatids of each dyad from each other. The final result is that the four cells produced by the meiotic divisions (Fig. 29, 12) each have one chroma* tid from each tetrad or one-half the number of chromosomes found before meiosis took place. This is expressed in another way by saying that the number of chromosomes has been reduced from the diploid to the haploid (monoploid) number.

Here we must note that it makes no difference whether the first meiotic division divided a tetrad reductionally or equationally. The second division always distributes the two chromatids of each dyad into different cells. Each of the four daughter cells has one chromatid from each tetrad, and therefore one representative fro either one of the two synaptic mates (A or a), but not from both!

Distribution of the chromosomes. — As each tetrad orients itself independently upon the spindle it is evident that it is a matter of chance which half of a tetrad, or of a dyad, goes to either pole of the spindle. Accordingly, if we had eight chromosomes, A, a, B, b, C, c, D, and d, these would unite in synapsis to form four double chromosomes, Aa, Bb, Cc, and Dd. These A:a B:6b C: D:d would form the four tetrads, A: 7 Bb’ CO: s, and Ded’ Fol


lowing the two meiotic divisions (equation and reduction, regardless of their order), the mature germ cells would have four chromosomes (the haploid number), but only one representative of each synaptic pair. The possible combinations are 2‘ or 16, namely, ABCD, ABCd, ABcd, Abcd, ABcD, AbCD, AbCd, aBCD, aBCd, aBcd, abcd, aBcD, abcD, abCD, and abCd (Fig. 30).

Fig. 30. — Showing the distribution of the chromosomes in fertilization and the following meiotic divisions. (After Wilson.)

Accordingly the number of different types of gametes which may be formed can be determined from the formula 2” when n is the haploid number of chromosomes characteristic of the species.

The chromosomes in fertilization. — Evidently when the egg and sperm unite in fertilization, the pronucleus contributed by each contains the haploid number of chromosomes. In this way © the diploid number characteristic of the species is restored. It is obvious that, unless the number had been reduced by meiosis, it would be doubled in each new generation.

In the second place, it is clear that each germ cell contributes a homologous set of chromosomes, and that in synapsis the chromosomes unite in homologous pairs. In the example referred to SEX CHROMOSOMES, X-O TYPE 69

above, chromosomes A, B, C’, D came from one parent and a, b, c, d came from the other. We can now visualize each synaptic pair as consisting of one paternal and one maternal chromosome.

During meiosis the paternal and maternal chromosomes are sorted out into different assortments in the different germ cells. During fertilization these different assortments are brought together in random recombinations. We have said that in an animal with 8 chromosomes we might have 2‘ or 16 different classes of gametes. In random fertilization this number would be squared, so that there would be 4‘ or 256 possible combinations. Many of these would be duplicates, so that the exact number of different classes of zygotes according to their assortment of chromosomes would be 34 or 81.


Fig. 31. — Chromosomes of Protenor. A, A’, male diploid group. B, B’, female diploid group. The X-chromosomes are indicated by X. (After Wilson.)


Sex chromosomes, X-O type. — In many animals, such as the insect Protenor, the male has one chromosome less than the female, the numbers in Protenor being 13 and 14, respectively (Fig. 31). If the synaptic pairs are assembled, it is clear that the male has six pairs of ordinary chromosomes (autosomes) and an extra one, the X-chromosome. The female has six pairs of autosomes and a pair of X-chromosomes. In the female the chromosomes unite in synapsis, form a tetrad, and are segregated in the meiotic divisions so that every egg has a complete set of autosomes and one X-chromosome (A + X). In the male, on the other hand, the single X-chromosome has no synaptic mate and so goes on the spindle of the first meiotic division as a dyad, which is carried to one pole of the spindle entire. In the second meiotic division the dyad is divided as usual. The end result is that only half the spermatids receive an X-chromosome, and two classes of sperms are formed, either with or without an X-chromosome (A + X or A +0). If a sperm with an X-chromosome fertilizes the egg, the female combination (2A + 2X) is restored.


Fig. 32. — Diagram showing history of the X-chromosome during meiosis and fertilization. (After Wilson.)

If a sperm without the X-chromosome penetrates the egg, the male combination (2A + X) is formed (Fig. 32).

Sex chromosomes, X-Y type. — But the sexes do not always differ in chromosome number, for in many animals, like the insect Lygaeus (Fig. 33), the X-chromosome of the male is furnished with a synaptic mate which differs from it in size, form, and probably composition, and is therefore known as the Y-chromosome. The male forms a tetrad ae and the sperms therefore have either an X-chromosome or a Y-chromosome. Fertilization by a sperm bearing the X-chromosome results in the development of a female (2A + 2X), whereas if a sperm bearing a Y-chromosome enters the egg the embryo will give rise to a male (2A + XY).

Sex chromosomes, W-Z type. — As an exception to the general rule among the vertebrates, the birds have dissimilar sex chromosomes in the female. The cytological details are difficult to interpret but the theoretical explanation is that the female has two dissimilar sex chromosomes known as W and Z, while the male possesses two similar sex chromosomes of the Z type (Fig. 34B). In the meiosis of the odcyte, therefore, a tetrad wis is formed, and the ovum receives either a W-chromosome or a Z-chromosome. The spermatocyte forms a tetrad a and all sperms carry one Z-chromosome. In this group, therefore, it is the ovum which determines the sex of the embryo rather than the sperm. This explanation agrees with the data obtained from genetics.


Fig. 33. — Chromosomes of Lygaeus. A, A’, male diploid group. B, B’, female diploid group. X and Y indicate the X- and Y-chromosomes respectively. (After Wilson.)


CHROMOSOMES OF THE AMPHIOxuUS.— The diploid number is 24,

CHROMOSOMES OF THE FROG.— The diploid number is 26, and the sex chromosomes of the male are of the X-Y type (Fig. 34A). 72 THE CHROMOSOMES AND THE GENES

CHROMOSOMES OF THE CHICK. — The diploid number is 35 or 36. The sex chromosomes have not been positively identified, but genetic evidence indicates that the sex chromosomes of the female are of the O-Z or*the W-Z type (Fig. 34B).

CHROMOSOMES OF MAN. — The diploid number, according to the most recent researches, is 48. The sex chromosomes are of the X-Y type (Fig. 34C). It is interesting to note that with 48 chromosomes the possible types of oe number 274


Fig. 34. — Metaphase plates of male diploid chromosome groups. A, frog (after Witschi). B, chick (after Hance). C, man (after Painter).

or 16,777,300, and that from these 3% zygote-recombinations are possible.

B. The Genes

It has already been said that the behavior of the chromosomes itself might suggest that these bodies are concerned with the transmission of hereditary characters. We shall now turn our attention to the laws of heredity as worked out by plant and animal breeders and learn how the data of genetics agree with the data of cytology.

The unit of genetics is the gene. These genes are arranged in linear order in the chromosomes, presumably bound together by the chromonemata, and possibly identified with the chromomeres. They exist in great numbers; in the fruitfly Drosophila it is estimated that there are between 2000 and 3000. Ordinarily ultramicroscopic, it has been reported recently by Belling (1930) and by Bridges (1934) that they have been able to identify these units in material of exceptionally favorable nature. The genes are known by the effects their presence induces, and named according to the most obvious of these effects. Thus the Drosophila SEGREGATION 73

has a gene for (or a gene which induces among other effects) the normal type of wing. But there have arisen, among the millions of fruitflies raised by geneticists, some with abnormal types of wings, such as a vestigial wing. In thisease there is said to be a gene for (or a gene which induces among other effects) the vestigial type of wing.

Dominance. — Among the original discoveries of Mendel was the fact that, if two organisms with alternative characters were mated, the offspring would show either one or the other of the characters concerned. This is known as the law of dominance. When a Drosophila with normal wings is mated to one with vestigial wings, all the offspring have the normal type of wing (Fig. 35). Therefore the gene for normal is said to be dominant to the gene for vestigial, which, conversely, is said to be recessive to the gene for normal.

It is customary among geneticists to use the initial letter of the name for the abnormal character as a symbol for the gene inducing its appearance, as well as a symbol for the gene inducing the alternative (allelomorphic) normal character. The two are distinguished by using a capital letter for the dominant gene, a lower-case letter for the recessive gene. In this case, then, the symbol of the gene for vestigial is v, and the symbol of the gene for normal is V.

Every adult has two haploid sets of chromosomes, and therefore a pair of every kind of chromosome. If both members of a pair have the same gene (w or VV) they are said to be homozygous; but if one chromosome has the dominant, and the other has the recessive gene (Vv), they are said to be heterozygous.

The individuals that are mated together in-the first instance are known as the parental generation (P,); their offspring are known as the first filial generation (F1); the next generation is the second filial generation (Ff); and so on.

Segregation. — In the experiment where a normal long-winged fly was mated with a vestigial-winged fly, the long-winged parent must have had two chromosomes each containing the dominant gene V, for all the offspring (F'1) showed this dominant character. The vestigial-winged parent must have had two chromosomes containing the recessive gene v. In the maturation of the gametes all the sperms received V, while the eggs all received



Fig. 35. — Diagram to show the effects of crossing two flies differing in respect to one pair of genes. V is used for the dominant gene for the normal character long wings; v is used for the recessive mutant gene for vestigial wing. (From Curtis and Guthrie, after Morgan et al.)

The F, flies have the genetic constitution Vv, that is to say, they are heterozygous. When they are mated to each other the eggs will receive a chromosome containing the gene V or the gene v, and the same is true of the sperm.


The F, flies will consist of three genetic groups (genotypes) because of random fertilization, namely, homozygous long-winged flies (VV), heterozygous long-winged flies (Vv), and vestigialwinged flies (wv). From Fig. 36, it will be seen that the ratio will be one homozygous long-winged fly to two heterozygous long-winged flies, and to one vestigial-winged fly. Or one may say that there are two recognizable classes of adults (phenotypes), in the ratio of three long-winged flies to one vestigial-winged fly. This is the famous Mendelian ratio applied to the inheritance of one pair of Gametes as allelomorphic characters, or as we should say today, to one pair of genes.


Fig. 36. — Diagram to show the segregation of the genes caused by the distribution of the chromosomes to the gametes and zygotes of the Fi and F; generations. Y, and v as before.




Evidently Mendel’s law of segregation may be stated in terms of the gene theory as follows: allelomorphic genes are segregated during maturation into different gametes.

Assortment. — It is an amazing coincidence that Mendel studied the inheritance of seven pairs of allelomorphic characters in 76 THE CHROMOSOMES AND THE GENES

the edible pea, a species which has seven pairs of chromosomes, and that the genes for each pair of characters were located in a different pair of chromosomes.

When a Drosophila with vestigial wings and normal gray body color is mated to a fly with normal long wings and ebony body color, the F, flies are gray-bodied and long-winged. Evidently the gene for gray body (£) is dominant to the gene for ebony body (e). That the genes for these characters are independent of those affecting wing length is shown when the hybrid F, flies are mated together. Four classes of phenotypes result in the F, generation: 9 long-winged, gray-bodied; 3 long-winged, ebonybodied; 3 vestigial-winged, gray-bodied; and 1 vestigial-winged, ebony-bodied. This ratio of 9: 3:3 :1 breaks down to 3 long to 1 vestigial, and 3 gray to 1 ebony, demonstrating mathematically that two pairs of factors are involved.

It is evident that the problem involves the segregation of two pairs of chromosomes (Fig.37). The genetic constitutions of the P; flies were wHE and V Vee, respectively. The gametes receive one chromosome from each pair of synaptic mates, so the genetic constitution of the eggs is v# and that of the sperms Ve (or vice versa).

The F;, flies have the formula VvKe, and their gametes, because the chromosome pairs are assorted independently, will belong to four classes: VE, Ve, vE, and ve.

The F, flies as seen from the checkerboard diagram will fall into 16 combinations, which by canceling the duplicates reduce to 9 genotypes (VVEE, VVEe, VvEE, VuEe, VVee, Vee, wKE, vvEe, and vvee), and 4 phenotypes as listed above.

Mendel’s law of assortment may be phrased in terms of gene theory as follows: different pairs of allelomorphic genes when located in different pairs of chromosomes are assorted independently during maturation into different gametes.

It may be noted that if n stands for the number of pairs of genes located in different pairs of chromosomes, then 2” represents the number of gamete classes formed by the F: generation; 2" represents the number of phenotypes in the F, generation; 3” the number of genotypes in the F, generation; and 4" the number of combinations in the Punnett square. The number of individuals in each phenotype is obtained by expanding the 3 : 1 formula as follows: (8:1), (9:3:3:1), (27:9:9:9:3:3:3:1)


Zygotes from which P, developed

Gametes of P,


to long-winged, gray~bodied flies

©) Zygotes of F, that give rise

a C<_)

axe |(HB)|(HO)| (HH) | (HO






VVEB VVEe VvEBD VvEe Zygotes of F, long long long long gray . gray gray gray 9 long-winged, VVEe VVee VvEe Vvee one veth ed ©) long long long long gray" bo: : gray ebony gray ebony 8 long-winged, bony-bodi VvEE | vVvEe wEE vvEe ebony-bodied long long vestigial vestigial 8 vestigial-winged, gray gray gray gray gray-bodied VvEe ‘Vvee vvEe vvee 1 vestigial-winged, ly long long vestigial vestigial ebony-bodied gray ebony gray ebony







Fig. 37. — Diagram to show the assortment of two pairs of genes due to the distribution of two pairs of chromosomes. E, gene for gray body; e for ebony body; V, and v as before. (From Curtis and Guthrie.)


Linkage. — The characters with which Mendel worked segregated freely, showing that their genes were not borne in the same chromosome. Later studies have shown that some characters do not segregate, and this leads to the assumption that their genes are carried in the same chromosome and therefore are inherited together.

When a Drosophila with gray body color and long wings is mated to one with black body color and vestigial wings, the F1 flies are gray-bodied and long-winged. Note that the gene for black (6) will act very differently from the gene for the similar color ebony (e). If the F; flies are bred together a very confusing ratio appears in the F, generation: practically all the flies are gray-bodied and long-winged or black-bodied and vestigial-winged like the P, generation, but there are only a few individuals representing the other classes we might expect under Mendel’s law of assortment. If we make a reciprocal cross between a long-winged black-bodied fly and a vestigial-winged gray-bodied fly, the F, flies are all of these two (P;) types with few exceptions. This continued association of two genes through several generations is called linkage and suggests that the associated genes are located in the same chromosome.

This theory may be tested by back-crossing (Fig. 38) a male of the F; generation (BbVv) to a double recessive female (bbw). All her eggs will have the recessive genes (bv). We can then test the constitution of the sperm by examining the progeny of this cross (here called F, for convenience), for all the F, flies must have the genes (bv) from the mother. The flies of this generation are either gray-bodied and long-winged (BbVv) or black-bodied and vestigial-winged (bbvv). This seems to show that the genes B and V were located in one chromosome while b and v were located in the synaptic mate.

Crossing over. — Now for the exceptional (cross-over) flies noted above. There is no crossing over in the maturation of the male F, fly, but how about the female? When we mate (Fig. 39) a female F, fly (BbVv) to a double recessive male (bbvv), the progeny (F,) fall into four classes: 41} per cent gray-bodied and long-winged (BbVv), 413 per cent black-bodied and vestigialwinged (bbvv), 84 per cent gray-bodied and vestigial-winged (Bbvv), and 83 per cent black-bodied and long-winged (bbV»).


Obviously there has been an exchange of some sort between the chromosomes of the female F, fly. Both cytological and experimental evidence seem to indicate that this crossing over takes place in the prophase of the first meiotic division (Fig. 29, 4). Although there are still difficulties in determining exactly how the crossing over takes place between the four strands, it is generally agreed that the actual crossing over takes place between two of them. The idea of linkage between genes in the same chromosome suggested the idea that the genes form a longitudinal series in each chromosome. This is supported by the behavior of the chromosomes in ordinary somatic mitosis, in synapsis, and in crossing over (Fig. 40).


Fig. 38. — Diagram to show the inheritance of two pairs of genes when located in one pair of chromosomes, (linkage). In this case the male /; fly is back-crossed to a double recessive female. B, gene for gray body; }, for black body; V, and v as before. (After Morgan.)



Fig. 39. — Diagram to show the inheritance of two pairs of genes when located in one pair of chromosomes between which crossing over takes place. In this case the female F; fly is back-crossed to a double recessive male. Symbols as in Fig. 38. The figures at the bottom of the illustration indicate the percentage of each phenotype in the entire hatch. (After Morgan.)


Finally Sturtevant (1913) suggested that the percentage of crossing over between two pairs of linked genes might represent a function of the distance between the loci of the genes in the chromosome. Accordingly, maps have been constructed, by © Morgan and his co-workers, on the general assumption that one per cent of cross-overs is represented on the map by a distance of one unit between the genes involved. Without going into further details of the methods used in constructing these maps, for there are many complicating factors, a glance at the accompanying chart (Fig. 41) will show the progress that has been made in this direction.


Fig. 40. — Diagram to illustrate: A, splitting of a chromosome in somatic mitosis; B, union of two chromosomes in synapsis; C, union of two chromosomes in synapsis accompanied by crossing over. (After Wilson.)


Sex-linked inheritance. — One of the most striking evidences that genes are borne in the chromosomes is afforded by what is known as sex-linked (criss-cross) inheritance. This is illustrated in Drosophila by the inheritance of white eye color, an allelomorph of red, the normal eye color. If a white-eyed male is mated with a red-eyed female (Fig. 42), the F, flies of both sexes will have red eyes. But if these F, flies are bred together the F, generation will be made up of red-eyed females, (50 per cent) red-eyed males (25 per cent), and white-eyed males (25 per cent). It looks at first like an ordinary 3 to 1 Mendelian ratio, except for this curious distribution of eye color in the two sexes. 100) a m 3 eltow (B) 7 0 telegraph(W) 7 0. roughoid (E) ¥ Wes airy win 2. Star (E) | t ‘ vhs Pethet cay T 34 aristaless (8) F So broad (W) + 6.4 expanded (Ww) + |. prune Ce) : fT. (8 white(E) Ve facet te) 4 12+ Gull WW) \ 134 Notch CE Yt Truncate (W) 45 Abnormal(B) + !4.t dachsous (B) T.\55 echinus(E) +416. Streak () +\ 169 bifid (W) Ty 75 ruby CE) SB eres 0) Tt clu + 1 + N72 Geltex cw) 20. divergent (w) 20. cut (W) 21. singed (H) aot @) 275 tan (B) + 3. achs (8 4 26. sepia CE F 217 lozenge (E) yT 265 hairy & T 35. Ski- I (w) T 33. vermillion(E) 361 miniature(w) +41. Jammed(W) 4 35. rose (CE) + 362 dusky (W) + 362 cream-M (E) J 38% furrowed (E) 1464 Minute-e (H) 401 Minute-h (H) 148.5 black (B) $ 402 tilt (W) + 43. sable @) 48.1 jaunty (Ww) 404 Dichaete (H) + 444 garnet (E) 422 thread (8 _T 546 purple (Ee) + 44. scarlet () 5 4575 cinnabar(E)~. 48. pink (E) 4.2 small win . 44 + 545 redmentangtW) 02 safranin(E) Ff, sn maroon (e) i 366 forked a BO. curled (W) F 57. Ba . . L ¥ be srall ° 7 O44 pinkwingw) T B48 Bairy win supr r 59. fuse F 58.5 spineless(H) 4d 596 +67. vestigial(W) = P 62. Beadex CW) + 68+ telescope W) F..587 bithorax (B) | “59.5 bithorax-b + 65. cleft (W) die Lobe Ce) tT. 62. stripe (B) - hope | 63.1 glass C) | 474% gap Ww) 662 Delta WW) a> +4 10. bobbed(H) +755 Curved (w) 4+ 695 hairless CH) + 107 ebon F 337 peony 4d ‘757 cardinal (E) 4835 fringed) T 76.2 white ocelli ) —>! +90. x \ “ 9 humpy (8) + 914 rough @) v 4+ 995 arc (W) 4 93. crumpled Ww) + 1005 lexus Ww) + 938 Beaded Ww. T 102+ fethal-ta 94.1 Painted (W) I 05. brown (E mW 05+ blistered(w) | 1007 glare ©) « 4 106. purpleoid, {e) F101. Minute CH) gid Teeckt) spec F075 balloon ew) + 106.2 Minute-g (H)

Vv

bent (W) shaven (B) eyeless (E) rotated (8) Minute-I¥ (H)


Fig. 41.— The chromosomes of Drosophila melanogaster and map showing the positions of many genes as determined from cross over ratios. theses indicate part of body affected: B, body; E, eye; H, hair; W, wing: -’ Arrows indicate position of attachment point. exact position of genes in Y still undetermined.

1925 and Stern 1929.)

Letters in paren All genes in IV are closely linked. The (From Sharp after Morgan et al.

(82)


In the reciprocal mating, a red-eyed male to a white-eyed female (Fig. 43), the F: generation is made up of red-eyed females


Fig. 42. — Diagram to show the inheritance of one pair of genes when located in the X-chromosome (sex-linkage). W, gene for red eye (dominant, normal); w, gene for white eye (recessive, mutant). The empty hook-shaped chromosome represents the Y-chromosome. N. B. In the text the X-chromosome, when bearing w, the gene for white eyes, is designated by a small x. In this cross, red-eyed female is mated to a white-eyed male. (After Morgan et al.)

and white-eyed males (criss-cross inheritance). When these F, flies are bred together, there are four classes of flies in the F, generation: red-eyed males and females, and white-eyed males and females (25 per cent in each class). This is not a Mendelian ratio, but it can be explained on the assumption that the gene for white eye color (and its allelomorphs, of which there are several) is located in the X-chromosome.


Fig. 43. — The reciprocal cross to that shown in Fig. 42. A white-eyed female is mated to a red-eyed male. Symbols as in Fig. 42. (After Morgan et al.)


Let us use the symbol X for an X-chromosome bearing a gene for red, x for an X-chromosome bearing a gene for white, and Y for the Y-chromosome. In the first genetic experiment, formulas for the parental generation are X X (red female) and x Y (white male). All the eggs receive an X, the sperms either SEX-LINKED INHERITANCE 85

zor Y. Consequently the Ff: generation is made up of flies with the formula Xx (heterozygous red female) and XY (red male). The eggs of this generation receive X or x, the sperms X or Y.

A

Two pairs of homologous chromosomes showing positions of allelomorphic genes.

uae Re Sab

a

Crossing over: The chromosomes of the pair shown in A may twist about one another as in C and break in the plane of the dotted line so that comparable sections are exchanged as shown in D.

ac wR bank oe Ob bath

RQ

Deletion: One member of the chromosome pair shown in A may twist on itself as in K and break in the plane of the dotted line so e that an internal section containing gene c is lost, or deleted, as shown in F.

~ bab >

ae 9 Qa 86 >

Inversion: One member of the chromosome pair shown in A may twist on itself as in G and break in the plane of the dotted line so that the section containing genes B and C is inverted as shown in H.

bs &Q >


a 8° &@ &

Duplication and Deficiency: If one member of the chromosome pair shown in A comes to lie across the other as shown in I and a break occurs in the plane of the dotted line, the chromosome on the left in J will have a duplication and contain both gene d and gen D, while the chromosome on the right will have a deficiency of the section contain- I J ing gene D.

tea a @& Q

Translocation: One member of the chromosome pair shown in A may come to lie across one member of the chromosome pair shown in B, as seenin K. If a break occurs in the plane of the dotted line, sectiotis of non-homologous chromosomes are exchanged, or translocated, as shown in L. K L


Fia. 44.— Diagrams to show crossing over and various chromosomal aberrations. (From Curtis and Guthrie.)


So the F, generation is composed of flies with the following combinations: XX (homozygous red females), Xx (heterozygous red females), XY (red males), and xY (white males).

In the other experiment the parental formulas are xx (white female) and XY (red male). The eggs receive an x, the sperm X or Y. Hence there are two classes in the F; generation, xX (heterozygous red females) and xY (white males). The eggs receive either an x or an X, the sperms receive either zor Y. The four combinations possible in the F, generation are xX (heterozygous red female), xx (white female), xY (white male), and XY (red male).

Chromosomal aberrations. — Crossing over takes place between the two X-chromosomes, but apparently not between the X-chromosome and the Y-chromosome in Drosophila. We have already noted the fact that in this little fruitfly crossing over does not take place in the male.!' But crossing over by no means exhausts the possibility of effecting new combinations of genes by the behavior of the chromosomes during the maturation of the germ cells. Exact as the mechanism of meiosis may seem, many possibilities of disturbance have been discovered by genetic and cytological methods.



Fig. 45. — Diagrams showing I, normal disjunction of X-chromosomes in odgenesis, and fertilization by two types of sperms; II, non-disjunction, both X-chromosomes remaining in egg; III, non-disjunction, both X-chromosomes passing to polocyte. A, one haploid set of autosomes. (From Curtis and Guthrie.)


1 There is some recent evidence to show that such crossing over can be induced by high temperatures.



The accompanying diagram (Fig. 44) illustrates graphically some of the aberrations which may take place during meiosis. These result in the appearance of unexpected individuals with new combinations of genes ,


Fia. 46. — Intersexes and supersexes in Drosophila, occurring in the progeny triploid females. A, female-type intersex. B, male-type intersex. C, superfemale. D, supermale. a, b, and c are the chromosome groups characteristic of A, B, and C respectively. ‘ (From Curtis and Guthrie, after Morgan et al.)

Non-disjunction. — A special type of chromosomal aberration is one in which the two members of the synaptic pair may fail to separate during the meiotic divisions, so that one egg receives, for example, two X-chromosomes (A + 2X), while another receives none (A) (Fig. 45). When fertilized by a sperm with an Xchromosome, the egg with two X-chromosomes, if it develops into an adult, will be a superfemale (2A + 3X) differing markedly from her sisters (Fig. 46C). When fertilized by a sperm with a Y-chromosome, the egg without any X-chromosomes (2A + Y) dies. The other possible combinations are shown in the diagram.


In some cases all the chromosomes fail to disjoin so that an egg receives a diploid set of chromosomes (2A + 2X). When fertilized by an A + X sperm it becomes a triploid female (8A + 3X). The eggs formed by these triploid females may have the formula 2A + XorA+2X. If an egg of the first type (2A + X) is fertilized by sperm carrying an X-chromosome (A + X), the zygote will have the formula 3A + 2X. Such a zygote develops into an abnormal fly known as an intersex (Fig. 46A), male in some respects and female in others. Superfemales (2A + 3X) may also arise from the egg of the second type (A + 2X) being fertilized by an A + X sperm. Supermales (3A + XY), on the other hand, arise from the fertilization of a 2A + X egg by an A + Y sperm (Fig. 46D). It would appear from these formulas as though the determination of sex depended on some sort of ratio between the genes in the X-chromosomes and the autosomes, and Bridges (1921) has formulated a theory of genic balance to account for the observed results.

Gynandromorphs. — Intersexes must not be confused with gynandromorphs, which are individuals with one part of the body male and the rest female. Bilateral gynandromorphs in Drosophila (Fig. 47) arise from female zygotes (2A + 2X), but during

nat the first cleavage division one of the X


Fig. 47. —Gynandromorph_ ghromosomes is lost on the mitotic spindle. in Drosophila melanogaster. Note eosin eye and the result is that one of the daughter cells miniature wing on right has the female complex (2A + 2X) while as compared to red eye the other has the male complex (2A + X).

and long wing on left. . °

This fly is male on the S0Metimes such an aberration takes place

right side and female on in a later cleavage division so that there

the left. (After Morgan

ig only a small area of male cells.

and Bridges.) Teratology. — All students of embryology are familiar with the fact that development does not always proceed normally. Abnormal embryos are known as monsters, and their study forms the subject matter of the embryological subscience known as teratology. It is clear from the sections just preceding that many of these monsters must be due to chromosomal aberrations with consequent disturbance of the genic balance. Others, as will be noted in Chapter VII, are due to environmental factors.

Mutations. — So far we have considered the genes as though they were immutable. But the question naturally arises as to the origin of the genes which are allelomorphic to the so-called normal genes. In Drosophila the abnormal genes, or mutants as they are called, arose in laboratory cultures. It has been discovered that the rate of mutation, i.e., the number of mutants arising in a given number of flies, may be increased by high temperatures (Plough) and by irradiation (Miller). When one of these genes is altered in any way to become a mutant, the course of development is disturbed. Most mutant genes disturb the course of development so greatly as to cause death (lethal mutants). A smaller number produce visible changes when present in each chromosome of the synaptic mates (recessive mutants). A few produce visible changes if contained in a single chromosome (dominant mutants). Accordingly, every species of animals contains a certain number of mutant genes (400 in Drosophila). As these enter into new genetic combinations according to the behavior of the chromosomes in meiosis and fertilization, they give rise to individual differences in development. But the greater number of stable or non-mutant genes holds development true to the specific type.

One of the outstanding problems in experimental embryology still awaiting solution is the question how the genes actually determine the course of development. But the modern student of embryology accepts the general theory that it is the complement of genes, from the egg and sperm respectively, which initiates and largely controls the development of the individual.

Summary

The egg and sperm are the material contributions of the parents to the new individual. The equivalent structures of the egg and the sperm are their nuclei. Each nucleus contains the haploid number of chromosomes. The fertilized egg has two haploid sets, or the diploid number. In somatic mitosis the chromosomes are split longitudinally and divided equally among the daughter 90 THE CHROMOSOMES AND THE GENES

cells, so that each daughter cell contains an assortment precisely equivalent to that of its sister cell and the mother cell. In the course of the meiotic divisions the diploid number of chromosomes is reduced to one haploid set. This is accomplished through the union of the homologous members of the two sets in synapsis. Each synaptic pair forms a tetrad of four chromatids, the members of which are distributed independently among the mature germ cells. In this way different classes of gametes are formed with varying chromosomal complexes.

The chromosome is built up from a thin thread, the chromonema, which binds together the genes, the units of heredity, provisionally located at nodes of the chromonema called chromomeres. These genes, ordinarily ultra-microscopic, are self-reproducing units which seem to accelerate definite chemical reactions without losing any of their own substance in the process. The course of development is largely controlled by the activities of these genes. These activities may be disturbed during meiosis by chromosomal aberrations, thus altering the genic balance and modifying the course of development, in some cases so much as to cause death. The genic balance may also be altered by point mutations or changes in the constitution of an individual gene recognizable through the effects produced.

Either aberrations or point mutations when not lethal may be transmitted in heredity. The distribution of these aberrant chromosomes or mutant genes in meiosis and fertilization is the material basis for heritable differences arising in the course of development of individuals belonging to the same species.

References

Cowdry, E. V. (ed.) 1924. General Cytology, Sections X, XI.

Morgan, T. H. 1913. Heredity and Sex. —— 1919. The Physical Basis of Heredity. “—— 1922. The Mechanism of Mendelian Inheritance, 2nd Ed. A— 1934. Embryology and Genetics. fA and others. 1928. The Theory of the Gene. Sharp, L. W. 1934. Introduction to Cytology, Chaps. 17-24,

Wilson, E. B. 1925. The Cell, etc., Chaps. 9-12.


Chapter V Cleavage And The Germ Layers

The fertilized egg (zygote) is a complete and balanced cell.

It has two entire sets of chromosomes, each with a full comple “ment of genes, one set from each parent. These nuclear elements are contained in a cell body whose cytoplasm is principally maternal in origin and which has a definite organization as indicated by its polarity. We are now to examine the way in which the embryo develops from the fertilized egg.

It is customary to distinguish three steps in the early development of the embryo. First is the period of cleavage in which the egg undergoes a number of mitotic cell divisions at each of which the number of cells (blastomeres) increases while the size of the cells decreases. The period ends with the embryo in the form of a blastula, a sphere or disc in which the blastomeres are not stratified into different layers.

Second comes the period of gastrulation in which the blastomeres arrange themselves into an outer and inner layer of cells, known as ectoderm and endoderm, respectively. This twolayered embryo is called a gastrula.

’ Third is the period in which a middle layer, including the mesoderm and the notochord, is formed between the ectoderm and endoderm. Although this layer sometimes develops during gastrulation, it is customary to distinguish a period of mesoderm (chorda-mesoderm) formation. This distinction is not always valid, nor is it important, for, as will be seen, the material which is to form the middle germ layer can sometimes be distinguished in gastrulation, cleavage, or even in the fertilized egg.

A. Cleavage

As there are different types of eggs according to the amount and distribution of the yolk, so there are different types of cleavage according to the pattern formed by the dividing egg.

Rules of cleavage. — Certain rules have been formulated to express the simpler geometrical relationships of the blastomeres.


The first are those of Sachs: (1) cells typically tend to divide into equal parts; (2) each new plane of division tends to intersect the preceding one at right angles. Sachs’s rules are supplemented, and to some extent explained, by those of Hertwig: (1) the typical position of the nucleus (and hence of the mitotic figure) tends towards the center of its sphere of influence, i.e., of the protoplasmic mass in which it lies; (2) the axis of the spindle typically lies in the longest axis of the protoplasmic mass, and division therefore tends to cut this axis transversely.

Methods of cleavage. — The rate of division is governed by the rule of Balfour: the rate of cleavage is inversely proportional to the amount of yolk present. This leads to a distinction between two types of cleavage. In the first type the cleavage planes divide the egg completely into separate blastomeres. This is known as holoblastic cleavage, and is characteristic of isolecithal and moderately telolecithal eggs. In the second type the cleavage planes do not pass through the yolk and so the separate blastomeres come to lie upon a mass of undivided yolk. This is known as meroblastic cleavage and is typical of extremely telolecithal eggs. It is generally true that isolecithal eggs have equal holoblastic cleavage (Fig. 48A). Moderately telolecithal eggs have unequal holoblastic cleavage (Fig. 48B), and extremely telolecithal eggs have meroblastic cleavage (Fig. 48C).


Fig. 48. — Diagram to show main types of cleavage in vertebrates. A, equal holoblastic. B, unequal holoblastic. C. meroblastic.


Cell lineage. — It must not be thought that cleavage results in a mass of identical blastomeres. Painstaking examination of dividing eggs has shown that in the normal development of favorable material the origin and fate of every blastomere can be determined accurately. The genealogical history of the blastomeres is known appropriately as cell lineage. One of the most clean-cut examples, in forms allied to the vertebrates, is the cell lineage of the tunicate Styela (Cynthia), worked out by Conklin in 1905. The accompanying diagram (Table 6) shows the cell lineage up to the 32-cell stage with the ultimate fate of each of the blastomeres.

In reading this chart the student should understand the system used in naming the blastomeres, which is illustrated most easily by means of the 8-cell stage (Fig. 49). The blastomeres which will give rise to structures on the right side of the embryo are underlined. The blastomeres formed at the animal hemisphere are in lower-case letters; those at the vegetal hemisphere are in capital letters. Those formed at the antero-dorsal side of the embryo are given the designation A or a; those at the posteroventral side are named Borb. The first exponent is the number of the cell generation, counting the fertilized egg as the first generation, the blastomeres of the first cleavage as the second generation, etc. The exponent after the decimal point indicates whether the cell is in the first, second, third, etc., row from the vegetal pole. Thus the cell labelled A‘! is antero-dorsal, left side, vegetal hemisphere, of the fourth generation, and in the row next to the vegetal pole.


Fig. 49. — Cleavage of Styela (Cynthia) egg. A, 4-cell stage from left side. B, same stage from animal pole. C, 8-cell stage from left side. D, same stage from animal pole. For explanation of lettering see text. (Irom Richards, after Conklin.)


TABLE 6 Ceuvt Lineage or Styela (Cynthia) arteR Conknin (1905)

(Ist) (2nd) (8rd) (4th) (5th) (6th) Generation (1) (2) (4) (8) (16) (32) Number of cells

5 a®8 Tctoderm (epidermis) a ao7 “ “

4.2 a 98-6 “ “

5.3 a5 “ (neural plate)

A®4 Chorda-neural plate

A&3 _Endoderm

A®2 Chorda-neural plate

A®! Hindoderm

b&8 Ectoderm

b®7 “

bé-6 “cc;

bé5 “cs

B&+ Mesoderm (gray crescent) Be3 “cc 6c “cc Be “ (yellow “ ) Be! Endoderm

|

|

| _ Eaa | ats Ectoderm (epidermis)

|

|

Ab.2

Abt AB? (Left) ( ps4

b®3 B3 Bs2

Be

5.4 a g8-7

4.2 a 6“ ‘6

353 ass = ass “ (neural plate)

A®4 Chorda-neural plate

A®3 Endoderm

A’? Chorda-neural plate

A®! Endoderm

b*8 Ectoderm

b&7 “

bes “

bes “

Bé+ Mesoderm (gray crescent) Bes ‘“ 6 ‘“ Be “ (yellow “ ) Be! Endoderm

A52 Atl ~ ~~ Aba

AB! ~ b*2


b53

Bs


Bt


B51

The first cleavage is bilateral; i.e., it divides the egg, with its presumptive organ regions, into a right blastomere (AB?) and a left blastomere (AB’). At the second cleavage each of these is divided into an antero-dorsal blastomere (A? and A‘) and a postero-ventral blastomere (B? and B*). The third cleavage plane (Fig. 49C, D) separates the smaller cells of the animal hemisphere (a*?, b*”, a*?, b*?) from the larger cells of the vegetal hemisphere (A4}, Bt, Atl, B*?).

By the sixth generation (32-cell stage) the organ-forming regions have been segregated into different blastomeres as follows:

Animal hemisphere:

14 Ectoderm, epidermis. 2 Ectoderm, neural plate.

Vegetal hemisphere: 4 Ictoderm and mesoderm, chorda-neural plate. 4 Mesoderm, gray crescent. 2 Mesoderm, yellow crescent. 6 Endoderm cells.

The cell lineage of many types of invertebrates has been investigated in a similar manner, and as a result it is now generally recognized that during cleavage the successive generations of blastomeres show a progressive differentiation. Larlier or later, the presumptive organ regions of the fertilized egg are segregated into different groups of blastomeres, each group forming a presumptive organ region of the blastula (page 102).

Later (Chapter VII), experiments will be described which indicate that individual blastomeres may, under different conditions, give rise to parts of the embryo other than those which they produce in the normal course of development.

CLEAVAGE: THE AMPHIOXxUS. — In the egg of the amphioxus (Fig. 50), which is isolecithal, cleavage is holoblastic and almost equal. The first cleavage commences as a depression at the animal pole, which later assumes a groove-like form and elongates until it becomes a wide meridional furrow extending around the egg. This constriction deepens until the two hemispheres are completely divided, when each blastomere rounds up into a spherical shape. The second cleavage also commences at the animal pole and is meridional but at right angles to the first, following the second rule of Sachs. The third plane of cleavage 96 CLEAVAGE AND THE GERM LAYERS

is at right angles to both the first and second and hence would be equatorial if the egg were completely isolecithal. But as the yolk is a little concentrated at the vegetal pole, the nucleus, following Hertwig’s first rule, is in the center of the protoplasm, i.e., on the egg axis slightly nearer the animal pole. So the third cleavage plane is nearer the animal pole and accordingly is latitudinal. The quartette of cells in the animal hemisphere is therefore smaller than those in the vegetal hemisphere. The smaller cells are called micromeres; the larger ones, macromeres. The fourth division divides each of the eight existing blastomeres in two. There are two planes of cleavage, each meridional, at right angles to the third, and also at right angles to each other. Sometimes the cleavage planes of the fourth division are parallel to each other instead of being at right angles. This makes the bilateral symmetry of the dividing egg quite obvious. In the fifth cleavage 32 cells are produced, again by two planes of cleavage, at right angles to the planes of the fourth, but this time latitudinal and parallel to each other. From this time on cleavage becomes more and more irregular. The early cleavages have been fairly regular; each has divided the entire egg mass; and the blastomeres, with the exceptions noted, have been almost equal. The blastomeres round up as each cleavage is completed, and a jelly is secreted between them. In this way a small cavity called the segmentation cavity or blastocoel is formed.



Fig. 50. — Cleavage of the amphioxus egg. A, before cleavage. B, commencing first cleavage, from posterior side. C, second cleavage, from vegetal pole. D, third cleavage, from left side. KE, fourth cleavage, from vegetal pole. F, fifth cleavage, side view, segmentation cavity indicated by dotted lines. 166. (After Conklin, 1932.)


Conklin (1933) states that comparison of the cleavage of the amphioxus with that of the tunicates shows a general resemblance between the two in the distribution of the organ-forming substances to the blastomeres, in the generally bilateral type of cleavage, and the order of division; but in all respects the tunicate egg is the more precise and the more precocious in differentiation.


CLEAVAGE: THE FROG. — The frog’s egg (Fig. 51) is telolecithal with holoblastic unequal cleavage. Here the first division commences as a depression at the animal pole, which elongates and extends around the egg as a shallow furrow until the ends meet at the vegetal pole. The constriction extends inwards and eventually bisects the egg into two blastomeres which round up very slightly. The plane of second division is also meridional and through the animal pole but at right angles to the first. The first two cleavage planes intersect each other at the animal pole; but as the blastomeres round up, the planes no longer form a cross, but two blastomeres are pushed away from each other, while the other two are in contact forming a short polar furrow between them. The third cleavage is latitudinal, about 20° above the equator, and the micromeres are considerably smaller than the macromeres. Theoretically the fourth and fifth planes of cleavage bear the same relationships to the earlier ones as do those of Amphioxus, but actually they are more irregular. The two planes of the fourth cleavage often fail to pass through the vegetal pole and hence become vertical rather than true meridional planes. As these planes originate in the animal hemisphere, the micromeres are divided before the macromeres, so that a 12-cell stage intervenes between the 8-cell and 16-cell stages. Similarly, following Balfour’s rule, the latitudinal cleavage plane in the animal hemisphere of the he fifth division appears before the corresponding


Fig. 51. — Cleavage of the frog’s egg. A, third cleavage. B, fourth cleavage (12 cells). C, fifth cleavage. D, sixth cleavage. 1, I’, later stages. (After Morgan.)


“a plane in the vegetal hemisphere, so that there is a 24-cell stage before the 32-cell stage is attained.

The cell lineage of the frog’s egg has not been followed in detail as

A B has that of the tunicate or amphi Fie. 62.—The gray crescent of the Oxus. It is known, however, that frog’s egg in early cleavage. A, first the first cleavage plane ordinarily cleavage, posterior view. W}.third divides the gray crescent into two

cleavage, from left side. dagen trical hal .

diagrammatic. bs symmetrical halves (Fig. 52A), so

that cleavage is normally bilaterally symmetrical from the outset. The blastomeres receiving the gray crescent material will give rise to notochord and neural plate in later development.

CLEAVAGE: THE CHICK. — In telolecithal eggs with meroblastic cleavage such as that of the fowl, only the protoplasm of the egg, i.e., the blastodisc, is divided, and the cleavage planes do not segment the yolk (Fig. 53). The first furrow commences at the animal pole and extends outwards towards the edges of the blastodisc. The second is formed by two furrows, at right angles to the first, one in each blastomere, which grow towards the first furrow and also towards the edge of the blastodisc. They may join the first furrow at approximately the same point or at separate points, in which case a polar furrow is formed. These four cells are incomplete, as the furrows do not extend all the way to the yolk nor to the edge of the blastodise, but remain connected both below and at their margins. From this point on, tleavage is irregular. Some cleavage planes are circular and cut off central cells from marginal. These may be compared with the latitudinal planes of the holoblastic type. Others are radial, like the first and second. Still others are tangential and divide the central cells into upper and lower layers, as in the frog’s egg.


Fig. 53.— Cleavage of the hen’s egg. A, first cleavage. B, second cleavage. C, third cleavage. D, later cleavage. All from animal pole. Approx. X12. (A, B, D, after Kélliker; C, after Patterson.)



CLEAVAGE: MAN AND OTHER MAMMALS. — The cleavage of the human ovum has not yet been observed, but in the egg of the monkey (Fig. 54) and rabbit (Fig. 55) the cleavage is clearly of the equal holoblastic type. In the rabbit the first cleavage takes place 100 CLEAVAGE AND THE GERM LAYERS

about: 22} hours after coitus. It is equal and complete. The second cleavage follows in about 3 hours. Here the two cleavage spindles frequently lie at right angles to each other so that the four blastomeres assume the form of a cross. Cleavage is now irregular, 5-, 6-, 7-, and 8-cell stages appearing in order. The 8-cell stage is attained about 32 hours after coitus. There is now considerable difference in size, the largest blastomere being almost twice the size of the smallest. The 16-cell stage is reached in another hour and a half. In reaching this stage the cleavage of one blastomere is tangential so that there is always one cell completely enclosed. In later cleavages more tangential cleavages occur, and this, with the shifting of the blastomeres upon each other, results in a solid mass of cells called a morula.


Fig. 54. — Cleavage of the monkey’s egg. A, first cleavage. B, second cleavage. C, third cleavage. 170. (After Lewis and Hartman in Arey.)



Fig. 55. — Cleavage of the rabbit’s egg. A, fertilized egg (note albumen layer). B, first cleavage. C, second cleavage. D, third cleavage. I, fourth cleavage. F, fifth cleavage. 180. (After Gregory.)


The blastula. — The period of cleavage terminates in the appearance of the blastula, but this does not mean that cell division comes to anend. The blastula is generally defined as_a hollow sphere of blastomeres surrounding a cavity, the blastocoel. But this definition does not fit the blastulae formed by meroblastic cleavage. So we shall distinguish three classes of blastulae. The first is of the hollow sphere type (coeloblastula) and is the result of holoblastic equal cleavage*(Fig. 56A). A variety of this type, in which the blastocoel is displaced towards the animal pole, is the result of holoblastic unequal cleavage (Fig. 56B).


Fig. 56.— Diagrams of vertebrate blastulae. A, coeloblastula following holoblastic equal cleavage (amphioxus). , coeloblastula following holoblastic unequal cleavage (frog). CC, discoblastula following meroblastic cleavage (chick). D, blastocyst (mammals.)


The second type of blastula (discoblastula) is the result of meroblastic cleavage in which the blastomeres rest in a flat disc, the blastoderm, on the undivided yolk mass (Fig. 56C). A segmentation cavity later combines with a yolk cavity, formed by the digestion of the yolk underlying the blastoderm, to form a blastocoel. Such a blastocoel is roofed with cells but has a floor of yolk. The third type of blastula is found only in mammals and is called a blastocyst (Fig. 56D). The solid morula forms a blastocoel which enlarges until it almost separates an outer layer of cells (trophoblast) from an inner cell mass (the embryonic knob). Presumptive organ regions of the blastula. — As might be inferred from the results of cell-lineage studies, the regions of the blastula will give rise to different parts of the embryo in normal development. In the tunicate and amphioxus, Conklin has mapped out the presumptive organ regions of the blastula, and Vogt and his students, by means of a most ingenious technique, have accomplished the same result for the amphibian blastula. Experimental evidence (Chapter VII) indicates that in the tunicate and amphioxus the organ-forming regions are definitely determined whereas in amphibians, the regions have a greater plasticity and may give rise to parts of the embryo quite different from those formed in normal development. BLASTULA OF THE AMPHIOXxuS. — In the development of the amphioxus we find a good example of the coeloblastula (Fig. 57). The blastomeres are arranged in a single layer around the enlarged blastocoel which is entirely cut off from the exterior. The blastomeres at the animal pole are micromeres; those at the vegetal pole are macroPresumptive — Mees ; the cells at the equa- . torial belt are transitional in Presumptive type. ; Fia. 57. — Blastula of the amphioxus. Sag- The cells which are to form ittal section. X220. (After Conklin.) the mesoderm are rounded and in active mitosis. They are arranged on a crescent on one side of the egg while those which will form the chorda-mesoderm make up a corresponding crescent on the other. The endoderm cells are the larger cells of the vegetal hemisphere.


BLASTULA OF THE FROG. — The blastula of the frog (Fig. 58) resembles that of the amphioxus in all essential characters, but shows minor differences due largely to the greater amount of yolk present. In the first place, the blastoderm is no longer one layer of cells in thickness. Tangential divisions have increased the number of cells so that at the animal pole the blastoderm may be approximately four cells deep. Furthermore, the greater differ ence in size between the micromeres of the animal pole and the macromeres of the vegetal pole result in the blastocoel’s occupying an eccentric position entirely within the limits of the animal hemisphere.


Fig. 59. — Diagrams of the Triton egg showing movement of surface areas stained with nile blue and neutral red during gastrulation. Areas on surface shown with sharp outline, those on interior without outline. (After Vogt.)



Fig. 58. — Blastula of the frog. Vertieal section. (After Brachet.)


The blastula of the frog shows certain regional differentiations. Thus the cells of the animal hemisphere are smaller than those of the vegetal hemisphere. Morgan has pointed out that those arising in the region of the gray crescent are definitely smaller, i.e., dividing more rapidly, than those in any other meridian.

Vogt has demonstrated the fate of different regions of the blastula in normal development by marking them with such harmless dyes as nile blue and neutral red. The stain persists long enough so that the migration of the dyed cell groups can be traced through gastrulation and even later (Fig. 59).

He has succeeded in mapping out the surface of the blastula into presumptive organ regions, as seen in the diagram (Fig. 60).


Fig. 60. — Diagrams to show presumptive organ regions of the frog blastula. A, from left side. B, from dorsal surface. The cross indicates the position of the vegetal pole. (After Vogt, 1929.)


BLASTULA OF THE CHICK. — The blastula of the chick is a discoblastula. The blastoderm consists of an inner mass of micromeres completely separated from one another by cleavage planes, and an outer ring of macromeres which are partially separated from one another by incomplete radial cleavage planes only. These latter cells are in direct protoplasmic continuity by means of an outer ring of undivided cytoplasm and a thin lower layer of undivided cytoplasm passing beneath the inner mass (Fig. 61). This undivided cytoplasm is called the periblast. The micromeres of the inner mass are separated from the underlying divided periblast by means of a thin cleft which is the original blastocoel.


Fig. 61. — Section of early chick blastula. Compare Fig. 53D. (After Patterson.)


The blastoderm expands over the yolk, new cells being added to the inner cell mass from the outer ring of cells. The periblast, contributing its cytoplasm to the formation of new cells in the outer ring, soon uses up all the material contained in the thin lower layer. Meantime its outer ring, now called the germ wall, expands outward. With the disappearance of the lower layer of periblast, the cells of the inner mass form the roof of a cavity which includes the original blastocoel plus the space originally occupied by the lower layer of periblast. These cells form an area known as the area pellucida because it can be detached from the yolk without carrying any yolk particles and hence appears more transparent. The cells of the outer ring and the germ wall make up the area opaca, so-called because particles of yolk adhere to them when removed from the egg and render them less transparent. 106 CLEAVAGE AND THE GERM LAYERS

BLASTULA OF MAN AND OTHER MAMMALS. — No human embryo in the blastula stage has been recorded, so a description of the blastocyst of the rabbit will be given in its place. About 75 hours after coitus and while the egg is still in the oviduct, a cleft, the blastocoel, appears in

Embryonic knob the morula apparently

Albumen Embryonic duc to the formation of some fluid. This extends rapidly until an outer layer of cells, the trophoblast, is separated from an inner cell mass, the embryonic knob. The


Blastocoel ae separation is almost comTrophoblast ’ . , B plete (Fig. 62A), extend.

Fig. 62. — Sections of rabbit blastocysts. 200. ing about 270 of the (After Gregory.) possible 360°. By this time the blastocyst has reached the uterus and the secretion of fluid is greatly increased, expanding the blastocoel and stretching the trophoblast cells. The embryonic knob flattens against one pole (dorsal) of the trophoblast, and the entire blastocyst increases greatly in size (Fig. 62B). This flattening of the embryonic knob is not characteristic of all mammalian blastocysts.

B. Gastrulation

The vertebrate blastula becomes converted into a two-layered embryo, or gastrula, through the migration of cells from the exterior to the interior of the embryo. In so doing the blastocoel is obliterated and replaced by a new cavity, the gastrocoel (archenteron), which communicates to the exterior by means of an opening, the blastopore. The cells left on the exterior form the outer germ layer commonly known as ectoderm (ectoblast, epiblast). Those on the inside, lining the gastrocoel, form the inner germ layer, usually called the endoderm (entoderm, entoblast, hypoblast). But, as will be seen later, they may also include cells which will give rise to the middle germ layer, the chordamesoderm, consisting of the mesoderm (mesoblast) and notochord (chorda dorsalis). In such cases the inner layer may be called mesendoderm (see page 115). The different types of blastulae resulting from different types of cleavage naturally give rise to different types of gastrulae (Fig. 63) according to the means by which the endoderm is segregated from the ectoderm.

Students of gastrulation distinguish five types of cell migrations which will be described briefly here, and developed more fully in later paragraphs.

1. Invagination (Fig. 638A). Typical of the cocloblastula resulting from equal holoblastic cleavage. The cells of the animal hemisphere move inward in a continuous shect, obliterating the blastococl, until they come to rest against the cells of the animal hemisphere, thus giving rise to a new cavity, the gastrocoel, which opens to the exterior by means of the blastopore. This process is made possible by the continued growth of cells at the lip of the blastopore which roll inward (involution, see 3) as invagination continues.



Fig. 63. — Diagrams of vertebrate gastrulation. A, by invagination (amphioxus). B, by epiboly and involution (frog). C, by involution (chick). D, by delamination (mammal).


2. Epiboly (Fig. 63B). Typical of the coeloblastula resulting from unequal holoblastic cleavage. The cells of the animal hemisphere grow over the cells of the vegetal hemisphere, creating a gradually narrowing circular fold, the lip of the blastopore. This process also involves the growth and rolling inward of cells at the moving lip (involution, sec 3) to form the roof of the gastrocoel.

3. Involution (Fig. 63B, C). Typical of the discoblastula resulting from meroblastic cleavage. The cells at one region of the disc roll inward and spread out under the disc to form the roof of a gastrocoel. The region where involution takes place is the dorsal lip of the blastula. Involution also accompanies invagination and epiboly (sec 1 and 2).

4. Delamination (Fig. 63D). Typical of the blastocyst in mammals. The lower cells of the embryonic knob split off as a loose layer which later reorganizes itself to enclose a gastrocoel.

5. Concrescence (Fig. 64). As the blastopore narrows, cells which originally lay along the right and Iecft halves of the dorsal lip converge towards each other. And, since the dorsal lip is also growing backward, these cells will form the right and left sides of an axial (antero-posterior) streak.


Fig. 64. — Diagrams showing four stages in the process of concrescence. (After Lillie.)


GASTRULATION IN THE AMPHIOXuS. — The first indication of gastrulation is a flattening of the macromeres of the vegetal hemisphere (Fig. 65A). These cells divide less frequently and become more columnar, while the others divide more frequently and become more cubical or spherical in shape. This change in the shape and rate of division, says Conklin (1932), is apparently the principal cause of invagination (Fig. 65B), although it may be due also in part to the resorption of material from the blastocoel jelly, or to exosmosis, for the contents of the blastocoel become less viscous as gastrulation proceeds.

In later stages of gastrulation the gastrula increases in length, owing to the backward growth of the lips of the blastopore (Fig. 65C). While this process is taking place cells are being rolled from the exterior to the interior (involution). The lips of the blastopore grow unevenly, the ventral lip finally turning upward to reduce the blastopore to a very small opening (Fig. 65D). Conklin expressly denies that this narrowing of the blastopore is caused by the growing together of the right and left halves of the dorsal lip (concrescence). The cells left on the exterior after gastrulation is complete are ectoderm. Those which have been carried to the interior are endoderm, and presumptive mesoderm. The segregation of the notochord and mesoderm cells is discussed in Section C of this chapter.


| Presumptive 7 chordax neural plate Presumptive mesoderm Presumptive endoderm


Fig. 65. — Sections of amphioxus embryos during gastrulation. A, blastula (6 hours after fertilization). B, gastrula (93 hours). C, gastrula (12 hours). D, gastrula (14 hours). Animal pole indicated by presence of polocyte. Anteroposterior axis shown by arrow. All sagittal sections. 180. (After Conklin, 1932.)


In late gastrulation the cells of the ectoderm develop cilia, by means of which the embryo rotates within its fertilization membrane.


GASTRULATION IN THE FROG. — The first stage in the gastrulation of the frog is the formation of a groove on the dorsal side of the embryo in the region of the gray crescent (Fig. 66A). Along this groove, cells are pushed into the interior (involution), while at the same time the cells immediately above the groove are growing down over the surface of the embryo to cover them (epiboly). In this way a two-layered fold is produced, the dorsal lip of the blastopore (Fig. 66D). 110 CLEAVAGE AND THE GERM LAYERS



Fig. 66. — Three stages in the gastrulation of the frog’s egg. A, dorsal lip stage, from vegetal pole. B, do., sagittal section. C, lateral lip stage, from posterior surface. D, do., sagittal section E, ventral lip (yolk-plug) stage, from posterior surface. ¥, do., sagittal section. (B, D, I, after Brachet.)


As the two-layered fold grows down over the cells of the vegetal hemisphere, it extends laterally, thus forming the lateral lips of the blastopore (Fig. 66B). And, since it is covering a spherical surface, the ends of the fold eventually meet to form the ventral lip (Fig. 66C). Epiboly and involution take place at all points on the lip of the blastopore, but chiefly at the dorsal lip, which moves approximately 90° around the egg. At this time the egg presents the appearance of a black sphere with a small white circular area, known as the yolk plug (Fig. 66C).

Within the egg, two distinct phenomena have been taking place. First, the cells turned inward by involution at the dorsal lip have spread out to form the roof of a wide but shallow cavity, the gastrocoel. Second, small cells have arisen from the large yolkladen cells of the vegetal hemisphere, and these form the floor of the gastrocoel. They join the cells resulting from involution at the anterior end of the gastrocoel (Fig. 66E).

There is now an extensive displacement of the interior cells, resulting from the growth forward of the gastrocoel, and the consequent thinning of its floor. It is still uncertain whether the floor is pushed across the blastocoel, thereby obliterating it, or whether the thin floor is ruptured so that the blastocoel is added to the enlarging gastrocoel (Fig. 66F). In either event the center of gravity in the egg is now altered so that it rotates about a horizontal axis in such a way that the blastopore is carried back to a point a little beyond its starting point, 100°.

The blastopore is now in its definitive position and marks the posterior end of the embryo. The dorsal side, already marked by the appearance of the dorsal lip, is uppermost. In the concluding stages of gastrulation the blastopore narrows to a small slit. This narrowing is brought about by the growing together of the right and left halves of the dorsal lip (concrescence) as epiboly and involution continue.

The cells of the inner layer during later stages of gastrulation appear to be split into two separate layers. The one of these which lines the gastrocoel is endoderm. The other lying between the endoderm and the ectoderm is the chorda-mesoderm. The mode of origin of the latter will be described in the following section.


GASTRULATION IN THE CHICK AND PIGEON. — The blastula of the chick is a disc of blastomeres lying over the undivided yolk.

It is divided into an interior area pellucida and an outer area

opaca. This outer area is extending itself in all directions over the undivided yolk (epiboly). The account which follows is based on gastrulation in the pigeon.

Three zones are distinguishable in the area opaca. First, there is a margin of overgrowth where the cells are completely separated from the yolk. Second comes a zone of junction, whose deeper cells are not separated from the yolk. The third division is the inner zone, whose cells, completely separate from the yolk, are being added to the area pellucida.

The first indication of gastrulation is the thinning of the blastoderm at the posterior end and the complete separation of the cells from the yolk at that region (Fig. 67A). In other words, there is a crescentic area, almost a quarter of the cir Endoderm cymference, of the blastoderm which lacks the zone of junction completely.

Fig. 67. — Surface views showing three stages Here the cells roll inward in the gastrulation of the hen’s egg, from ,. luti Fie. 68 d the animal pole. (After Patterson.) (invo ution) (Fig. ) an

multiply until they have spread completely under the upper layer to roof in the old blastocoel and convert it into the new gastrocoel, whose floor is made up of undivided yolk. The slit-like opening where the zone of


Dorsal lip


Dorsal lip

junction disappeared is the blastopore, and the rim along which involution took place is the dorsal lip.

There is very little overgrowth at the dorsal lip while involution is taking place, and consequently the edges of the blastoderm on either side swing around to enclose the lip region in the advancing


Fig. 68. — Sagittal section through early gastrula of pigeon (36 hours after fertilization). Posterior half of section only. d.b., dorsal lip of blastopore. (J*‘rom Richards after Patterson.)

germ wall. In this way the blastopore is compresscd laterally and concrescence takes place.



GASTRULATION IN MAN AND OTHER MAMMALS.— No human embryo nas been observed before the separation of the germ layers. The account which follows is based on the pig. From the lower surface of the embryonic knob, individual cells detach themselves to form a sheet (Fig. 69) which rapidly establishes


Fig. 69. — Section to show an early stage in the gastrulation of the bat’segg. (After Van Beneden.)

itself as a layer immediately inside the trophoblast, enclosing most of the old blastocoel. We may now consider the trophoblast and the remainder of the embryonic knob as ectoderm and the inner layer as endoderm. The cavity which it encloses is comparable to the gastrocoel plus the yolk sac of the egg-laying mammals.

The cells of the trophoblast immediately overlying the embryonic knob (Rauber’s cells) now disappear, and the embryonic knob flattens out to become the embryonic disc. This disc lies at the surface and constitutes part of the wall of the blastocyst.

In the primates, judging from studies on the lemur, Tarsius, and from the appearance of the earliest human embryo (Fig. 70), the endoderm does not grow out around the entire trophoblast,


Fig. 70. — Diagrams to show three stages in the gastrulation of the human egg during implantation. The uterine wall indicated by hatching. (Hypothetical based on Teacher; the embryo in C based on Miller.)

but forms a very small vesicle immediately under the embryonic knob. The cavity of this vesicle may be considered a gastrocoel but is more generally known as the ‘‘yolk sac.”

C. The Middle Germ Layer (Chorda-Mesoderm)

During or immediately following gastrulation a third germ layer appears between the ectoderm and endoderm. This layer consists of the notochord (chorda dorsalis), an axial supporting rod found only in the vertebrates and their allies the protochordates, and two sheets of mesoderm on each side of the notochord. Later wandering ameboid cells, originating from the mesoderm and known collectively as the mesenchyme, make their appearance.

The student should note that in many elementary texts the middle germ layer is called the mesoderm and that the notochord is variously derived from mesoderm, endoderm (amphioxus and frog), or ectoderm (chick and mammals). This terminology dates back to the phylogenetic period of-embryology (Chapter I), when THE LATER HISTORY OF THE GERM LAYERS 115

it was supposed that a blastula composed of undifferentiated blastomeres gave rise to a gastrula with two separate (primary) layers, and that the mesoderm and the notochord arose separately from one or the other of the so-called primary layers, primitively from the endoderm. Today it is generally recognized that the notochord arises in the same manner and at the same time as the mesoderm. To avoid the clumsy phrase, mesoderm and notochord, many writers are now employing the term chorda-mesoderm for the middle germ layer, and restricting the term mesoderm to the middle germ layer exclusive of the notochord, a usage employed in this text. The compound word mesendoderm (mesentoderm) is now used by many writers to include both the endoderm and the chorda-mesoderm when these layers lie beneath the ectoderm but have not yet segregated from each other.

In collateral reading the student will sometimes encounter the word endo-mesoderm used in connection with mesoderm “ originating from” or, better, associated with, endoderm in early development. Similarly the word ecto-mesoderm is employed to designate mesoderm “ originating from,” or associated with, ectoderm in early development. Other writers use the terms peristomial mesoderm, meaning mesoderm appearing in the region of the blastopore, and gastral mesoderm for mesoderm appearing to arise from the invaginated endoderm. But inasmuch as the middle germ layer can often be traced to definite blastomeres during early cleavage, this distinction is of small importance.

It is well established, however, that among the vertebrates the movement of the presumptive chorda-mesoderm to its definitive position in the roof of the gastrocoel is intimately associated with the formation and closure of the blastopore. This is true no matter whether the blastopore is a large circular opening as in the amphioxus and the frog, or reduced ‘to a primitive streak by concrescence as in the chick and man.

The later history of the germ layers. — With the segregation of the three germ layers, the presumptive organ regions are now located in one or another of the three. But it must not be supposed that the organs of the adult are exclusively ectodermal, endodermal, or mesodermal. On the contrary, most of them contain material from at least two, and sometimes all three. In Part III will be found an account of the development of the different organ systems, classified according to the germ layer from which arise the tissues associated with their special functions. Meantime the following table is presented.

TABLE 7 DERIVATIVES OF GERM LAYERS

Eetoderm Chorda-mesoderm Tendoderm

A Notochord 2B) Mesoderm


1. Epidermis of skin and 1. Epithelium of coclom 1. Mpithelia of digesall openings into the body | and exocoel tive tube, including thy2. Epithelia of eye, ear, 2. Nephric (exeretory) | mus gland, thyroid gland, and nose system parathyroid gland, in3. Nervous system, in- 3. Genital (reproduc- | ternal respiratory — orcluding interrenal glands, | tive) system gans, volk sac, and alpituitary gland (in part), 4. Suprarenal gland lantois pineal gland 5. Blood-vaseular sys4. Epithelium of amnion | tem and chorion 6. Connective — tissue

including skeleton 7. Musculature 8. Dermis of skin



THE MIDDLE GERM LAYER IN THE AMPHIOXUS. — As mentioned in earlier sections, Conklin (1933) has been able to distinguish the mesoderm cells in the amphioxus in the blastula stage (ig. 57), where they form a crescent of small rounded blastomeres in the region where the ventral and lateral lips of the blastopore will form. The notochord cells, associated with those which will later give rise to the neural plate, occupy a corresponding chordaneural crescent at the dorsal lip. After the invagination of the endoderm the cells of the mesoderm and notochord form the lip of the blastopore, the notochord cells at the dorsal lip, mesoderm at the ventral and lateral lips. As the lips of the blastopore grow backward, these cells are carried to the interior by involution (Fig. 65).

When the ventral lip grows upward, the mesodermal cresent is tilted up behind so that its arms run in an antero-posterior direction to form the angles between the roof and sides of the gastrocoel (Fig. 71). In the meantime the notochord cells, also carried into the interior, form a flat plate between the two arms of the mesoderm. Thus the roof of the gastrocoel is composed of three strips of chorda-mesoderm, mesoderm on each side, notochord in the middle. A longitudinal groove in the notochord plate deepens, and the folds on either side come together to form a solid cord separate from the ectoderm above and the mesoderm on either side. The mesodermal grooves (Fig. 72) also become deeper. Transverse constrictions meantime appear in the lateral grooves, which divide them into a series of pouches (enterocoels). Finally these pouches are constricted off from the gastrocoel and become the paired somites (Fig. 73).



Fig. 71. — Optical hemi-sections of amphioxus gastrula (14 hours after fertilization). A, left inside. B, posterior to show notochord and mesodermal groove inside. (After Conklin, 1932.)


Fig. 72. — Sections of amphioxus embryo (19 hours after fertilization). A, sagittal section. B, transverse section. 166. (After Conklin.)


The endoderm, which formerly occupied the floor and anterior end of the gastrocoel, extends to form new sides and a new roof.

The gastrocoel, now for the first time completely lined with endoderm, is the primordium of the digestive tube.

The cells of the chorda-neural crescent remaining on the exterior of the embryo give rise to the neural plate on the dorsal surface. They are covered by the ventral lip of the gastrula as it grows over the dorsal side of the embryo. Beneath this covering there appears a longitudinal groove with a fold on either side. These folds arch up and meet in the ventral line to form the neural tube.


Fig. 73. — Sections of amphioxus larva (244 hours after fertilization). A, frontal section. B, transverse section. X166. (After Conklin.)


THE MIDDLE GERM LAYER IN THE FROG. — As noted in earlier sections, we owe to Vogt (1929) the identification of the various regions on the amphibian blastula. This identification was accomplished by staining small regions of the blastula surface with harmless dyes and tracing their movements during and after gastrulation (Fig. 59). He finds that the material first to be turned in at the dorsal lip is endoderm. Immediately anterior and dorsal to this is a crescent-shaped area which will give rise to the notochord. On either side of this are the horns of a crescent extending from the other side of the blastula which will become mesoderm. Immediately anterior to the chorda crescent is the crescent-shaped area of the neural plate, the two together being equivalent to the chorda-neural crescent of the amphioxus. The mesodermal crescent also corresponds to the mesodermal crescent of the amphioxus except that its arms already extend dorsally.


In the gastrulation of the tailed amphibia (urodeles), the material turned in at the dorsal lip is notochord and mesoderm, so that the roof of the gastrocoel is chorda-mesoderm as it is in the amphioxus, and endoderm cells must grow up from the sides and floor to form a new roof.


In the frog, however, the first material to roll in at the dorsal lip of the blastopore is endoderm and notochord (Fig. 60). When the material from the mesodermal crescent rolls in, instead of following the endoderm, it wedges in between the endoderm and ectoderm (Fig. 66F), so giving the appearance of splitting off from the endoderm in the roof of the gastrocoel. The roof and sides of the gastrocoel are, therefore, endodermal except for a narrow dorsal strip represented by the notochord (and a narrow strip beneath it, the hypochord). When the notochord (and hypochord) separate from the roof, this small gap is closed by endoderm and the roof is completely endodermal.


As the endoderm, notochord, and mesodermal regions are turned in around the lips of the blastopore the overgrowth of


the lips covers the large yolkladen cells from which the floor of the gastrocoel is produced. Meantime the expanding cells from the ectodermal region of the blastula occupy the region formerly held by the material which has been turned in.


Fig. 74. — Diagrams showing direction of displacements during amphibian gastrulation. A, from posterior surface. B, from left side. Thick lines on exterior surface. Thin lineson interior. (After Vogt, 1929.)

Now the dorsal lip of the blastopore is the one at which epiboly and involution take place most rapidly. Consequently materials on the right and left of the mid-dorsal region are stretched towards the medial line to take the place of the material lost by involution (Fig. 74). In this way the two arms of the mesodermal crescent move together to form parallel strips on either side of the notochord. Similarly the two horns of the neural crescent move together to form parallel strips which eventually enclose the blastopore at the posterior end, while the neural plate itself occupies a longitudinal dorsal position on the gastrula. All the rest of the surface is now material which will form the epidermoet . Mis of the skin. ( myocoel ) The mesoderm continues Intermediate its growth between the ectoderm and endoderm



Fig. 75. — Transverse sections to show three stages in the origin of the notochord and mesoderm in the frog embryo. (After Brachet.)



Lateral (Fig. 75) until it forms a Cecoleen} continuous sheet except at the blastopore. The material on either side of the notochord is separated by Fie. 76. — Diagram of a transverse section of transverse constrictions vertebrate embry 0 te show the regions of the into blocks or somites, corresponding to the somites of the amphioxus. Next comes an intermediate zone from which the gonad and kidney will arise. The remainder splits into an outside (somatic) layer closely applied to the ectoderm, and an inner (splanchnic) layer similarly applied to the*endoderm. The space between (Fig. 76) is the coelom.

The neural plate develops a longitudinal groove, surrounded at the anterior end and sides by ridges known as the neural folds. The embryo has now reached the stage known as the neurula (Fig. 112).


THE MIDDLE GERM LAYER IN THE CHICK.— Mesoderm formation in the chick takes place after the egg has been laid and incubation begun. At about the sixteenth hour (Patten) the blasto- por ee derm is considerably lengthened Pa ser, in an antero-posterior direction, {4 and has an axial thickening known 3 as the primitive streak (Fig. 77). 3 This streak represents the dorsal lip of the blastopore laterally compressed through concrescence as explained on page 108. The germ wall has grown together behind the primitive streak and is ad- Vie. 77. — Blastoderm of the chick at

15 hours of incubation. (After vancing out over the yolk. Ina puyar) more advanced embryo the primitive streak is differentiated into a primitive groove in the middle, primitive folds on either sides, a primitive pit at the


Fig. 78. — Blastoderm of chick to show early stage in development of notochord. A, surface view at 20 hours (after Duval). B, transverse section, left half only. C, sagittal section. (B, C, after Lillie.)

anterior end of the groove, and a primitive (Hensen’s) node in front of the pit where the primitive folds unite (Fig. 78). 122 CLEAVAGE AND THE GERM LAYERS

Sections reveal that from the sides and posterior end of the primitive groove, cells are growing outward, between the ectoderm and the endoderm, to form a sheet of mesoderm. At the anterior end a narrow strip of cells grows forward to form the notochord.

During the remainder of the first day of incubation the area pellucida increases in length, particularly in the region directly in front of the primitive streak. This appears to displace the primitive streak rearwards, and during this time the streak actually shortens.

The mesoderm growing out to the sides is carried forward in this movement and so comes to lie close to the advancing notochord. Furthermore, two horns of mesoderm grow forward, later to curve in and meet in front of an area which contains ectoderm and endoderm only (proamnion). The mesoderm on either side of the notochord thickens to form a segmental zone, so called because it will shortly be divided by transverse constriction into somites, exactly as in the frog. Six pairs of somites are present at the end of the first day (Duval). There is a zone of intermediate mesoderm. The remaining or lateral mesoderm, growing out into the area opaca, splits tangentially into an outer somatic and an inner splanchnic layer, as in the frog. In the splanchnic mesoderm, thickenings appear in the inner region of the area opaca. They mark the primordium of the area vasculosa (Fig. 79).

The ectoderm and endoderm of the clear area give rise to a crescentic fold at the anterior end which is called the head fold as it is the primordium of the head of the embryo. It contains a pocket of endoderm known as the fore-gut, distinguished by the possession of a cellular floor. There is an opening known as the anterior intestinal portal between the fore-gut and the midgut, whose floor is the undivided yolk.

The ectoderm in front and to either side of the notochord is the neural plate. It develops a groove and folds shortly before the end of the first day, and at 24 hours of incubation the folds have met in the region of the brain to form a tube but have not as yet fused together.


THE MIDDLE GERM LAYER IN MAN. — The earliest human embryo is the ‘ Miller’ ovum (Fig. 69). This specimen, supposed to be about 13-14 days old, consists of an outer vesicle, the trophoblast, containing two smaller vesicles, one of which, lined with endoderm, represents a small gastrocoel (yolk sac) the other of ectoderm surrounds a cavity (the amnion, Chapter V). Where the two vesicles are in contact a circular disc of ectoderm and endoderm pressed together represents the embryonic disc. In later specimens this embryonic disc develops a primitive streak, quite as in the chick blastoderm (Fig. 80). Notochord and mesoderm develop in much the same way, somites appearing at the end of the first month. A head fold and neural groove appear in similar fashion.


Fig. 79. — Diagram showing embryonic and extra-embryonic areas of chick embryo at 24 hours of incubation. Above, surface view; below, transverse section.



Summary

During cleavage the fertilized egg is divided into a large number Notochord of daughter cells or blastomeres which arrange themselves about

Primitive a cavity to form the blastula. The pattern of cleavage and the form of the blastula vary accord ay ing to the amount and distribution

Fig. 80. — Surface view of embryonic of the yolk in the fertilized egg.

dise in human embr i 4 ‘ :

has been cut away, wo. * After The presumptive organ Tegions of

Heuser.) the fertilized egg are segregated

into different groups of cells which compose the presumptive organ regions of the blastula.

During gastrulation, the blastomeres are reorganized into different strata or germ layers about a new cavity, thus forming a gastrula. The method of gastrulation varies according to the type of blastula formed after cleavage. The two layers segregated during gastrulation are usually known as the ectoderm and endoderm, but it must be recognized that one or the other of these so-called primary layers includes the presumptive mesoderm as well.

In the concluding period of germ-layer formation, the middle germ layer or chorda-mesoderm, including the notochord and the mesoderm proper, is segregated from the other germ layers to occupy a middle position between them.

While the germ layers are being segregated from each other the primordia of certain organs are arising from their respective presumptive regions. Thus the notochord is separated from the mesoderm proper, the neural plate from the presumptive epidermis. In the mesoderm proper, the somites begin to take form, and the somatic layer separates from the splanchnic to form the coelom.

References

Brachet, A. 1921. Traité d’embryologic, Books 3, 4, and 5.

Conklin, E. G. 1905. The Organization and Cell Lineage of the Ascidian Egg. Jour. Acad. Nat. Sci. Phila., 2nd Series, Vol. XIII.

—— 1932. The Embryology of Amphioxus. Jour. Morph. 54:69-151.

—— 1933. The Development of Isolated and Partially Separated Blastomeres of Amphioxus. Jour. Exp. Zool. 64:303-375.

Cowdry, E. V. (ed.) 1924. Gencral Cytology, Section 9.

Gregory, P. W. 1930. The Early Embryology of the Rabbit. Publ. Carnegie Inst. Wash. 407:141~-168.

Hertwig, O. (ed.) 1906. Handbuch, etc., I, Chaps. 2 and 3.

‘Huxley, J. S., and de Beer, G. R. 1934. The Elements of Experimental Embryology, Chap. 2.

Jenkinson, J. W. 1913. Vertebrate Embryology, Chaps. 5 and 6.

Kellicott, W. E. 1913. General Embryology, Chaps. 7 and 8.

Kerr, J. G. 1919. Textbook of Embryology, II, Chap. 1.

Korschelt, E., and Heider, K. 1902-1910. Lehrbuch, etc., Chaps. 7 and 8.

Lillie, F. R. 1919. The Development of the Chick, 2nd Ed.

MacBride, E. W. 1914. Textbook of Embryology, I, Chap. 17.

Patten, B. M. 1929. The Early Embryology of the Chick, 3rd Ed.

1931. The Embryology of the Pig, 2nd Ed.

Wilson, E. B. 1925. The Cell, etc., Chaps. 13 and 14.


Chapter VI Embryonic Form And Extra-Embryonic Structures

After the germ layers have been segregated, the primordia of several great organ systems are already localized. Before proceeding to an account of the way in which the organ systems develop from the different germ layers (organogeny), we must examine the way in which the vertebrate body assumes its form. This is found to be closely connected with certain structures (adnexa) which develop also from the germ layers and play an important part in embryonic (and fetal) life, but which are discarded before hatching (or birth). These extra-embryonic structures are the yolk sac, the amnion, chorion, and allantois, as well as a structure found only in the mammals, the placenta.

A. The Form Of The Body

The general form of the vertebrate body is cylindrical, while the form of the vertebrate egg is spherical. There are in general _-_Nearal tube two methods of growth ( &_ Notochord Amniotic by means of which the cae cylindrical shape is at .. tained. In the first, y.’ characteristic of smallyolked eggs with a spherical gastrula, the Fia. 81. — Diagrammatic transverse sections show- main factor is growth in

ing effects of yolk on form of embryo. A, small length, along the anteroyolked embryo (frog). B, large yolked embryo posterior (cephalo-cau (chick). (After Assheton.) dal) axis. Inthesecond type, which is characteristic of large-yolked eggs, the embryo is modeled from a flat disc into the form of a cylinder connected with a great yolk sac by some sort of pedestal or stalk. Much of this modeling is done by the outgrowth of the head and the tail re spectively, especially among the anamniote vertebrates, but there 126 GENERAL PLAN OF THE BODY 127

is also some actual undercutting, especially evident among the Amniota. This undercutting is accompanied by the formation of amniotic folds, as will be seen in the development of the chick.

diagram of cross-sections through the body of a small-yolked embryo (Fig. 81A) and a large-yolked embryo (Fig. 81B) will make clear the difference between the cylindrical embryo and the plate-like embryo before it has been remoulded. In the amniote vertebrates with a large-yolked egg the embryo develops from

e)


B c

Fig. 82. — Diagrams to show growth in length by concrescence. Arrows indicate direction of growth. (After Assheton.)

material at the edge of the blastoderm, and as this is rolled together in concrescence the embryo increases in length (Fig. 82).

vy General plan of the body. — The body of the vertebrate is basically a tube within a tube, i.e., a digestive tube within a body tube (Fig. 76).

The digestive tube is endodermal in origin and originates from the gastrocoel. Here again the small-yolked form has a tubular — intestine from the beginning. It is only necessary to form anterior and posterior openings, for the blastopore either closes or is roofed in by the neural folds. The new openings arise from ectodermal pits, the stomodgum at the anterior end, the proctodeum at the posterior end. In general these openings are not completed until after the yolk has been wholly consumed. The gastrocoel of large-yolked embryos has only a roof and sides of endoderm, for the floor is composed of the yolk. Hence the rolling in or undercutting of the body commencing at the head end, and later at the tail end, forms a pocket at each end, the fore-gut and hind-gut respectively. The mid-gut is the remainder of the open gastrocoel connected with the developing yolk sac by means of the yolk stalk. 128 EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

Between the two tubes lies the mesoderm. The ventral mesoderm of small-yolked embryos (lateral of large-yolked forms) splits into a somatic and splanchnic layer. The first of these is closely applied to the ectoderm to form the somatopleure; the second is associated with the endoderm to form the splanchnopleure. The space between is the coelom or body cavity. Other and lesser antero-posterior tubes such as the neural tube, formed from ectoderm, and axial blood vessels, e.g., the aorta, formed from mesoderm, are indicated in the figure and will be discussed in later chapters.

Metamerism. — With growth in length is associated a second factor in the development of the vertebrate body, that of metamerism. This is first indicated by the appearance of metameres



Oral gland

Pronephric elevation

Somites


Fig. 83. — Diagrams of early embryos to show similarities in body form. A, frog (after W. Patten). B, chick (after Kerr). C, man (after His).

such as the enterocoels in the amphioxus or somites in the true vertebrates. In later organogeny are found further evidences of. metamerism in the nervous system, nephric system, vascular system, and others. However, the primary metamerism of the body is shown in the mesoderm. The somites are formed successively, commencing at the anterior end and therefore affording a basis of classifying the early embryos of any species by the number of these units present (Fig. 83). BODY FORM OF THE FROG 129

The head. — The vertebrate body is distinguished by a wellmarked region at the anterior end, containing the mouth, visceral arches, special sense organs (nose, eye, and ear) and the highly developed brain. Herein the amphioxus differs from the vertebrates, for it has so little head that some zoélogists make a special group (Acraniata) to contain it.

The anterior end of the body is already determined in the vertebrate egg (animal pole). It is the surface opposite that of the blastopore, or in front of the primitive streak. It is the region where the neural folds first arise and where they first meet. It is the first part of the body to be freed of the yolk in the largeyolked embryos. A glance at the diagrams of carly embryos (Fig. 83) will suffice to prove that this is the most highly differentiated part of the body.

In the Amniota the head is inclined ventrally at the region of the branchial arches. This cervical flexure causes a constriction (Fig. 83) which is the primordium of the neck, a region found only in reptiles, birds, and mammals.

The tail. — All vertebrate embryos, even those of species in which the adult is tailless (frog, man), develop a well-marked tail in early development. This region is characterized by the absence of a digestive tube and coelom. It develops early in the anamniotes, where it is of great use to the free-swimming larva, but more slowly in the amniotes.

The appendages. — The paired appendages of vertebrates arise as buds (Fig. 83C) which later develop into fins or limbs. Limb buds do not appear in the amphioxus or the cyclostomes. In all other vertebrates which do not possess paired appendages in the adult condition, it is said that limb buds appear in the embryonic life and are resorbed later.

BODY FORM OF THE FROG. — The spherical egg of the frog, being only moderately telolecithal, is converted into the cylindrical shape of the embryo principally through the growth of the head and of the tail.

In the head region the neural plate is much wider than elsewhere, and when the neural folds close in to form the neural tube the brain will be larger than the spinal cord. On either side of the head the optic vesicles, the primordia of the eyes, push out from the brain and make well-marked bulges. The ectoderm im130 EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

mediately external to each optic cup will later give rise to the lens of the eye. Anterior to each eye is a depression in the ectoderm, the nasal (olfactory) pit. These pits are the primordia of the nose. Posterior to each eye a similar otic (auditory, acoustic) pit originates, the primordium of the inner car. On the ventral side, folds of ectoderm give rise to the ventral sucker (mucous gland) in the form of the letter V. Between the limbs of the V there appears an ectodermal pit called the stomodeum or primordium of the mouth. On the ventral side of the body, just ante


Fig. 84. — Growth of the frog embryo. A, late neurula, 2.4mm. B, embryo of 3 mm. C, embryo of 6 mm., just hatched. D, young larva, external gill stage, 9mm. H#, larva, internal gill stage, 11 mm. (Measured alive and drawn after preservation. X10.)

rior to the base of the tail, a similar pit, the proctodeum, is the primordium of the cloacal opening.

On the sides of the head five dorso-ventral grooves appear (in the order I, V, II, III, IV). These are the visceral (branchial,

gill ”?) grooves, some of which will later break through into corresponding outpushings from the fore-gut, the visceral (pharyngeal, “ gill’) pouches, to form the visceral (pharyngeal, “ gill ’’) clefts. For the present we need simply note that they separate six transverse bars or ridges which are known as the visceral BODY FORM OF THE FROG 131

arches. Each visceral arch contains an aortic arch. (See Table 8.) Arch I (mandibular) contributes to the formation of the jaws. Arch II (hyoid) contributes to the gill cover (operculum) and to the support of the tongue. Arches III, IV, and V are often known as branchials 1, 2, and 3, respectively. On arches III, TV, and V develop outgrowths which become the external gills TABLE 8 PHARYNGEAL DerivaTIVES



Pouches Aortic Grooves TL Arches ‘lefts : ; (From endoderm) Arehes Clefts Arches — |(From ectoderm) Visceral Aortic arch I arch [

(mandibular)












Visceral Visceral Visceral pouch I cleft I groove I (hyomandibular) (spiracle of clasmobranchs) Visceral Aortic arch IT arch II (hyoid) Visceral Visceral Visceral pouch II cleft IT groove II Visceral Aortic arch LIT arch IIT (1st branchial) Visceral Visceral Visceral pouch ITI cleft IIL groove II] Visceral Aortic arch 1V arch 1V (2nd branchial) Visceral Visceral Visceral pouch IV cleft IV groove IV Visceral Aortic arch V arch V (8rd branchial) Visceral Visceral Visceral pouch V cleft V groove V Visceral Aortic arch VI arch VI (4th branchial) Visceral Visceral Visceral pouch VI cleft VI groove VI

(vestigial in frog) (lacking in frog) (lacking in frog) 132. EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

(branchiae). That on V is rudimentary. Later a fold grows from arch II to cover the external gills completely on the right, but with an opening on the left known as the atriopore (“ spiracle”’). While this is taking place the grooves between arches II, III, IV, V, and VI break through into the corresponding visceral pouches to form the visceral clefts. Internal gills (demibranchs) develop in the clefts, and the external gills disappear. Meantime the mouth has opened and developed horny jaws.

The tail arises by the backward growth of the tissue in the neural folds (Bijtel) at the point where they united over the blastopore. The notochord and neural tube grow backward, carrying epidermis and muscle-forming material with them. Dorsal and ventral folds make the tail fin.

The paired limbs arise as limb buds. The anterior buds arise first but are concealed beneath the operculum. The one on the left side appears first, pushing through the atriopore.

BODY FORM IN THE CHICK. — The body of the chick is cut off from the blastoderm by the outgrowth of a head fold accompanied by an undercut, the subcephalic pocket, which appears during the first day of incubation. This fold extends backward in the form of an inverted U as the lateral folds arise. These are also accompanied by undercuts known as the lateral sulci. Finally there is a posterior tail fold accompanied by a subcaudal pocket appearing on the third day. Outgrowth at the folds with some undercutting as well causes the body of the embryo to stand up from the surrounding blastoderm to which it is attached by a short pedestal, the umbilical stalk. The head bends down sharply at the cephalic flexure, but pressing against the yolk, it turns or twists toward the right so that the left side of the head rests on the yolk. The ventral bend is known as flexure, the dextral twist is known as torsion. Flexure and torsion commence in the middle of the second day of incubation, and continue in a caudal direction until, at the end of the fourth day, the chick lies completely on its left side.

The primordia of the brain and sense organs arise much as they do in the frog. A stomodeum appears early in the third day of incubation, the proctodeum during the fourth day. Four visceral grooves (in the order I, IJ, III, IV) and five arches appear between the end of the second and beginning of the fourth day of incubaet Te teta mene em mena

BODY FORM IN THE CHICK 133

‘ .

‘ ‘


D

A, 25 hours of incubation. B, 38 hours of D, 68 hours of incubation. Compare


C

Fig. 85. — Growth of the chick embryo.

incubation. C, 48 hours of incubation. Figs. 200, 206, 212, 218, respectively. A, B, approx. <9; C, D, approx. X4.

(After Duval.) 184 EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

tion. Only the first three clefts actually open into the fore-gut, and these are soon closed again.

The tail arises from the backward growth of the tail fold but never attains any great length.

The limb buds appear during the third day of incubation.

BODY FORM IN MAN. — Human embryologists distinguish three periods during intra-uterine development: the period of the ovum, from fertilization to germ-layer formation, two weeks; the period of the embryo, until the embryo has assumed a definitely human appearance, the end of the second month; and the period of the fetus. It is the second of these with which we are concerned.

By the end of the third week the head fold is formed, and at the fifth the tail fold is developed. Neural folds are formed and unite


. A B C

Fia. 86. — Growth of the humanembryo. A, neural folds (after Ingalls). B, neural tube commencing, seven somites (after Payne). C, ten somites, (after Corner).

much as in the chick (Fig. 86). The primordia of eye, ear, and nose-also_originate in a similar manner. Five visceral grooves are formed, by the end of the fifth week, separating six visceral _arches, but although the visceral pouches appear and unite with the grooves, true visceral clefts are not formed. By the end of the seventh week, the visceral grooves have disappeared. A cephalic flexure appears in the fifth week. The neck (cervical) THE DISPOSITION OF THE YOLK IN THE FROG 135

flexure develops in the week following and accelerates the disappearance of the visceral grooves.

A tail is developed from the tail fold which is quite prominent during the six and seventh weeks of development but is overgrown and resorbed during the eighth.

Limb buds make their appearance toward the end of the fifth week.

B. THE YOLK SAC

Yolk sacs are found in the development of all large-yolked eggs, among both anamniotes and amniotes. As the name implies, this structure is a larger or smaller bag protruding from the body and connected with it by a yolk stalk.

Origin and development. — The yolk sac develops from the outer margin of the blastoderm which advances under the vitelline membrane and around the yolk mass until the yolk is completely enclosed (Fig. 82).

Function and fate. — It contains the yolk, which, in meroblastic cleavage, is not divided among the blastomeres. But it plays a far more important part in development than simply acting as a reservoir for food reserves. (It is lined with endoderm just like that of the intestine, and is furnished with arteries, veins, and capillaries, which make up the area vasculosa. The endodermal lining digests the yolk, and the vitelline veins carry the digested food to the developing embryo. We may think of the yolk sac as an extra-embryonic intestine. \ It is interesting to note that in some viviparous elasmobranchs, like the dogfish, the yolk sac continues to be of use, even after the yolk is consumed. Pressed against the wall of the uterus it absorbs the uterine “milk” which this organ secretes (much like a tertiary egg envelope) and conveys it to the embryo through the vitelline veins. A similar device is seen among the marsupials (page 144).

he yolk sac is usually drawn up into the body when the umbilicus closes and is later resorbed.)

THE DISPOSITION OF THE YOLK IN THE FROG. — The frog has no yolk sac, for the yolk is divided among the large blastomeres which later make up the floor of the intestine. The mass of these cells, however, creates a bulge on the ventral surface of the embryo (Fig. 84) which resembles externally a small sac. 136 EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

THE YOLK SAC OF THE CHICK. — The yolk sac of the chick is formed by the advancing edge of the blastoderm. Looking down on the blastoderm of the chick at the end of the first day of incubation (Fig. 79), one distinguishes a series of concentric rings. Proceeding from the periphery inward, we note first the area vitellina externa, consisting of the margin of overgrowth and the zone of junction (page 112). Then comes the area vitellina interna in which we can distinguish the ectoderm and endoderm, the latter closely applied to the yolk. Finally there is distinguished the area vasculosa into which the mesoderm has pushed, splitting, as it advances, into the somatic layer (next the ectoderm) and the splanchnic layer (next the endoderm). Between the somatic and splanchnic layers lies the exocoel (extra-embryonic coelom), as the coelem is called when it extends beyond the boundaries of the embryo. The blood vessels of the area vasculosa develop in the splanchnic mesoderm. The exocoel separates the splanchnopleure (endoderm and splanchnic mesoderm) from the somatopleure (somatic mesoderm and ectoderm), so that it can be said that the yolk sac of the chick consists of splanchnopleure. By the end of the fourth day of incubation the yolk is completely covered except for a small area at the vegetal pole, known as the yolk sac umbilicus (Fig. 89C, D). When the chick hatches, the empty yolk sac still attached to the intestine is drawn into the coelom and gradually disappears.

THE YOLK SAC OF MAN. — In man, as in other mammals, the yolk sac arises in connection with gastrulation. The endoderm growing out from the lower surface of the embryonic knob apparently reorganizes itself to form a very small gastrocoel or yolk sac. The roof of this gastrocoel forms the roof of the digestive canal; the anterior end is set off (with the head fold) to make the fore-gut; the posterior end is set off (with the tail fold) to make the hind-gut. The remainder constitutes the small yolk sac (Fig. 86A). This sac is later squeezed between the amnion and chorion (Fig. 90), and loses its connection with the intestine, through the degeneration of the yolk stalk.

In other mammals (Fig. 68) the endoderm grows completely around the interior of the trophoblast and forms a larger yolk sac. In the mouse, where the embryonic knob hangs well down in the cavity of the blastocyst, this results in the knob’s being covered AMNION AND CHORION 141

Body stalk



Amniotic cavity f Allantois



Yolk sac Chorion A B Chorion laeve


~ Amniotic


cavity Umbilical cord Allantoi¢e stalk “t) Chorion ‘Placenta frondosum D

Fig. 90. — Diagrams to show development of extra-embryonie structures in human embryo. Four stages illustrated by sagittal sections. (After Corning.)

Trophoblast

Rauber’s cells



Ectoderm 4 — Endoderm

Amniotic a folds 2 Primitive ,

streak — Betoderm




Fig. 91. — Amnion formation in the bat’s egg. A, primary amniotic cavity. B, origin of amniotic folds. (After Van Beneden.) 142 EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

ate. The embryonic disc thus comes to form part of the blastocyst wall.

The amnion and chorion are formed by amniotic folds (Fig. 91). The internal limb of each fold is formed of somatopleure derived from the embryonic disc and will form the amnion as in the chick. The outer limb of each fold, however, is formed of ectoderm derived from the trophoblast associated with somatic mesoderm and gives rise to the chorion. The mesoderm growing out from the primitive streak, and delaminating into somatic and splanchnic layers, becomes the lining of the exocoel.

D. THE ALLANTOIS

The development of an amnion and chorion is always accompanied by the appearance of another sac, the allantois. This extra-embryonic structure appears as an evagination from the hind-gut and is therefore lined with splanchnopleure. It grows out through the exocoel of the umbilical stalk into the exocoel of the chorion, which it usually fills. It is filled with an allantoic fluid which receives the nitrogenous wastes of the embryo in the form of uric acid (Needham), and may be thought of in the first instance as an extra-embryonic urinary bladder. As it fills the chorion, its walls, being composed of splanchnic mesoderm in the outer layer, easily fuse with the mesodermal layer of mesoderm of the amnion, chorion, and yolk sac, whenever these structures come together. Furthermore, it has an area vasculosa served by the allantoic (umbilical) veins and arteries. This area vasculosa when applied to the chorion is the region where the blood is nearest to a source of atmospheric oxygen. Here an exchange of gases, carbon dioxide for oxygen, takes place, and the allantois may be considered as an extra-embryonic lung.

In the cleidoic egg of reptiles, birds, and egg-laying mammals, the allantois also takes part in the formation of an albumen sac wherein this material is digested. In the marsupials and placental mammals it contributes to the formation of a placenta (hemiplacenta in marsupials) whereby digested food is obtained from the maternal circulation. These functions of the placenta will be discussed in the sections following.

ALLANTOIS OF THE CHICK. — The allantois (Fig. 92) arises towards the end of the third day as an evagination from the floor of ALLANTOIS OF THE CHICK 143

the hind-gut. It grows out between the yolk and the wall of the subcaudal pocket into the exocoel (Fig. 89B). Here it expands greatly until by the end of the ninth day it has filled the entire exocoel. Its outer wall unites with the chorion (Fig. 89C) to form a chorio-allantois, its inner wall unites with the amnion above and the yolk sac below.

Now the chorion, carrying with it an inner fold of allantois, grows down beyond the yolk-sac umbilicus (page 136), and around

Yolk sac



Area vasculosa

Allantois

Fia. 92. — The embryo chick and its extra-embryonic structures on the sixth day of incubation. X13. (After Duval.)

the mass of albumen, which has become more viscous through the loss of water and is displaced towards the lower side of the egg. The albumen is enclosed in a double-walled sac of chorion with the allantois between the two walls of the sac (Fig. 89D). The layer next to the albumen is the ectoderm of the chorion, but the mesoderm of the allantois supplies the blood vessels. It is interesting to observe that it is the ectoderm of the albumen sac which absorbs the albumen, whereas in the yolk sac it is the endoderm which carries on this function.

By the twelfth day of incubation the albumen sac is closed except at the yolk-sac umbilicus where it has an open connection 144. EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

with the yolk sac. On the sixteenth day the albumen is consumed. On the seventeenth the yolk-sac umbilicus closes by the constriction of a ring of mesoderm derived from the old edge of the blastoderm. The yolk sae with the remains of the albumen sac still attached is retracted into the body cavity of the chick on the nineteenth day of incubation, aided by contractions of the amnion and the inner wall of the allantois.

ALLANTOIS OF MAN AND OTHER MAMMALS. — In most of the mammals there is a well-developed allantois, arising like that of the chick, growing into the exocoel, and uniting with the chorion to participate in the formation of the placenta, but the human allantois is rudimentary. It arises as a minute tubular evagination which develops from the endodermal roof of the gastrocoel even before the formation of the tail fold. It grows out into the body stalk, a mass of mesoderm connecting the embryo with the chorion (Fig. 90) for a short distance, but never gets so far as the chorion. However, the allantoic (umbilical) blood vessels continue down the body stalk to the chorion where they form a chorionic area vasculosa in the region of the developing placenta.

THE PLACENTA

Before discussing the human placenta it will be helpful to review the different types of placentation recognized in mammals. Two types are distinguished according to the degree of union between the trophoblast and the lining of the uterus (mucosa); a second basis of distinction is whether the wall of the allantois comes in contact with the chorion or not.

Indeciduate type. — The first_type of placenta is called indeciduate. In this type, found in several groups particularly the ungulates, the trophoblast is closely applied to the mucosa but both retain their integrity. The blood vessels of the placenta absorb food material excreted by the mucosa and exchange carbon dioxide for oxygen by diffusion.

Marsupials. — Among the marsupials are found both nonallantoic and allantoic hemiplacentae. In the opossum, Didelphys (Fig. 938A), the enlarged yolk sac is pressed against the trophoblast, which in turn is closely applied to the mucosa, forming folds which project into depressions in the uterine wall. The absorbed nutriment is conveved to the embrvo bv means of INDECIDUATE TYPE 145

the area vasculosa of the yolk sac. In Perameles (Fig. 93B), an allantoic hemiplacenta is formed by the union of the allantoic sac with the trophoblast. Where this hemiplacenta touches the





Allantoic cavity

Exocoel

cavity

Fig. 93.— The extra-embryonic structures of marsupials. Diagrammatic. <A, Didelphys. B, Perameles. (After Jenkinson.)

mucosa the epithelium of the latter thickens and is invaded by maternal capillaries. The trophoblast is said to be resorbed so that the capillaries of the allantois come into intimate connection with those of the uterus. It should be mentioned in this connection that Perameles also possesses a well-developed area vasculosa

Allantoic cavity

Amniotic Exocoel cavity

Wall of uterus

Yolk sac

Allantoie cavity

stalk


Fig. 94. — Diagram of extra-embryonic structures in the pig. (After Smith.)

on the yolk sac. It is very probable, therefore, that both yolk sac and allantoic circulations are concerned with the nutrition of the developing young.

Ungulates. — In the ungulates there is a well-developed allantoic placenta of the indeciduate type (Fig. 94). The blastocyst elongates, and over its surface appear projections of the tropho146 EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

blast which contain a core of mesoderm. These projections, villi, grow into corresponding depressions of the uterine wall, called crypts. The allantois meantime has filled the exocoel, and capillaries from the allantoic arteries and veins penetrate the mesodermal cores of the villi. These capillaries are brought very near those of the uterine wall, but the blood remains separated from that of the mother by (1) the endothelial lining of the maternal capillaries, (2) the connective tissue of the mucosa, (3) the epithelium of the mucosa, (4) the trophoblast, (5) the mesoderm of the villi, and (6) the endothelial lining of the fetal capillaries (Fig. 99A, B). At birth the villi are pulled out of the crypts, and the placenta, with the remaining embryonic membranes, is discharged as the “ after-birth.”

Deciduate type. — The second type of placentation is called deciduate. In this type the trophoblast attacks the mucosa and



Amniotic cavity

Exocoel

Wall of uterus

Fia. 95. — Diagram of extra-embryonic structures in the dog. Sagittal section. (After Jenkinson.)

erodes part of the lining.. It is characteristic of the majority of the clawed mammals (unguiculates) and primates. In the first group the placenta is allantoic; in the primates, non-allantoic. Carnivores. — In the carnivores (Fig. 95) is found a deciduate placenta of the allantoic type. The blastocyst elongates although not to the extent it does in the ungulates. During this time PLACENTA OF MAN 147

the epithelium of the uterus is cast off. At the circular zone of the uterus which is in contact with the equator of the blastocyst the epithelium of the uterus fails to regenerate. Into this zonary area grow the villi of the trophoblast which become penetrated by the allantoic capillaries. The villi send out branched processes, each with its capillaries, which surround the maternal capillaries. Thus the maternal blood is separated from that of the fetus by (1) the endothelium of the maternal capillaries, (2) a varying amount of maternal connective tissue, (3) the trophoblast, (4) a varying amount of chorionic connective tissue, and (5) the endothelial lining of the fetal capillaries (Fig. 99C). At birth a certain amount of maternal tissue is torn away with the placenta.

PLACENTA OF MAN. — In the human placenta there is the most intimate contact between the maternal and fetal circulation.

Amniotic cavity (. (9 3 Allantoic cavity ARAN ANG v bp \ QS! NC Kes LP Yolk sac WON p ZA cavity Exocoel a f


Fig. 96. — Diagram of extra-embryonic structures in man. (After Kolliker.)

The placenta is non-allantoic. It will be recalled that the embryonic knob retains its connection with the trophoblast as the body stalk. Into the body stalk grows the small evagination from the hind-gut which represents the endodermal lining of the allantois (Fig. 90). It:never comes in contact with the trophoblast and soon degenerates. The limiting sulci of the amnion approach each other and become the walls of the umbilical cord. 148

EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

This encloses (Fig. 96) the body stalk, yolk stalk, allantoic stalk, as well as the two umbilical arteries and two umbilical veins which


Fia. 97. — Diagram to show the uterine deciduae (After JXollmann.)

(human).

vessels.

grow out from the body of the embryo towards the trophoblast. These umbilical blood vessels represent the allantoic vessels of all other amniotes. Later the umbilical veins fuse, and all this tissue assumes common connective tissue characteristics with the exception of the walls. of the blood vessels.

The deciduae. — It will be remembered that the blastocyst burrows into the uterine wall, eroding epithelium, connective tissue, and blood

As the embryo increases in size, this erosion continues

and the embryo sinks into the compact layer of the mucosa and

comes in contact with the spongy layer. The mucosa grows around the burrowing embryo, shutting it off from the cavity of the uterus. There may now be distinguished (Fig. 97) three regions in the mucosa: (1) the decidua basalis, to which the blastocyst is attached; (2) the decidua capsularis, which cuts off the blastocyst from the uterine cavity; and (8) the decidua vera,


Chorion

car


eS Seite

Fig. 98. — Human embryo 11 mm. in length, about 6 weeks old, to show extra-embryonic structures.

x1}. (After Arey.)

including the remainder of the uterine lining. THE CHORION 149

The chorion. — The trophoblast, while entering the uterine wall, becomes differentiated into an outer syncytial layer and an inner cellular layer. During the process of implantation, nutrition is obtained by the syncytial layer, which sends out projections or false villi into the maternal tissue. Thereafter mesodermal cores grow into the false villi converting them into the true villi which later receive capillaries from the umbilical blood vessels.

Chorionic _— epithelium ~yu gy Se






\ Chorionic Uterine PS ») epithelium epithelium Bt (| Uterine . ay) E ~ epithelium Uterine cs VN Uterine capillary ~~" =~ + SS capillary




Chorionic ~~ epithelium

Uterine Villus

capillary Lacuna

=— Decidua

Sy

Fig. 99. — Sections through placentae of A, pig; B, cow; C, cat; and D, human. (Semi-diagrammatic after Grosser.)

Some of these bore into the uterine wall to become fixation villi. The others, losing their syncytial layer, remain in the space between the trophoblast of the chorion and the maternal tissue as nutrition villi (Fig. 98). These are bathed in maternal blood which is brought into the intervillous space and carried thence by the eroded uterine capillaries. Only those villi which are in 150 EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTUBES

contact with the decidua basalis persist; the others degener ite, thus differentiating the chorion into the chorion frondosum, with villi, and the chorion laeve, devoid of the same. In the human

Amnion 6,

Villi of chorion

Decidua basalis


Fused decidua vera and

capsularis

Fia. 100. — Diagram of fetus (near term) to show relationships of extra-embryonic structures and deciduae. (After Ahlfeld.)

placenta the maternal blood is separated from the fetal blood stream by only (1) the cellular layer of the trophoblast, (2) the chorionic connective tissue of the villi, and (3) the endothelia of the fetal capillaries (Fig. 99D). SUMMARY 151

Parturition. — The history of the extra-embryonic structures as well as that of the deciduae is terminated by birth (parturition). Owing to the absence of an allantoic sac the amnion enlarges to fill the exocoel. Later, growth of the fetus results in pressing the ch nfs eve and decidua capsularis against the decidua vera and

i, ating the uterine cavity (Fig. 100). At birth the placenta, carrying with it the decidua basalis, and the attached membrane, whfch represegee the fused amnion, chorion laeve, decidua capsularis, and decjghaa vera, are cast off as the caul or ‘‘after-birth.”

Y p

i SUMMARY

The method by which the external form of the vertebrate embryo is assumed is closely connected on the one hand with the shape of the gadtppla, and on the other with the presence or absence of certain extra-embryonic structures, the yolk sac, amnion, chorion, and allantois.

With growth in length we associate the occurrence of metamerism, or the serial repetition of parts, and the formation of a head and a tail. The paired limbs arise as buds.

The yolk sac is found only in embryos developing from extremely telolecithal eggs. It is lined with endoderm and functions as an extra-embryonic intestine. The splanchnic layer of the mesoderm adjacent to it develops an area vasculosa which conveys the digested yolk to the body of the embryo.

The amnion and the chorion arise typically from folds of somatopleure which fuse above the embryo, thus giving rise to an inner membrane, the amnion, and an outer one, the chorion. The amnion, lined with ectoderm internally, contains amniotic fluid in which the embryo develops. The chorion, lined with somatic mesoderm internally, contains the exocoel, a continuation of the embryonic coelom. Neither of these membranes has any vascular system of its own. They are found only in the development of reptiles, birds, and mammals.

The allantois always develops in amniote embryos. It arises as a ventral evagination of the hind-gut and typically grows out into the exocoel which it completely fills. It functions as an extra-embryonic bladder and lung, and because of its vascular area may act (in connection with the chorion) as an organ of nutrition, e.g., as an albumen sac. - 152 EMBRYONIC FORM AND EXTRA-EMBRYONIC STRUCTURES

In mammals the blood vessels of the allantois invade the chorion giving rise to the placenta, an organ where substances may be exchanged by diffusion between the maternal and fetal blood streams. The placenta is connected to the embryo by the umbilical stalk, whose walls are formed by the amnion. In some mammals, such as the carnivores and primates, parts of the uterine wall, the deciduae, are concerned in the formation of the placenta, and cast off with them at birth.

References

Allen, FE. (ed.) 1932. Sex and Internal Seerctions.

Assheton, R. 1916. Growth in Length.

Hertwig, O. (ed.) 1906. Handbuch, cte., I, Chaps. 6-8.

Jenkinson, J. W. 1913. Vertebrate Embryology, Chaps. 8 and 9.

Kerr, J. GG. 1919. Textbook of Embryology, 1, Chaps. 7 and 8.

Lillie, F. R. 1916. The Development of the Chick, 2nd Ed., Chap. 7.

“Marshall, F. H. A. 1922. The Physiology of Reproduction, 2nd Td. Meisenheimer, J. 1921--1930. Geschlecht und Geschlechter im Tierreiche. Needham, J. 1931. Chemical !mbryology, III, Sections 20-22, 24, and Epilegomena.

Chapter VII Experimental Vertebrate Embryology

Recent progress in vertebrate embryology has resulted so largely from the application of the experimental method that even the beginning student must acquaint himself with some of the methods used and the results so far obtained. Within the limits of this text only a few of the important fields in which the experimental method has been employed can be mentioned, and the student must be referred to more extended treatises for further information concerning this relatively new and important branch of embryology.

The amphioxus and the frog have long been used by experimental embryologists, and more recently successful methods have been devised for the experimental study of the developing egg of the hen. Triton, in Germany, and Ambystoma, in this country, are urodele amphibia whose eggs have been particularly favorable for experimental embryology. The eggs of mammals, difficult to obtain, and, so far, impossible to orient during the carly stages of embryology, have been employed to a lesser extent.

The experimental embryologist alters the conditions under which the egg develops in the hope of determining the factors involved in particular developmental processes. It is appropriate that we conclude our study of carly embryology with a short account of some of the experiments which bear directly on the organization of the fertilized egg, on differentiation during cleavage and the formation of the germ layers, and on the direct effects of environmental factors upon development.

A. THE ORGANIZATION OF THE FERTILIZED EGG

The fertilized egg, as we have seen, is the product resulting from the union of two germ cells, the egg and the sperm. It contains two pronuclei, of maternal and paternal origin, respectively, as well as a mass of cytoplasm which is almost ex clusively maternal in origin. The nuclei contain the parental 153 154 EXPERIMENTAL VERTEBRATE EMBRYOLOGY

contributions of genes, the units which together determine the hereditary characters of the developing individual. How the genes produce their effects is not known, but it is certain that they must act directly upon the cytoplasm. Accordingly we may turn first to experiments dealing with the nuclei of the fertilized egg, and second, to those concerned with the organization of the cytosome itself.

1. Tue NuciueEar ORGANIZATION

The fact that the fertilized egg has the diploid number of chromosomes and of genes, while the two gametes have the haploid number, naturally leads to the question whether the diploid number is necessary to continued development. A considerable number of experiments bear directly upon this question.

Artificial parthenogenesis. — The frog’s egg can be induced to develop by puncture with a finely pointed glass needle (Loeb and others). These artificially parthenogenetic eggs have given rise to tadpoles and frogs. Apparently the number of chromosomes is redoubled (diploid number), perhaps by a division of the chromosomes without a corresponding division of the cell. But the genes are exclusively maternal in origin.

Irradiated sperm. — Sperms of the amphibian Triton, treated to an appropriate dosage of radium emanations, have their nuclei injured in such a way that they are unable to form normal pronuclei (Hertwig). But they retain their mobility and are able to penetrate the egg and induce development. The sperm head remains in the cytoplasm and passes to one or another of the developing blastomeres but takes no part in mitosis and ultimately degenerates. The number of chromosomes in the larval cells is usually haploid, although redoubling may occur.

Irradiated eggs. — Eggs of Triton have also been irradiated to kill the egg nucleus and then fertilized with normalsperms. These eggs develop with the haploid number of chromosomes, showing that either pronucleus, maternal or paternal, is adequate for development.

Fertilization of enucleate eggs.— In some marine invertebrates, e.g., the sea urchin, the egg can be broken into fragments by shaking. Naturally only one fragment will contain the nucleus, but the enucleate fragments can be fertilized and will FERTILIZATION OF ENUCLEATE EGGS 155

give rise to dwarf but otherwise normal larvae. This phenomenon is known as merogony. A similar result can be obtained in telolecithal vertebrate eggs such as those of Triton, where several sperms normally enter the egg. After the entrance of the sperm

Egg nucleus



Sperm nuclei

Fig. 101. — The experimental production of haploid larvae in Triton. A, fertilized egg with two sperm nuclei. B, same after constriction separating part of egg with diploid nucleus (right) from part with haploid nucleus formed by supernumerary sperm (left). C, showing relatively more advanced diploid embryo (right) and less advanced haploid embryo (left). D, diploid larva. , haploid larva. (After Spemann.)

it is possible to constrict the egg into two halves, by means of a fine hair loop, in such a way that the female pronucleus lies in one half (Fig. 101). This half will eventually have the diploid number of chromosomes, for a sperm pronucleus will conjugate with 156 EXPERIMENTAL VERTEBRATE EMBRYOLOGY

the egg pronucleus. The other half will have only the haploid number. Both halves will develop into larvae, one of which will have haploid and the other diploid nuclei. Species hybrids. — Many experiments have been made in the attempt to fertilize the egg of one species with a sperm from another species. Often as in the teleost fish f 5 \ (Moenkhaus), both pronuclei take part in the oF i 2 subsequent cleavage, although frequently the


i Wry chromosomes from the two pronuclei (Fig. 102) i i : form separate groups on the mitotic spindle i il | | ; (gonomery). But in other cases Hertwig has i | ft : shown that the male pronucleus takes no part in i | § subsequent cleavages, so that the embryo reall

| : 1 y y

| Q develops parthenogenetically. Jd! a i Natural interspecifie hybrids in both plant and No animal kingdom are more common than for Fie. 102. -—Chro- merly believed. Usually these interspecifie hymosomes in ana- brids are infertile, as the mule and many types phase of first of hybrid bony fish, but they often grow to larger cleavage of a hy- oto. ane . " : : . . brid fish, Menidia 512° and are more active (hybrid vigor) than the eggand Fundulus parents. sperm, illustrat- |The equivalence of the pronuclei. — Although,

Chien eo Mncake as we have seen in Chapter IV, the pronuclei may

haus.) differ from one another in regard to individual

genes, the experiments mentioned above indi cate that a single set of genes, paternal or maternal, is adequate

for the development of an egg. It must be recognized that the

experimental haploid animals are frequently less vigorous than normal diploid forms.

2. THE ORGANIZATION OF THE CYTOPLASM

Polarity. — The primary expression of the egg’s organization is the polarity already impressed upon it in the ovary (page 37). That this polarity is itself not due to gravity is shown by the fact that frog eggs which are kept in motion during early development give rise to normal embryos (Morgan, Kathariner). But polarity is not immutable, for many experiments in which the eggs of frogs have been made to develop in an inverted position (Born, Pfliiger, Morgan) show that the yolk streams down through the egg, and ASYMMETRY 157

cleavage begins in the relatively yolk-free region which was formerly the vegetal pole.

Gradient. — There scems to be good reason to suppose that the polar axis represents a metabolic axial gradient (Child), for when dilute solutions of lethal chemicals, e.g., potassium cyanide, are applied to the frog’s egg (Bellamy), disintegration begins at the animal pole and continues toward the vegetal pole, which is the last part of the egg to be affected.

Cytoplasmic materials. — In some animals there seems to be a definite stratification of materials in the egg along the polar axis, but when this stratification is disturbed by whirling the eggs about in a centrifuge, the eggs develop with the original polarity undisturbed. On the other hand, in telolecithal eggs like that of the frog, centrifuging distorts the cytoplasmic framework (Conklin).

Bilaterality. — The animal pole marks the anterior end of the developing amphibian embryo. Its dorsal side is marked by the gray crescent which appears on the side opposite the point of entry of the sperm. Many observations (Jenkinson and others) show that the point of entry marks a second dorso-ventral axis and establishes the bilaterality of the developing embryo. But in parthenogenetic eggs (when development is initiated by puncture) the point of entrance of the needle seems to have no constant relation to the subsequent bilaterality of the egg. This would indicate (Huxley and de Beer) that the egg has an underlying bilaterality of its own which is not strong enough to withstand the stronger stimulus afforded by the entrance of the sperm but is apparent in parthenogenesis.

Bellamy has described a second axial gradient in the frog’s egg shown by the action of potassium cyanide in which the high point centers in the gray crescent. ‘This is the dorso-ventral axis of the embryo, which is therefore normally determined by the entrance point of the sperm.

Asymmetry. — The vertebrate embryo is not, strictly speaking, bilaterally symmetrical. <A third axis or gradient from one side to the other (usually left to right) is often apparent, as seen in the development of the atriopore on the left side of the tadpole, the fact that the heart of the chick develops on the right side, and the fact that the head turns to the right in torsion. The stomach in 158 EXPERIMENTAL VERTEBRATE EMBRYOLOGY

all vertebrates is twisted to the left of the mid-line, and many other examples might be mentioned. When this asymmetry is reversed we have the phenomenon known as situs inversus, and this condition can be reproduced experimentally by developing the egg in a lateral temperature gradient and in other ways. Thus the egg of the hen when overheated on the left side develops situs inversus. It has been shown by Spemann that, when two blastomeres which would ordinarily produce the right and left sides of an embryo are separated by a hair loop, the left-hand blastomere gives rise to a normally asymmetrical embryo, while the right-hand blastomere gives rise either to an embryo with normal asymmetry or to one with situs inversus.

These few examples of experiments on the fertilized egg indicate that the egg is a complex system with a definite organization indicated by its three axial gradients corresponding to its three spatial dimensions, viz., an antero-posterior gradient (polarity), a dorso-ventral gradient, and frequently a left-right gradient. Furthermore, the system contains two complete sets of chromosomes and genes, either one of which is adequate in further development.

B. ORGANIZATION OF THE EMBRYO DURING CLEAVAGE

Cell-lineage studies seemed to indicate that the dividing egg is becoming a mosaic of blastomeres, cach set apart from the others to form a specific portion of the embryo. Roux (1888) was the first to realize that this might be tested experimentally. He destroyed one of the 4-blastomeres of the frog’s egg and observed that the other gave rise to a }-embryo, which later regenerated the missing portion.

Later investigators devised a number of methods by which blastomeres could be separated from each other, by shaking them, cutting them apart with fine needles, constricting them with fine threads, or placing them in artificial calcium-free sea water. Blastomeres of marine eggs in this medium separate immediately, and when returned to normal sea water continue their development without further separation (Herbst).

Regulation and mosaic eggs. — The results of their experimentation seemed to indicate that in some eggs, e.g., those of the amphioxus (Fig. 103), either of the }-blastomeres might, when REGULATION AND MOSAIC EGGS 159

separated, give rise to complete embryos (Wilson). These were called regulation eggs and were said to have indeterminate cleavage. In others, such as Styela (Conklin) or the molluse Dentalium (Wilson), the }-blastomeres give rise only to }-embryos (Fig. 103). These were called mosaic eggs and were said to have determinate cleavage.

Experiments on frog’s eggs had been inconclusive until recently an improved technique has made it possible to separate blastomeres of the two-cell stage completely (Schmidt, 1980, 1933).

eee ! 4

Fig. 103. — Diagram to show the fate of isolated blastomeres from mosaic and regulation eggs. A, mosaic egg of Dentalium. At left, a complete embryo produced by entire egg: at right, partial embryos produced by the }-blastomeres when artificially separated. B, regulation egg of Amphiorus. At left, embryo pro duced by entire egg; at right, perfect dwarf embryos produced by 34-blastomeres. (After Wilson.)


These experiments show that each of the 3-blastomeres can give rise to a complete and perfect larva, provided only it contains some of the gray crescent region. If, on the other hand, the egg is so constricted that the first cleavage divides it into an animal and a vegetal half, the animal half, containing the gray crescent, Fig. 104. — Embryos arising from separated 3-blastomeres of the newt’segg. A, the constriction separates the dorsal and ventral halves of the embryo. B, the constriction separates the right and left halves. C, perfect embryo arising from the dorsal }-blastomere. D, mass of cells arising from ventral }-blastomere. E, two perfect embryos arising from right and left 3-blastomeres respectively. (After

Spemann.) (160) REGULATION AND MOSAIC EGGS 161

gives rise to a complete embryo, while the vegetal half, lacking this region, is unable so to organize itself (Fig. 104). The importance of the gray crescent as the seat of the organizer is discussed on page 169. This seems to indicate that Roux’s results were due to the presence of the injured blastomere inhibiting complete development on the part of the uninjured blastomere. In this connection it is interesting to note that Witschi (1927) has


Fig. 105. — Experiment demonstrating equality of nuclei formed during cleavage (Triton). A ligature has been tied around the fertilized egg restricting the nucleus to the right-hand portion. A, 16-cell stage, one nucleus passing into left-hand portion. B, ligature tightened to separate the two portions. C, perfect embryos formed by the separate portions. The nucleus of a ith-blastomere equivalent to that of a complete zygote. (After Spemann.)

described a case in which two eggs were found in a single chorion. Each of them was flattened on the side next to its neighbor and in later development showed deficiencies in the corresponding region.

A beautiful demonstration that it is the cytoplasm and not the nucleus which is concerned with differentiation during cleavage is afforded by an instructive experiment of Spemann. If the egg 162 EXPERIMENTAL VERTEBRATE EMBRYOLOGY

is tied off before cleavage so that the nucleus is confined in one of its halves (Fig. 105), all cleavage planes will be restricted to that’ half until eventually a cleavage plane, in this case at the fourth cleavage, coincides with the plane of constriction. The nucleus which enters the previously enucleate half is naturally one which would serve a ;-blastomere. If the loop is now tightened until the two haves are completely separated, the portion containing this single nucleus will give rise to an embryo like the one from the portion containing the fifteen nuclei and exactly like one arising from a complete fertilized egg.

Pressure experiments. — Further examples of the regulative power of some eggs may be seen in pressure experiments. If the eggs of the frog are placed between glass plates during cleavage, the third cleavage planes will be meridional instead of latitudinal, and the fourth cleavage plane is latitudinal (Fig. 106). Now if




Fia. 106. — Diagram to show new relationship of blastomeres in frog’s egg resulting from pressure during cleavage. A, normal 8-cell stage. B, 8-cell stage formed under pressure. C, normal 16-cell stage. D, 16-cell stage formed under pressure. Cells normally in animal hemisphere shown in stipple. (Suggested by a diagram in Wells, Huxley and Wells.)

the eggs are released, their later development will be quite normal even though the blastomeres are occupying positions unlike those which they hold ordinarily.

Double embryos. — Still another example may be seen in the eggs of Triton. If these are freed from the egg envelopes, the blastomeres at the two-cell stage assume a dumb-bell shape. Mangold discovered that, by placing one embryo in the two-cell stage over another (Fig. 107), a double embryo resulted almost exactly similar to a single embryo in the four-cell stage, and would MONOVULAR TWINS AND MONSTERS 163

develop as such, provided only that the gray-crescent regions of the two fell in the same plane. Otherwise double monsters resulted. We shall see the importance of the gray-crescent region more clearly in a later section dealing with the organizer which develops in this region.

Chemo-differentiation. — It is quite clear from these experiments that the developing egg of the regulation type possesses a very great plasticity in the early stages of development as compared to the mosaic type illustrated by the egg of the tunicates. It may be assumed that the difference between these two lia. 107. — Double embryo arising types lies in the time at which defi- from fusion of 2-cell stages of

. : Triton alpestris (above and below) nite organ-forming substances are and Triton taeniatus (right and segregated in the cytoplasm of the left) when laid over each other egg. Conklin has demonstrated crosswise. Note that a new that these rogions are segrogated {Hwee ip under ny al after fertilization in the egg of the — geidel.) tunicate, whereas in amphibian eggs the only segregated region is that of the gray crescent. Huxley (1924) has suggested the term chemo-differentiation for the segregation of organ-forming substances. A good example is seen in the first division of the egg of Dentaliwm, the mollusc referred to above where a polar lobe passes completely to one or the other of the first 4-blastomeres. The cell receiving this lobe gives rise to the apical organ, mesoderm, foot, and shell. Here the very first division of the fertilized egg is determinate and dependent upon the segregation of the organ-forming substance found in the polar lobe (Fig. 108).

Monovular twins and monsters. — The extreme plasticity of the vertebrate egg as seen by the fact that either two separate individuals or duplicate monsters may be formed from the complete or partial separation of blastomeres suggests an explanation of identical twins and the duplicate monsters which play so large a part in the study of teratology. It is generally accepted that identical, as distinguished from fraternal, twins are the product 164 EXPERIMENTAL VERTEBRATE EMBRYOLOGY

of a single fertilized egg which has divided completely during early embryology, whereas the duplicate monsters, ranging from Siamese twins to monsters in which one individual is but a parasite upon the body of the other, result from incomplete separation. These identical twins are always of the same sex. Ordinary or




Ja Wy


Fig. 108. — Diagram to show possible distribution of organ-forming substances in mosaic and regulation eggs. *A, immature egg. B, mature egg showing stratified organ-forming substances. C, cleavage with equal division of chromosomes. D, segregation of one organ-forming substance in left-hand 3-blastomere. EF, equal division of organ-forming substances between the 3-blastomeres. (After Wilson.)

fraternal twins (triplets, etc.) are supposed to be the product of separate eggs which ovulated and were fertilized at about the same time. Such twins are frequently of different sexes. In this connection we might mention the free-martin, a sterile female twinned with a male, not infrequent among cattle, and supposed to result from one of two eggs which develop a common chorion and therefore a common blood stream. It is supposed that a male PLASTICITY (DEPENDENT DIFFERENTIATION) 165

hormone circulating in the common blood stream inhibits the normal development of the female twin, so resulting in the production of the sterile free-martin (Lillie).

C. ORGANIZATION OF THE EMBRYO DURING GERM-LAYER FORMATION

The amphibian embryo is remarkably hardy and during the early stages of development will endure very severe operations. The work of Harrison in this country and of Spemann in Germany has resulted in the perfection of a method of removing portions of an embryo (micro-dissection) and grafting them into a new environment, where they will continue development. The embryo from which the portion is removed is known as the donor; the removed portion is called the graft (transplant); and, when the portion removed is transplanted into another embryo, the second embryo is termed the host.

The accompanying diagram (Fig. 109) will bring out some of the methods which have been developed in transplantation experiments. Thus the graft may be transplanted into another portion of the same embryo (homoplastic transplantation).!. It may be transplanted into another embryo of the same species (heteroplastic transplantation). It may even be transplanted into an embryo of another species or genus (xenoplastic transplantation).

Another method which has brought interesting results is to transplant the removed portion into a nutrient medium and allow it to develop there under sterile conditions (explantation). This is also known as cultivation “ in vitro,” which means in glass. Another ingenious technique is to transplant the graft into a cavity of another embryo and allow it to develop there. The example shown in the diagram is of a bit of embryonic tissue transplanted into the eyeball of a tadpole, which acts as a nutrient chamber. Hoadley and others have developed a technique of grafting chick-embryo tissue from a donor to the chorio-allantois of a host. Such a technique is called interplantation (implantation).

Plasticity (dependent differentiation). — In the amphibian egg, which is of the regulation type, it has been demonstrated that the

1 Some" investigators use autoplastic=homoplastic; homoplastic=heteroplastic; and heteroplastic = xenoplastic. 166 EXPERIMENTAL VERTEBRATE EMBRYOLOGY

presumptive organ regions of the blastula, (and until about the middle of gastrulation) are quite plastic, i.e., can be transplanted into other localities and will give rise to the organs appropriate



Cover glass


Fig. 109. — Diagrams to show different methods of transplantation, ete. A, homoplastic transplantation. B, heteroplastic transplantation (both donor and host of same species). C, xenoplastic transplantation (donor and host of different species). D, explantation (in vitro). E, interplantation. (Based on a diagram of Dirken.)

to the new locality. Thus, material which is presumptive epidermis can be transplanted into a region where it will become neural plate, mesoderm, orevenendoderm. Or on the other hand, material which is presumptive endoderm can be made to develop into ectoderm or mesoderm by transplantation. The only exception to this rule is the region where the dorsal lip is to form. PLASTICITY (DEPENDENT DIFFERENTIATION) 167

This will never give rise to anything except dorsal lip and the structures arising from the dorsal lip. This exception will receive special attention later (page 169).

Very instructive experiments are those in which material is transferred from a species with heavy pigmentation (Triton taentatus) to one with light pigmentation (Triton cristatus).


Fig. 110. — Xenoplastic transplantation between Triton taeniatus (dark), the donor, and Triton cristatus (light), the host to show early plasticity. A, immediately after transplantation. B, the transplant developing in the gill region. C, the gills of the transplant relatively more advanced. D, section through C in the gill region. (After Spemann.)

Here the graft preserves its racial character of pigmentation while otherwise conforming to the development of the host. Figure 110 illustrates such an example of xenoplastic transplantation. The light-colored graft from T’. cristatus has developed into part of the neural tube of the host, where it stands out by reason of its light color. In the reciprocal transplantation (Fig. 111), the dark graft from 7’. taeniatus has given rise to the right external gills of the host. 168 EXPERIMENTAL VERTEBRATE EMBRYOLOGY

The loss of plasticity (self-differentiation). — After gastrulation is well under way this plasticity seen in earlier stages is lost. The various regions of the embryo have become determined and


A B

Fig. 111. — Reciprocal transplant to that shown in Fig. 110. Here T. cristatus

(light) is the donor and 7. taeniatus (dark) is the host. A, after transplantation.

B, the transplant developing in the neural plate (region of the brain). C, section in later stage showing transplant developing in forebrain. (After Spemann.)

thenceforth will give rise only to the structures normally developing from them. In other words, the amphibian embryo does not undergo chemo-differentiation until this time. From now on it isa real mosaic. Figure 112 shows a neurula in which the various

Ear field Neural tube field



Eye field

Nose field

Lens field Hindlimb field

Balancer field . Heart jj,‘ Forelimb field

field fielg Fig. 112. — Diagram of an amphibia neurula showing organ fields as determined by transplantation experiments. (After Huxley and de Beer.)

organ fields are determined. If a bit of tissue is removed from the eye-field and transferred to the flank of another neurula (Fig. 113), it will give rise only to an eye, even in its new and abnormal environment.

Similar experiments have been carried on with the chick (implantation on chorio-allantois), and it has been proved that the eye-field, ear-field, limb-buds, and other regions will develop and give rise only to the respective organs.

Very striking results have been obtained by implanting portions of rat embryos on the chorio-allantois of the chick, and a considerable amount of self-differentiation has been demonstrated.


Fig. 113. — Self-differentiation in the toad Bombinator. A, donor in early neurula stage showing region from which graft was taken. B, host in late neurula stage. C, section through later embryo of host, showing graft forming optic cup in region normally occupied by pronephros. (After Spemann.)

The organizer. — The loss of its early plasticity by the embryo seems to be due to the presence of an organizer (organisator) as discovered by Spemann. In the amphibian embryo this is the dorsal-lip region, already mentioned. It will be recalled that this region alone of the presumptive organ fields of early gastrulation did not show the phenomenon of plasticity. Wherever it is transplanted it will become dorsal lip. But the most striking thing about this dorsal-lip region is that wherever it is transplanted it will bring about involution, and will transform the surrounding material into organ fields such as are ordinarily found about the dorsal lip. In a word, the grafted dorsal lip organizes a new, secondary, embryo about itself, quite independent of the embryo which is organized about the dorsal lip of the host (Fig. 114). The organizer itself undergoes involution beneath the surface of the host and becomes the notochord and the somite-mesoderm of the secondary embryo. The other structures, such as neural


Fig. 114. — Effect of transplanting organizer. A, dorsal view of host (Triton taeniatus) in neurula stage. B, right side view at same stage showing secondary neural plate induced by organizer (dorsal lip region) of the donor (T'’riton cristatus) shown in white. C, later stage showing primary embryo in side view and secondary embryo in dorsal view. D, transverse section through C. (After Spemann and Mangold.)

plate, eyes, ears, kidney, heart, etc., arise from host tissue which has been brought under the influence of the organizer. Even after gastrulation this influence is continued, as can be shown by the following experiment. A bit of gastrocoel roof (notochord and mesoderm, in the urodeles), when transplanted into the GRAVITY (AND CENTRIFUGAL FORCE) 171

side of the gastrocoel, will induce the formation of neural folds above it.

So great are the powers of induction possessed by the organizer that it can cause presumptive ectoderm to become mesoderm or endoderm, and conversely, presumptive mesoderm can be transformed into ectoderm.

It is noteworthy that the organizer can exert its influence even in xenoplastic transplantation, e.g., the organizer from a toad can induce the formation of a secondary embryo in.a newt. Apparently the effects of the organizer are physico-chemical in nature, for the dorsal-lip region can be narcotized, boiled, or even dried, and still induce the formation of a secondary embryo. It is suggestive that bits of agar after being in contact for some time with an organizer are themselves capable of producing induction. There is reason to believe that the glycogen (animal starch) content of the organizer has something to do with its effects, and quite recently, it has been reported that cephalin will bring out about the induction of a secondary embryo. Many parts of the adult vertebrate are capable of bringing about induction, but in the living embryo, the chemical substance responsible is found only in the organizer itself.

D. ENVIRONMENTAL FACTORS IN DEVELOPMENT

Many experiments have been carried on in the attempt to find the definite results produced on the developing embryo by changes in the environment. These investigations have established normal limits of temperature, etc., within which development can be completed. Within these limits, although development may be altered as to rate, etc., it is nevertheless carried on to a successful outcome. Beyond these limits the alterations are so profound as to produce monsters or cause death. Among the factors susceptible to experimental control are gravity, heat, light, the chemical constitution of the environment, and food.

Gravity (and centrifugal force). — It has been remarked (page 156) that the original polarity of the egg is not due to any effect of gravity. In telolecithal eggs, however, gravity may have some effect on the course of development. Thus frog’s eggs when forcefully inverted may give rise to duplicate monsters. The hen’s egg if not rotated at regular intervals fails to hatch. It has been 172 EXPERIMENTAL VERTEBRATE EMBRYOLOGY

shown (Dareste, 1877) that this is due to the failure of the yolk sac to complete its development. It adheres to the allantois and cannot be retracted into the body as in normal development.

The influence of gravity may be shown in an exaggerated manner by prolonged centrifuging. It was found by O. Hertwig that, if the frog’s egg is centrifuged during cleavage, the yolk is so concentrated in the vegetal hemisphere that the cleavage planes fail to cut through it and the end result is meroblastic cleavage suggestive of that seen in the chick (Fig. 115). Undivided yolk Heat. — The rate of development is directly affected by temperature. Thus for the egg of the frog (Rana fusca, Hertwig) the normal temperaFia. 115.— Vertical section ture is about 15°-16°C. From this

through blastula of a frog’s egg point up to about 20°—22° C., devel following centrifuging. (After Hertwig.) opment continues normally; beyond this limit it is abnormal, death ensuing rapidly at 30°C. Below 15° C., development is retarded progressively with the drop in temperature, and at 0°C. cleavage ceases completely.

For the hen’s egg, Kaestner determined the optimum temperature for normal development to be between 35° and 39°C. (95°-102° F.). The maximum temperature tolerated is 43°C., the minimum 28° C. (20°-21° C., Edwards).

Eggs of either frog or hen which have been exposed to extreme heat or cold and then returned to the optimum temperature often develop abnormally. A common type of monster is one in which neural plate and notochord are split (spina bifida).

Very striking results have been obtained by subjecting the eggs of the frog or the hen to a temperature gradient, that is, controlling the temperature so that one side is hotter or colder than the other. If the gradient runs along the polar axis, and the greater heat is applied to the animal pole, the result is that the embryos and larvae have overlarge heads; if the higher temperature is applied to the vegetal pole, the head region is subnormal. When the temperature gradient is applied laterally, the development of the heated side proceeds more rapidly than that of the cooled side.

It may be concluded that, within the limits of toleration, development is accelerated by increased temperatures and retarded by decreased temperatures.

Light and other forms of radiation. — In spite of a considerable number of experiments designed to determine the effects of definite intensities and wavelengths of light upon the developing embryo, the results are as yet too inchoate to be discussed in an elementary text.

Ultra-violet light, X-rays, and radium emanations in extreme dosage cause the cessation of development. In smaller dosage, they bring about anomalies (abnormalities in structure caused by disturbances in development). It should be remembered that the work of Miiller and others indicates that these agents accelerate the rate of mutation of Drosophila genes, and so induce genetic point mutations as well as developmental anomalies.

Chemical composition. — The chemical composition of the surrounding medium affects profoundly the nature of development. The embryo cannot develop without oxygen, for it cannot live without respiration. It has been pointed out by Morgan that frog’s eggs in the very center of the egg mass often develop abnormally (spina bifida, etc.). And it has long been known that the hen’s egg ceases development if the pores of the shell are closed by water glass, varnish, or other agents.

Water, too, is an essential. The growth of the embryo depends upon the absorption of water, and all embryos must undergo their development within a watery medium. Even the terrestrial embryo has its private pond in the amnion. A slowing up in the rate of development, accompanied by abnormalities and a large percentage of deaths, results from incubating hen’s eggs in a desiccator. The percentage of water in the frog’s egg increases steadily during the first two weeks of development.

A very striking series of experiments was carried on by Herbst on the development of the sea urchin in artificial sea waters which had been made up omitting one after another of the elements found in normal sea water. Jenkinson, summarizing the evidence says:

“The experiments which we have been considering are unique of their kind, and it is impossible to exaggerate their importance. For, whatever may be the ultimate explanation of the facts, there can be no doubt whatever that the most complete demonstration has been given of the absolute necessity of many of the elements occurring in ordinary sea water, its normal environment, for the proper growth and differentiation of the larva of the sea urchin. Nor is this all. Some of the substances are necessary for one part or phase of development, some for another, some from the very beginning, others only later on. Thus potassium, magnesium, and a certain degree of alkalinity are essential for fertilization, chlorine and sodium for segmentation, calcium for the adequate cohesion of the blastomeres, potassium, calcium and the hydroxyl ion for securing the internal osmotic pressure necessary for growth, while without the sulph-ion and magnesium the due differentiation of the alimentary tract and the proper formation of the skeleton cannot occur; the secretion of pigment depends on the presence of some sulphate and alkalinity, the skeleton requires calcium carbonate, cilia will only beat in an alkaline medium containing potassium and magnesium, and muscles will only contract when potassium and calcium are there.”

The addition of chemicals to the medium has resulted in many interesting disturbances in development. We can call attention here to two only. In the sea urchin it was found that the addition of lithium salts to sea water caused the embryo to undergo a very curious form of gastrulation, in which the endoderm and mesoderm were evaginated instead of being invaginated (Herbst). Such an embryo is called an exogastrula.

Quite recently, Holtfreter (1933) has induced exogastrulation in the egg of Triton by removing the egg envelopes and placing the developing egg in weak Ringer’s salt solution. In the cases where development continued for some length of time (Fig. 116), it was discovered that the embryo developed in two parts, an ectodermal portion with no differentiation, connected by a narrow isthmus to a mesendodermal portion in which differentiation proceeded, but inan abnormal fashion. The embryo is inside-out. The mesendodermal portion of the exo-embryo develops a typical notochord, somites, kidney, gonad, a heart (empty), and a digestive tube, in which all the typical regions are indicated, including visceral pouches. These results confirm those of transplantation and explantation experiments discussed in an earlier section. FOOD (INCLUDING HORMONES AND VITAMINS) 175

Food (including hormones and vitamins).— The amount and kind of food supplied to the developing young naturally affect the subsequent development. ‘Thus, if frog tadpoles are fed on an exclusively vegetarian diet, the intestine becomes much longer than when an exclusively meat diet is offered. Specific foods often result in equally definite changes in the body. Thus Gudernatsch discovered that frog tadpoles fed on thyroid tissue grew less rapidly but underwent metamorphosis much more rapidly than the controls. Thymus-fed tadpoles, on the other hand, had a retarded metamorphosis accompanied by excessive growth. Later investigations indicate that the effects of thyroid are due to a hormone formed by this gland (thyroxin), which is a definite factor in bringing about amphibian metamorphosis.


Fig. 116. — Exogastrulation in Ambystoma. A, B, exogastrulae showing direction of displacements during exogastrulation, compare Fig. 74. C, section of later exo-embryo. (After Holtfreter in Huxley and de Beer.)


It is interesting to note that by the use of thyroid or thyroxin the Mexican axolotl (Fig. 117) may be induced to undergo metamorphosis, when it becomes a normal Ambystoma tigrinum. Otherwise the axolotl becomes sexually mature in the larval condition (neoteny), and was, therefore, long thought to be a separate species.

In this connection we may refer briefly to the many experiments dealing with the effects of the various endocrine glands when given as food or as transplants and the effects produced when these glands are removed at their first appearance (extirpation). Without going into details, for the results of these experiments are sometimes ambiguous, we may say only that they


Fig. 117. — Metamorphosis in Ambystoma. A, neotenic larva (axolotl). B, metamorphosed adult. (After Diirken.)

indicate the importance of hormones in embryonic as well as in adult life.

The rdle of the vitamins in the metabolism of the embryo is too little understood at the present time for us to do more than allude to this subject. Vitamin E is often called the anti-sterility vitamin because its absence from the diet results in loss of the reproductive power. Adamstone (1931) in this laboratory has shown that the chick embryo produced by hens on a vitamin-Efree diet dies early in development following extensive disturbances in the blood-vascular system.

Summary

Experimental embryology demonstrates that development is epigenetic. Given a suitable inheritance of genes, and a favorable environment, development proceeds normally through stages of increasing complexity. Any alteration, either in the genetic complex or in the factors of the environment, will bring about alterations in development.

The fertilized egg shows a definite organization as seen in its polarity and symmetry. These seem to be the expression of axial gradients. Sooner or later the cytoplasm of the egg undergoes chemo-differentiation and develops organ-forming substances — sooner in mosaic eggs, later in regulation eggs.

Cleavage segregates the organ-forming substances as they are differentiated, with the result that the isolated blastomeres of mosaic eggs have a limited potency, those of regulation eggs have a greater potency.

During germ-layer formation, the presumptive organ regions are segregated into the different germ layers. Among the vertebrates this reorganization is effected by an organizer, which in the frog is associated with the dorsal lip of the blastopore, and in the chick with the homologous primitive streak.

Even in regulation eggs a mosaic stage is established during germ-layer formation. The different organ fields are now determined, the earlier plasticity disappears, and each field is capable only of self-differentiation.

References

Allen, E. (ed.) 1932. Sex and Internal Secretion.

Bertalanffy, L. von, and Woodger, J. H. 1933. Modern Theories of Development. Brachet, A. 1931. L’oeuf et les factors de l’ontogénése.

Brambell, F. W. R. 1930. The Development of Sex in Vertebrates.

de Beer, G. R. 1926. Introduction to Experimental Embryology.

Child, C. M. 1915. Individuality in Organisms.

Duesberg, J. 1926. L’oeuf et ses localisations germinales.

Dirken, B. 1932. Experimental Analysis of Development (trans). Fauré-Fremiet, M. E. 1925. La cinétique du développement.

Huxley, J.S., and de Beer,G.R. 1934. The Elements of Experimental Embryology. Jenkinson, J. W. 1909. Experimental Embryology.

1917. Three Lectures on Experimental Embryology. Korschelt, E. 1927-1931. Regeneration and Transplantation.

Morgan, T. H. 1928. Experimental Embryology.

1934. Embryology and Genetics.

Needham, J. 1932. Chemical Embryology. Newman, H.H. 1923. The Physiology of Twinning. “Russell, E.S. 1930. The Interpretation of Development and Heredity. Schleip, W. 1929. Die Determination der Primitiventwicklung.

Weiss, J. 1930. Entwicklungsphysiologie der Tiere.

Wilson, E. B. 1925. The Cell in Development and Heredity, 3rd Ed.

Shumway (1935): Preface - Contents | Part I. Introduction | Part II. Early Embryology | Part III. Organogeny | Part IV. Anatomy of Vertebrate Embryos | Part V. Embryological Technique


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