Book - Comparative Embryology of the Vertebrates 3

PART III The Development of Primitive Embryonic Form

The general procedures leading to the development of primitive embryonic body form in the chordate group of animals are:

( 1 ) Cleavage. Cleavage is the division of the egg into progressively smaller cellular units, the blaslomeres (Chap. 6).

(2) Blastulation. Blastulalion results in the formation of the blastula. The blastula is composed of a cellular blastoderm in relation to a fluid-filled cavity, the blastocoel. The blastoderm of the late blastula is composed of neural, epidermal, notochordal, mesodermal, and entodermal major presumptive organ-forming areas. In the phylum Chordata, the notochordal area is the central region around which the other areas are oriented (Chap. 7). The major presumptive organ-forming areas of the late blastula exist in various degrees of differentiation (Chap. 8).

(3) Gastrulation. This is the process which effects a reorientation of the presumptive organ-forming areas and brings about their axiation antero-posteriorly in relation to the notochordal axis and the future embryonic body (Chap. 9). During gastrulation the major organ-forming areas are subdivided into minor areas or fields, each field being restricted to the development of a particular organ or part. (Pp. 378, 446, 447.

(4) Following gastrulation, the next step in the development of embryonic body form is tubulation and extension of the major organ-forming areas (Chap. 10).

(5) As tubulation and extension of the organ-forming areas is effected, the basic or fundamental conditions of the future organ systems are established, resulting in the development of primitive body form. As the development of various vertebrate embryos is strikingly similar up to this point, the primitive embryonic body forms of all vertebrates resemble each other (Chap. II).

In the drawings presented in Part III, the following scheme for designating the major organ-forming areas existing within the three germ layers is adhered to:

EPIDERMAL ECTODERM

NEURAL ECTODERM

MESODERM

ENTODERM

NOTOCHORD

PRE-CHOROAL

PERIBLAST

PLATE

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6

cleavage (Segmentation) and Blastulation

A. General considerations

1. Definitions

2. Early history of the cleavage (cell-division) concept

3. Importance of the cleavage-blastular period of development

a. Morphological relationships of the blastula

b. Physiological relationships of the blastula

1 ) Hybrid crosses

2) Artificial parthenogenesis

3) Oxygen-block studies

4. Geometrical relations of early cleavage

a. Meridional plane

b. Vertical plane

c. Equatorial plane

d. Latitudinal plane

5. Some fundamental factors involved in the early cleavage of the egg

a. Mechanisms associated with mitosis or cell division

b. Influence of cytoplasmic substance and egg organization upon cleavage

1) Yolk

2) Organization of the egg

c. Influence of first cleavage amphiaster on polyspermy

d. Viscosity changes during cleavage

e. Cleavage laws

1 ) Sach’s rules

2) Hertwig’s laws

6. Relation of early cleavage planes to the antero-posterior axis of the embryo

B. Types of cleavage in the phylum Chordata

1. Typical holoblastic cleavage

a. Amphioxus

b. Frog (Rana pipiens and R. sylvatica)

c. Cyclostomata

2. Atypical types of holoblastic cleavage

a. Holoblastic cleavage in the egg of the metatherian and eutherian mammals

1 ) General considerations

2) Early development of the rabbit egg

a) Two-cell stage

b) Four-cell stage

c) Eight-cell stage

d) Sixteen-cell stage

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

e) Morula stage

f) Early blastocyst

3) Types of mammalian blastocysts (blastulae)

b. Holoblastic cleavage of the transitional or intermediate type

1) Amhystoma maculatum (punctatum)

2) Lepidosiren paradoxa

3) Necturus maculosus

4) Acipenser sturio

5) Amia calva

6) Lepisosteus (Lepidosteus) osseus

7) Gymnophionan amphibia 3. Meroblastic cleavage

a. Egg of the common fowl

1 ) Early cleavages

2) Formation of the periblast tissue

3) Morphological characteristics of the primary blastula

4) Polyspermy and fate of the accessory sperm nuclei

b. Elasmobranch fishes

1 ) Cleavage and formation of the early blastula

2) Problem of the periblast tissue in elasmobranch fishes

c. Teleost fishes

1) Cleavage and early blastula formation

2) Origin of the periblast tissue in teleost fishes

d. Prototherian Mammalia

e. Cleavage in the California hagfish, Polistotrema (Bdellostorna) stouti

C. What is the force which causes the blastomeres to adhere together during early cleavage?

D. Progressive cytoplasmic inequality and nuclear equality of the cleavage blastomeres

1. Cytoplasmic inequality of the early blastomeres

2. Nuclear equality of the early blastomeres

E. Quantitative and qualitative cleavages and their influence upon later development

A. General Considerations

1. Definitions

e period of cleavage (segmentation) immediately follows normal fertilization^ or any other means which activates the egg to develop. It consists of a division of the entire egg or a part of the egg into smaller and smaller cellular entities) In some species, however, both chordate and non-chordate, the early cleavage stages consist of nuclear divisions alone, to be followed later by the formation of actual cell boundaries (fig. 62).^The cells which are formed during cleavage are called blastomere^

cleavage of the egg continues, the blastular stage ultimately is reached. The blastula contains a cavity or blastocoel together with an associated layer or mass of cells, the blastodern^ The blastula represents the culmination and end result of the processes at work during the cleavage period. Certain aspects and problems concerned with blastulation are considered separately in the following chapter. However, the general features of blastular formation are described here along with the cleavage phenomena.

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2. Early History of the Cleavage (Cell-division) Concept

An initial appreciation of the role and importance of the cell in embryonic development was awakened during the middle period of the nineteenth century. It really began with the observations of Prevost and Dumas in 1824 on the cleavage (segmentation) of the frog’s egg. The latter observations represented a revival and extension of those of Swammerdam, 1738, on the first cleavage of the frog’s egg and of Spallanzani’s description in 1780 of the first two cleavage planes, “which intersect each other at right angles,” in the egg of the toad. Other studies on cleavage of the eggs of frogs, newts, and various invertebrates, such as the hydroids, the starfish, and nematodes, followed the work of Prevost and Dumas. The first reported cleavage of the eggs of a rabbit was made in 1838-1839, a fish in 1842, and a bird in 1847. (See Cole, ’30, p. 196.) Newport, in 1854, finally founded the new preformation by showing that the first cleavage plane in the frog’s egg coincided with the median plane of the adult body (Cole, ’30, p. 196).

In the meantime, the minute structures of the bodies of plants and animals were intensively studied, and in 1838-1839, the basic cellular structure of living organisms was enunciated by Schlciden and Schwann. Following this generalization, many studies were made upon the phenomenon of cell division in plant and animal tissues. These observations, together with those made upon the cleaving egg, established proof that cells arise only by the division of pre-existing cells; and that through cell division the new generation is formed and maintained. Thus it is that protoplasm, in the form of cells, assimilates, increases its substance, and reproduces new cells. Life, in this manner, flows out of the past and into the present, and into the future as a never-ending stream of cellular substance. This idea of a continuous flow of living substance is embodied well in the famed dictum of R. Virchow, “Omnis Cellula c Cellula,” published in 1858 (Wilson, E. B., ’25, p. 114).

The consciousness of life at the cellular level acquired during the middle period of the nineteenth century thus laid the groundwork for future studies in cytology and cellular embryology. Much progress in the study of the cell had been made since R. Hooke, in 1664, described the cells in cork. In passing, it should be observed, that two types of cell division, direct and indirect, were ultimately defined. For the latter, Flemming in 1882, proposed the name mitosis, while the direct method was called amitosis.

3. Importance of the Cleavage-Blastular Period of Development

The period of cleavage and blastular formation is a time of profound differentiation as well as one of cell division. For, at this time, fundamental conditions are established which serve the purposes of the next stage in development, namely, gastrulation. Experimental embryology has demonstrated

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

that optimum morphological conditions must be elaborated during the cleavage phase of development along with the developing physiology of the blastula.

a. Morphological Relationships of the Blastula

There are two aspects to the developing morphology of the blastula, namely, the formation of the blastoderm and the blastocoel.

During cleavage and blastulation, the structure of the blastoderm is elaborated in such a manner that the major, presumptive, organ-forming areas of the future embryonic body are segregated into definite parts or districts of the blastoderm. The exact pattern of arrangement of these presumptive, organforming areas varies from species to species. Nevertheless, for a particular species, they are arranged always according to the pattern prescribed for that species. This pattern and arrangement of the major, presumptive, organforming areas permit the ordered and symmetrical migration and rearrangement of these areas during gastrulation.

Similarly, the blastocoel is formed in relation to the blastoderm according to a plan dictated by the developing mechanisms for the species. One of the main functions of the blastocoel is to permit the migration and rearrangement of the major, presumptive, organ-forming areas during gastrulation. Consequently, at the end of the blastular period, the blastoderm and the blastocoel are arranged and poised in relation to each other in such a balanced fashion that the dramatic cell movements of gastrulation or the next period of development may take place in an organized manner.

b. Physiological Relationships of the Blastula

The development of a normal-appearing, late blastula or beginning gastrula in a morphological sense is no proof that proper, underlying, physiological states have been established. A few examples will be given to illustrate this fact:

1) Hybrid Crosses. When the sperm of the wood frog, Rana sylvatica, are used to fertilize the eggs of the ordinary grass frog, Rana pipiens, cleavage and blastulation appear normal. However, gastrulation is abortive, and the embryo soon dies (Moore, ’41, ’46, ’47).

2) Artificial Parthenogenesis. In the case of many embryos, chordate and non-chordate, in which the egg is stimulated to develop by means of artificial activation, the end of the blastular stage may be reached, but gastrulative processes do not function properly. A cessation of development often results.

3) Oxygen-block Studies. In oxygen-block studies, where the fertilized eggs of Rana pipiens are exposed to increased partial pressures of oxygen from the time of fertilization to the four- or eight-cell stage, the following cleavages and the morphology of the blastula may appear normal, but gastrulation does not occur. Similar oxygen-pressure exposures during the late blastular and early gastrular stages have no effect upon gastrulation. This fact suggests that

GENERAL CONSIDERATIONS

283

important physiological events accompany the earlier cleavage stages of development (Nelsen, ’48, ’49).

Aside from the foregoing examples which demonstrate that invisible changes in the developing blastula are associated with morphological transformations is the fact that experimental research has demonstrated conclusively that an organization center is present in the very late blastula and beginning gastrula. The organization center will be discussed later. However, at this point it is advisable to state that the organization center is the instigator and the controller of the gastr Illative processes, and gastrulation does not proceed unless it is developed.

The above considerations suggest that the period of cleavage and blastulation is a period of preparation for the all-important period of gastrulation. Other characteristics of this phase of development will be mentioned in the chapter which follows.

4. Geometrical Relations of Early Cleavage

a. Meridional Plane

The meridional plane of cleavage is a furrow which tends to pass in a direction which, if carried to completion, would bisect both poles of the egg passing through the egg’s center or median axis. The latter axis theoretically passes from the midpolar region of the animal pole to the midpolar region of the vegetal pole. The beginning of the cleavage furrow which follows the meridional plane may not always begin at the animal pole (fig. 140D) although in most cases it does (figs. 142A-C; 154A-C; 155A).

b. Vertical Plane

A vertical plane of cleavage is a furrow which tends to pass in a direction from the animal pole toward the vegetal pole. It is somewhat similar to a meridional furrow. However, it does not pass through the median axis of the egg, but courses to one side of this axis. For example, the third cleavage planes in the chick are furrows which course downward in a vertical plane; paralleling one of the first two meridional furrows (fig. 155C). (See also figs. 153D; 154E relative to the third cleavage furrows of the bony ganoid fishes, Amia calva and Lepisosteus (Lepidosteus) osseus.)

c. Equatorial Plane

The equatorial plane of cleavage bisects the egg at right angles to the median axis and halfway between the animal and vegetal poles. It is never ideally realized in the phylum Chordata, and the nearest approach to it is found, possibly, in one of the fifth cleavage planes of the egg of Ambystoma maculatum (fig. 149F) and the first cleavage plane of the egg of the higher mammals (fig. MSA).

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

d. Latitudinal Plane

The latitudinal plane of cleavage is similar to the equatorial, but it courses through the cytoplasm on either side of the equatorial plane. For example, the third cleavage planes of the egg of Arnphioxus (fig. 1401) and of the frog (figs. 141 E; 142F) are latitudinal planes of cleavage.

5. Some Fundamental Factors Involved in the Early Cleavage of the Egg

a. Mechanisms Associated with Mitosis or Cell Division

There are two mechanisms associated with cleavage or cell division:

( 1 ) that associated with the chromosomes and the achromatic (amphiastral) spindle, which results in the equal division of the chromosomes and their distribution to the daughter nuclei, and

(2) the mechanism which enables the cytoplasm to divide.

In ordinary cell division or mitosis these two mechanisms are integrated into one process. However, in embryonic development they are not always so integrated. The following examples illustrate this fact: (1) In the early development of insects, the chromatin materials divide without a corresponding division of the cytoplasm (fig. 62). (2) During the early cleavage phenomena of the elasmobranch fishes, the chromatin material divides before corresponding cleavages of the cytoplasm appear (fig. 158A). (3) In the later cleavage stages of teleost fishes, the peripheral cells of the blastoderm fuse and form a continuous cytoplasm; within this cytoplasm the separate nuclei continue to divide without corresponding cytoplasmic divisions and in this way form the marginal syncytial periblast (fig. 159J, L, M).

On the other hand, cytoplasmic division may occur without a corresponding nuclear division. This behavior has been illustrated in various ways but most emphatically by the work of Harvey (’36, ’38, ’40, ’51) which demonstrates that non-nucleate parts of the egg may divide for a period without the presence of a nucleus. (See, particularly, Harvey, ’51, p. 1349.) Similarly, in the early development of the hen’s egg, a cytoplasmic furrow or division occurs in the formation of the early segmentation cavity without involving a nuclear division (fig. 156C, E). This type of activity on the part of the cytoplasm illustrates the fact that the cytoplasm has a mechanism for cell division independent of the nuclear mechanism. Lewis (’39) emphasizes the importance of the production of a superficial plasmagel “constriction ring” which constricts the cytoplasm into two parts during cell division.

b. Influence of Cytoplasmic Substance and Egg Organization upon

Cleavage

1) Yolk. Since the time of Balfour, much consideration has been given to the presence or absence of yolk as a factor controlling the rate and pattern

GENERAL CONSIDERATIONS

285

of cleavage. Undoubtedly, in many instances, the accumulation of yolk materials does impede or alter the cleavage furrows, although it does not suppress mitotic divisions of the nucleus as shown in the early cleavages in many insects, ganoid fishes, etc. On the other hand, the study of cleavage phenomena as a whole brings out the fact that other intrinsic factors in the cytoplasm and organization of the egg largely determine the rate and planes of the cleavage furrows.

2) Organization of the Egg. An illustration of the dependence of the pronuclei and of the position of the first cleavage amphiaster upon the general organization of the cytoplasm of the egg is shown in the first cleavage spindle in Amphioxus and Stye la. In the eggs of these species, the amphiaster of the first cleavage always orients itself in such a way that the first cleavage plane coincides with the median plane of the future embryonic body. The first cleavage plane, consequently, divides the egg’s substances into two equal parts, qualitatively and quantitatively. The movements of the pronuclei and the first cleavage amphiaster are correlated and directed to this end.

Various theories have been offered in the past to account for the migrations . of the pronuclei at fertilization and for the position of the first cleavage amphiaster. All of them, however, are concerned with the cytoplasm of the egg or its movements, which in turn are correlated with the organization of the egg. (See Wilson, E. B., ’25, p. 426.)

A second illustration of the dependence of the chromatin-amphiaster complex on conditions in the cytoplasm is afforded by experiments of Hans Driesch in 1891 on the isolation of the blastomeres of cleaving eggs of the sea urchin. He found that the first cleavage of the egg occurred from the animal to the vegetal pole, resulting in two blastomeres. Now, if these blastomeres are shaken apart, the following cleavages in the isolated blastomeres behave exactly as if the two blastomeres were still intaet, indicating a definite progression of the cleavage planes. That is, there is a mosaic of cleavage planes determined in the cytoplasm of the early egg.

A third example of the influence of egg organization upon cleavage is afforded by the egg of higher mammals. In this group, the first cleavage plane divides the egg in many cases into a larger and a smaller blastomere. The larger blastomere then begins to divide at a faster rate than the smaller blastomere. This accelerated division is maintained in the daughter cells resulting from the larger blastomere. Here, then, is an egg whose yolk material is at a minimum. Nevertheless, the blastomeres which result from the first cleavage arc unequal in size, and the cellular descendants of one of these blastomeres divide faster than the descendants of the other blastomere. Some conditioning effect must be present in the egg’s cytoplasm which determines the size of the blastomeres and the rate of the later cleavages. Many other illustrations might be given from the studies on cell lineage. However, the conclusion is inevitable that under normal conditions the cause of the cleavage

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

pattern and the rate of blastomere formation is an internal one resident in the organization of the egg and the peculiar protoplasmic substances of the various blastomeres. This apparent fact suggests strongly that the egg in its development “is a builder which lays one stone here, another there, each of which is placed with reference to future development” (F. R. Lillie, 1895, p. 46).

c. Influence of First Cleavage Amphiaster on Polyspermy

In figure 138 is shown the behavior of the sperm nuclei during fertilization in the urodele, Triton. This figure demonstrates that the developing first cleavage amphiaster suppresses the development of the accessory sperm nuclei. Similar conditions appear to be present in the elasmobranch fishes, chick, pigeon, etc.

d. Viscosity Changes During Cleavage

“The viscosity changes that occur in the sea-urchin egg are probably typical of mitosis in general. There is marked viscosity increase in early prophase, then a decrease, and finally an increase just before the cell divides” (Heilbrunn, ’21 ). Similarly, Heilbrunn and W. L. Wilson (’48) in reference to the cleaving egg of the annelid worm, Chaetopterus, found that during the metaphase of the first cleavage the protoplasmic viscosity is low, but immediately preceding cell division protoplasmic viscosity increases markedly.

e. Cleavage Laws

Aside from the factors involved in cleavage described above, other rules governing the behavior of cells during division have been formulated. These statements represent tendencies only, and many exceptions exist. “The rules of Sachs and Hertwig must not be pushed too far” (Wilson, E. B., ’25, p. 985 ) .

1) Sachs’ Rules:

(a) Cells tend to divide into equal daughter cells.

(b) Each new cleavage furrow tends to bisect the previous one at right angles.

2) Hertwig’s Laws:

(a) The typical position of the nucleus tends to lie in the center of the protoplasmic mass in which it exerts its influence.

(b) The long axis of the mitotic spindle typically coincides with the long axis of the protoplasmic mass. In division, therefore, the long axis of the protoplasmic mass tends to be cut transversely.

6. Relation of Early Cleavage Planes to the Antero-posterior Axis of the Embryo

In the protochordate, Styela, the first cleavage plane always divides the yellow and gray crescent material and other cytosomal substances into equal

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right and left halves; it therefore achieves a sundering of the egg substances along the future median plane of the embryo. The second cleavage plane occurs at right angles to the first (Conklin, ’05, a and b). This condition appears to be true of other ascidians, such as Ciona, Clavelina, etc. (Wilson, E. B., ’25, p. 1012). The behavior of the early cleavage planes is similar in Amphioxus (Conklin, ’32). Cleavage planes such as the foregoing, which always divide the egg in a definite way have been described as “determinate cleavage” (Conklin, 1897). Study figures 116, 132 and 167.

The first cleavage plane in the eggs of some frogs (e.g., Rana fusca and Rana pipiens) shows a great tendency to bisect the gray crescent and thus divide the embryo into right and left halves. However, unlike Styela, and Amphioxus, the first plane is not definitely fixed; considerable deviation may occur in a certain percentage of cases in any particular batch of eggs. In the newt, Triturus viridescens, the first cleavage plane generally is at right angles to the median plane of the future embryo (Jordan, 1893). In Necturus maculosus the first cleavage plane may in some eggs coincide with the median plane of the embryo, while the second cleavage plane may agree with this plane in other eggs. In some eggs there is no correspondence between the first two cleavage planes and the median plane of the embryo; however, the planes always cut from the animal to the vegetal pole of the egg (Eycleshymer, ’04).

In the teleost fish, Fund ulus heteroclitus, in the greater percentage of cases, the long axis of the embryo tends to coincide with either the first or second cleavage planes (Oppenheimer, ’36). Other teleost fishes appear to be similar. In the hen’s egg the first cleavage plane may or may not lie in the future median plane of the embryo (Olsen, ’42).

In some species it appears that the unfertilized egg may possess bilateral symmetry. For example, in the frog, Rana fusca, the point of sperm entrance evidently has an influence in orienting the plan of bilateral symmetry, and, as a result, the gray crescent appears opposite the point of sperm contact with the egg. However, in Rana esculenta and in Discoglossus pictus, two other anuran species, there is no constant relationship between the point of sperm entry and the plan of bilateral symmetry of the egg (Pasteels, ’37, ’38). In the latter cases, unlike that of Rana fusca, the stimulus of sperm entry presumably does not influence the plan of bilateral symmetry which is determined previous to sperm entrance.

It is to be noted that there is a strong tendency in many of the above species for the first cleavage amphiaster to orient itself in such a manner as to coincide with the median plane of the embryo or to be at right angles to this plane. This fact suggests that the first cleavage amphiaster is oriented in terms of the egg’s organization. It further suggests that the copulation paths of the respective pronuclei, as they move toward each other, together with the resulting cleavage path of the pronuclei (fig. 1 39), arc conditioned by the inherent organization of the egg’s cytoplasm.

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

B. Types of Cleavage in the Phylum Chordata

Cleavage in the phylum Chordata often is classified as either holoblastic or meroblastic. These terms serve a general approach to the subject but fail to portray the varieties and problems of cleavage which one finds within the phylum. Under more careful scrutiny, three main categories of cleavage types appear with typical holoblastic cleavage occupying one extreme and typical meroblastic cleavage the other, while between these two are many examples of atypical or transitional cleavage types. Moreover, the phenomena of cleavage are variable, and while we may list the typical cleavage of any one species as holoblastic, transitional or meroblastic, under certain modifying circumstances the cleavage pattern may be caused to vary.

Holoblastic cleavage is characterized by the fact that the cleavage furrows bisect the entire egg. In meroblastic cleavage, on the other hand, the disc of protoplasm at the animal pole only is affected, and the cleavage furrows cut through this disc superficially or almost entirely. Superficial cleavage occurs typically in certain invertebrate forms, particularly among the Insecta. However, in a sense, the very early cleavages in elasmobranch fishes, certain teleost fishes, and in birds may be regarded as a kind of superficial cleavage.

1. Typical Holoblastic Cleavage

In typical holoblastic cleavage, the first cleavage plane bisects both poles of the egg along the median egg axis, that is, the first plane of cleavage is meridional. The second cleavage plane is similar but at right angles to the first, thereby dividing the “germ” into four approximately equal blastomeres. (See Sachs’ rule (a), p. 286.) The third cleavage plane in typical holoblastic cleavage occurs at right angles to the median axis of the egg and the foregoing two meridional planes. (See Sachs’ rule (b), p. 286.) As it does not cut along the equatorial plane, but nearer the animal pole, it is described as a latitudinal cleavage. Two meridional cleavage planes (see definition, p. 283) followed by a latitudinal plane (see definition, p. 284) is the cleavage sequence characteristic of the first three cleavage planes of typical holoblastic cleavage. The following chordate species exemplify typical holoblastic cleavage:

a, Amphioxus

In this cephalochordate there exists as typical a form of holoblastic cleavage as is found anywhere in the phylum Chordata. The process of cleavage or segmentation in Amphioxus hdiS been described in the studies of four different men; as such, these descriptions form four of the classics of embryonic study. These studies were made by Kowatewski in 1867; Hatschek in 1881, English translation, 1893; Cerfontaine, ’06; and Conklin, ’32. With the exception of certain slight errors of observation and interpretation, Hatschek’s work is a masterpiece.

Fig. 139. Penetration path of the sperm, copulation paths of the pronuclei, the cleavage path of the pronuclei, and first cleavage spindle. (A) Conditions such as found in the urodele, Triton. (B) Conditions such as found in the protochordate, Styelii. (C) Conditions such as found in the protochordate, Anipkioxus.

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291

The first cleavage furrow cuts through the egg along the median axis of the egg, starting at the postero-ventral side of the egg (fig. MOD, E). It, therefore, is a meridional plane of cleavage. The second cleavage plane cuts at right angles to the first plane, producing four equal cells (fig. 140E, F). The third cleavage involves four blastomeres. Its plane of cleavage is almost equatorial but slightly displaced toward the animal pole, and therefore, more truly described as latitudinal plane of cleavage. This cleavage plane divides each of the four blastomeres into a smaller micromere at the animal pole and a larger macromere at the vegetal pole. Eight blastomeres are thereby produced (fig. 140G-I). In certain cases and at least in some varieties of A mphioxus, the four micromeres may not be placed exactly above the macromeres, but may be rotated variously up to 45 degrees, forming a type of spiral cleavage (fig. 140J). (See Wilson, E. B., 1893.) The fourth cleavage planes are meridional, and all of the eight cells divide synchronously. The result is sixteen cells, eight micromeres and eight macromeres (fig. MOJ, K). The fifth planes of cleavage are latitudinal and simultaneous (fig. MOL). The

. Fig. 140. Early cleavage and blastulation in A mphioxus. (K after Hatschek, 1893; all others after Conklin, ‘32.) (A) Median section through egg in the plane of bilateral

symmetry, one hour after fertilization. Second polar body at animal pole; egg and sperm pronuclei in contact in cytoplasm containing little yolk. Approximate antero-posterior axis shown by arrow. D. and V. signify dorsal and ventral aspects of future embryo. MS. = mesodermal crescent. (B) Sperm and egg nuclei in contact surrounded by astral rays. Sperm remnant, SR., shown at right. (C) First cleavage spindle in posteroventral half of the egg (see fig. 139C). Arrow shows median plane of future embryo and also the median plane of the egg. Observe that the spindle is at right angles to this plane of the egg. (D) Egg in late anaphase of first cleavage. Cleavage furrow deeper at postero-ventral side of the egg. MS. = mesodermal crescent. (E) Two-cell stage. Arrow shows median plane of embryo. MS. = mesodermal crescent now bisected into two parts. (F) Four-cell stage at conclusion of second cleavage, IVi hours after fertilization. (G) Four-cell stage at beginning of third cleavage. Posterior cells, P., slightly smaller than anterior cells. (H) Animal pole above, vegetal pole below. Cell at left is posterior, the one at right anterior. Spindles show third or horizontal cleavage plane. (I) Eight-cell stage, IV 2 hours after fertilization. Posterior cells, below at right, contain most of mesodermal crescent. Arrow denotes antero-posterior axis of embryo. (J) Late anaphase of fourth cleavage. (K) Sixteen-cell stage viewed laterally. There are eight micromeres and eight macromeres. (L) Side view of 32-cell stage, VA hours after fertilization. Every nucleus in anaphase or metaphase of sixth cleavage. (M) Left side of 64-cell stage. Arrow denotes antero-posterior axis of embryo. (N) Blastula, 31A hours after fertilization. Animal pole above, vegetal below. Entoderm cells at vegetative pole are larger, are full of yolk, and are dividing. Blastocoel is large. (O) Eighth cleavage period with more than 128 cells, 4 hours after fertilization. Antero-posterior axis of future embryo shown by arrows. Polar body indicates animal pole of original egg. Dorsal and ventral aspects indicated by D. and V., respectively. MS. = mesodermal crescent. (P) Section of blastula, AV 2 hours after fertilization. Entoderm cells have nuclei shaded with lines. (Q) Section of blastula, 5 Vi hours after fertilization. (R) Section of blastula, 6 hours after fertilization. Mesoderm cells lighter a^d on each side of entoderm cells. Section nearly transverse to embryonic axis. (S) Section of blastula at stage of preceding but in a plane as in (Q). MS. = mesodermal crescent. (T) Pear-shaped, late blastula. Pointed end is mesodermal; entoderm cells have cross-lined nuclei. D. and V. indicate dorsal and ventral aspects of embryo. See also fig. 167.

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plane nearest the animal pole divides each of the eight micromeres into an upper and a lower micromere, while the plane which furrows the eight macromeres divides each into upper and lower macromeres. Thirty-two cells are, thus, the result of the fifth cleavage planes. The lowest of the macromeres are larger and laden with yolk material (fig. MOL). The sixth cleavage planes are synchronous and approximately meridional in direction in all of the 32 cells, resulting in 64 cells (fig. MOM). The blastocoelic cavity is a conspicuous area in the center of this cell mass and is filled with a jelly-like substance (fig. MON). Study also figure 167.

When the eighth cleavage furrows occur, the blastocoel contained within the developing blastula is large (fig. MOP). As the blastula continues to enlarge, the blastocoel increases in size, and the contained jelly-like substance assumes a more fluid condition (fig. MOQ-S). The fully formed blastula is piriform or “pear-shaped” (fig. MOT). (See Conklin, ’32.)

The cleavage pattern of the urochordate, Styela partita, is somewhat similar to that of Amphioxus, but considerable irregularity may exist after the first three or four cleavages. In Styela the ooplasm of the egg contains differently pigmented materials, and yellow and gray crescentic areas are visible at the time of the first cleavage. (See fig. 132.) These different cytoplasmic areas give origin to cells which have a definite and particular history in the embryo. Observations devoted to the tracing of such cell histories are grouped under the heading of “cell lineage.” Cell-lineage observations are more easily made in the eggs of certain species because of definitely appearing cytoplasmic areas, where colored pigments or other peculiarities associated with various areas of the egg make possible a ready determination of subsequent cell histories. The general organization of the egg of Amphioxus, regardless of the fact that its cytoplasmic stuffs do not have the pigmentation possessed by the egg of Styela, appears similar to that of the latter (cf. figs. MOA; 167A). (See Conklin, ’32.)

b. Frog (Rana pipiens and R. sylvatica)

The egg of the frog is telolecithal with a much larger quantity of yolk than is found in the egg of Amphioxus. The pattern of cleavage in the frog, therefore, is somewhat less ideally holoblastic than that of Amphioxus.

The first cleavage plane of the frog’s egg is meridional (figs. MIC; M2A-C) . It occurs at about three to three and one-half hours after fertilization at ordinary room temperature in Rana pipiens. It begins at the animal pole and travels downward through the nutritive or vegetal pole substance, bisecting both poles of the egg. In the majority of eggs, it bisects the gray crescent. (See p. 287.) The second cleavage plane divides each of the first two blastomeres into two equal blastomeres; its plane of cleavage is similar to the first cleavage plane but is oriented at right angles to the first plane (figs. MID; M2D-E). The upper, animal pole end of each of the four blastomeres contains most

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of the dark pigment, while in the lower portion of each blastomere the yellowwhite yolk is concentrated. As a rule, the substance of the gray crescent is found in two of the blastomeres; the four blastomeres under the circumstances are not qualitatively equal.

The third or latitudinal cleavage plane is at right angles to both of the foregoing and somewhat above the equator, dividing each of the four blastomeres into an animal pole micromere and a larger vegetal pole macromere

Fig. 141. Normal development of Rana sylvatica. (A) Egg at fertilization. (B) Formation of gray crescent, sharply defined at one hour after sperm entrance. (C) First cleavage furrow meridional. (D) Second cleavage furrow meridional. (E) Third cleavage furrows, latitudinal in position. Four micromeres above and four macromeres below. (F) Fourth set of cleavage furrows, meridional in position, although some variation may exist and vertical furrows may occur. (G-I) Later cleavage stages. Pigmented pole cells become very small, and pigmented cells creep downward over vegetative pole area. (J) Appearance of dorsal blastoporal lip. (K) Blastoporal lips spread laterally, forming a broad, V-shaped structure. Pigmented cells proceed toward blastoporal lips. (L) Yolk-plug stage of gastrulation. (After Pollister and Moore, ’37.)

Fig, 142. Early development of Rana pipiens. (A) Polar view of first cleavage. The animal pole is considerably flattened at this time and tension lines are visible extending outward along either side of the furrow. (About V/2 hours after fertilization in the laboratory, room temperature 20 to 22® C.) (B) First cleavage furrow a little later.

(C) First furrow proceeds slowly though the yolk of vegetative (vegetal) pole. (D) Second cleavage furow meridional and at right angles to the first furrow. (E) Four-cell stage, view from animal pole. Observe short “cross furrow” connecting first and second cleavage planes. (F) Fourth cleavages meridional or nearly so. Taken from egg spawned in nature. Considerable variation may exist. Some cleavages may be vertical and not meridional. (G-I) Later blastula stage. (J) Stage just before appearance of dorsal lip of blastopore. (K) Dorsal lip of blastopore. (L) Yolk-plug stage of gastrulation.

294

295

Fig. 143. Stages in formation of the blastocoel in the cleaving egg of Rana pipiens taken from stained sections. (A) Eight-cell stage; blastocoel appearing particularly between micromeres. The macromeres form the floor of the developing blastocoel. (B, C) Later stages of formation of the blastocoel. Blastocoel situated at animal pole. Yolk-laden, vegetal pole cells form floor of the blastocoel while smaller, animal pole cells form its sides and roof. (D) Blastocoel at beginning of gastrulation.

(figs. 141E; 142F). The fourth set of cleavages, both in Rana sylvatica and Rana pipiens, in eggs that are spawned naturally, are oriented in a meridional direction (figs. 141F; 142F). These furrows first involve only the animal pole micromeres, but later meridionally directed furrows begin to develop in the yolk-laden macromeres (figs. 141F; 142F).

The cleavage of the various blastomeres of the egg to this point tends to be synchronous, and is comparable to that of Amphioxus. However, from this time on asynchronism is the rule and different eggs in a given lot manifest various degrees of irregularity. Exceptional eggs may occur in which the next two cleavage planes resemble the fourth and fifth series of planes in Amphioxus. But, on the whole, the micromeres divide faster than do the macromeres and thus give origin to many small, heavily pigmented, animal pole cells, while the macromeres or vegetal pole cells are larger and fewer in number. The smaller pigmented cells creep downward gradually in the direction of the larger vegetal pole cells (figs. 141G-I; 142G-K). The latter migration of the

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

Fig. 144. Cleavage in the rabbit egg. (After Gregory, ’30.) (A) One-cell stage. (B)

Two primary blastomeres, one larger than the other. (C) Eight-cell stage. (D) Sixteencell stage. (E) Morula stage of 32 cells. (F) External view of stage approximating that in (G). (G) Inner cell mass and blastocoelic cleft showing in embryo, about IV 2

hours after copulation. (H) Inner cell mass and blastocoelic space in embryo, approximately 90 hours after copulation. Entoderm cells have not yet appeared.

pigment cells is marked toward the end of the blastular period and during gastrulation. Cf. figs. 141 H~L; 142H-L.

The blastocoel within the mass of blastomeres of the cleaving egg of the frog forms somewhat differently from that in Amphioxus in that the cavity arises nearer the animal pole. The smaller micromeres of the animal pole, therefore, are more directly involved than the macromeres of the vegetal pole. Beginning at the eight-cell stage, a spatial separation is present between the four micromeres at the animal pole. The floor of this space or beginning blastocoel is occupied by the yolk-laden macromeres (fig. 143 A, B). As development proceeds, this eccentricity of position is maintained, and the

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blastocoel or segmentation cavity becomes an enlarged space filled with fluid, displaced toward the animal pole (fig. 143B-D). The contained fluid of the blastocoel of amphibia is alkaline, according to the work of Buytendijk and Woerdeman (’27), having a pH of 8.4 to 8.6.

For general references regarding cleavage in the frog, see Morgan (1897); Pollister and Moore (’37); Rugh (’51); and Shumway (’40).

c. Cyclostomata

Cleavage in the eggs of the genera of the family, Petromyzonidae, resembles very closely that of the frog. Further description will not be included. However, in the marine cyclostomes or hagfishes, the cleavage phenomena are strongly meroblastic. (See description of the marine cyclostomatous fish at the end of this chapter.)

2. Atypical Types of Holoblastic Cleavage

A variety of cleavage types is found in the eggs of many vertebrate species which do not follow the symmetrical, ideally holoblastic pattern exhibited in the egg of Amphioxus or even in the egg of the frog. In all of these atypical forms the entire egg ultimately is divided by the cleavage furrows with the possible exception of the eggs of the bony ganoid fishes, Amia calva and Lepisosteus osseus (and also in certain of the gymnophionan amphibia). In the latter species the yolk material at the yolk-laden pole of the egg is invaded by isolated nuclei which form a syncytium in the yolk material. Eventually this yolk material is formed into definite cells and incorporated into the gut area of the embryo.

a. Holoblastic Cleavage in the Egg of the Metatherian and Eutherian

Mammals

1) General Considerations. The eggs of metatherian and eutherian mammals are the most truly isolecithal of any in the phylum Chordata. They have also a cleavage pattern distinct from other chordate eggs. The first cleavage plane in the higher mammalian egg very often divides the egg into a larger and a slightly smaller blastomere (figs. 144B; 145 A, F; 147B, J). As shown by the work of Heuser and Streeter (’41) in the pig, the smaller blastomere is destined to give origin to the formative tissue of the embryo’s body, while the larger blastomere gives rise to auxiliary tissue, otherwise known as the nourishmenFobtaining or trophoblast tissue (fig. 145A-E). The smaller blastomere also contributes some cells to the trophoblast tissue. A similar condition of progressive specialization of the smaller and the larger blastomeres of the two-cell stage, producing two classes of cells, the one mainly formative and the other auxiliary or trophoblast, is present in the monkey (Heuser and Streeter, ’41) and probably in other higher mammals as well.

If one compares the early history of these two blastomeres with the early

Fig. 145. Early development of the pig. (A-E) Fate of the first two blastomeres. The langer blastomere of the two-cell stage gives rise to trophoblast tissue, whereas from the smaller blastomere, formative cells and trophoblast cells arise. (After Heuser and Streeter, Carnegie Inst., Washington, Contrib. Embryol., 20:3.) (F) Section of two cell stage. Specimen secured from oviduct of sow, killed two days, 3 Vi hours after ovulation. (G) Section of four-cell stage. Age is approximately IVz days. (H) Sixteen-cell stage, drawn from unsectioned specimen, protjably 3 Vi days old. (I) Blastular stage. Specimen secured from sow, days after copulation. (J-L) Stages showing the formation of the blastocoel. (J) About 4% days after copulation. (K) Six days, Wa hours after copulation. (L) Six days, 20 hours after copulation. (M) Beginning disintegration of trophoblast cells over the inner cell mass and separation of entoderm cells from the inner cell mass. (N) Trophoblast cells over inner cell mass almost absent, entoderm forms a definite layer below inner cell mass. (O) Trophoblast cells almost absent over the embryonic disc; entoderm layer continuous. (P-R) Stages shown in (M), (N), (O) respectively, showing the whole blastocyst. In (Q) the entoderm cells are shown migrating outward to line the cavity of the blastocyst.

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299

development of other vertebrate eggs, it is apparent that the nutritive (trophoblast) cells are located at one pole, while the formative cells of the embryo are found toward the opposite pole. The latter condition resembles the arrangement of formative cells and nutritive substances in teleost and elasmobranch fishes, in reptiles, birds, and prototherian mammals. This comparison suggests, therefore, that the first cleavage plane in the higher mammals cuts at right angles to the true median axis of the egg (cf. fig. 145A-E). If this is so, the first cleavage furrow should be regarded as latitudinal and almost equatorial, and the two blastomeres should theoretically be arranged as shown in figure 145A.

The determination of the animal and vegetal poles of the egg in this group of vertebrates is difficult by any other means than that suggested above. In many lower chordate species the polar bodies act as indicators of the animal pole, for they remain relatively fixed at this pole of the egg (e.g., Styela, Amphioxus, etc.). But in higher mammals the polar bodies “are never stationary, and there is evidently much shifting” (Gregory, ’30, relative to the rabbit), although in the two-cell stage, the polar bodies often appear between the two blastomeres at one end. It appears in consequence that the fates of the two blastomeres of the two-cell stage serves as a better criterion of egg symmetry at this time than is afforded by the polocytes. According to this view, the smaller blastomere should be regarded as indicating the animal pole, while the larger blastomere signifies the vegetative pole (fig. MSA).

With respect to the statements in the previous paragraph, it is well to mention that Nicholas and Hall (’42) reported that two early embryos may be produced by isolating the blastomeres of the two-cell stage in the rat, and one embryo is produced as a result of experimental fusion of two fertilized eggs. These experimental results suggest that the potencies of the two blastomeres are not so rigidly determined that two different kinds of development result when the blastomeres are isolated. In normal development, however, it may be that the innate potencies of the two blastomeres are not precisely the same. The ability to regulate and thus compensate for lost substances shown by many different types of early embryonic blastomeres, may explain the production of two early embryos from the separated blastomeres of the two-cell stage.

The second cleavage divides the larger blastomere into two cells, giving origin to three cells. Then the smaller blastomere divides, forming four cells. Cleavage from this time on becomes irregular, and five-, six-, seven-, eight-, etc., cell stages are formed.

Segmentation of the higher mammalian egg, therefore, is unique in its cleavage pattern. The synchrony so apparent in the egg of Amphioxus is lacking. Irregularity and individuality is the rule, with the auxiliary or nutritive pole cells dividing faster than those of the formative or animal pole cells.

Moreover, the blastomeres not only show their apparent independence of

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each other through their irregularity in division but also by their tendency to shift their position with respect to one another. One function of the zona pellucida during the early cleavage period appears to be to hold “the blastomeres together” (Heuser and Streeter, ’29). From the 16-cell stage on, the trophoblast or auxiliary cells begin to form the blastocoelic space, first by a flattening process and later by the formation of a cleft among the cells (fig. 145D). The growing presence of the blastocoel consigns the formative or inner cell-mass cells to one pole of the blastula (fig. 145J-L). A blastocoelic space thus is formed which is surrounded largely by trophoblast or nutritive cells (fig. 145K, L). The blastular stage of development of the mammalian embryo is called the blastocyst.

2) Early Development of the Rabbit Egg. The following brief description pertains to the early development of the rabbit egg up to the early blastocyst condition.

a) Two-cell Stage. The two-cell stage is reached about 22 to 24 hours after mating or 10 to 12 hours after fertilizcition. One cell has a tendency to be slightly larger than the other (fig. 144B). (Cf. also figs. MSA, F; 146A; 147B, J.)

b) Four-cell Stage. This stage is present about 24 to 32 hours after mating or 13 to 18 hours after fertilization. The larger cell divides first, giving origin to three cells; the smaller cell then divides. (Cf. figs. MSB, C; M6B, C; M7K, L. ) The mitotic spindles tend to assume positions at right angles to each other during these cleavages.

c) Eight-cell Stage. Eight cells are found 32 to 41 hours after mating. One member of the larger blastomeres of the four-cell stage divides, forming a five-cell condition, followed by the division of the second larger cell, producing six cells. (Cf. figs. MSC; M7M.) After a short period, one of the smaller cells segments, and thus, a total of seven blastomeres is formed. The last cleavage is followed by the division of the other smaller cell, producing eight blastomeres (fig-t- M4C; compare with fig. M7N). The mitotic spindles of each of these cleavages form at right angles to one another, thus demonstrating an independence and asynchrony. The latter conditions are demonstrated further by the fact that the blastomeres shift their position continually in relation to each other during these divisions.

d) Sixteen-cell Stage. The mitotic divisions increase in rate, and at about 45 to 47 hours after mating the 16-cell stage is reached (fig. M4D). The cells at the future trophoblast pole begin to flatten, and gradually certain blastomeres are enclosed within. In the macaque monkey, 16 cells are present at about 96 hours after fertilization.

e) Morula Stage. At about 65 to 70 hours after mating a solid mass of cells is present. This condition is known as the morula (mulberry-like) stage (fig. M4E, F). The trophoblast portion of the cell mass is more active in cell division.

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Fio. 146. Photomicrographs of cleavage in living monkey egp. (After Lewis and Hartman, Carnegie Inst.. Washington, Contrib. Embryol., 24.) (Figures borrowed from fig 33 Patten, ’48.) (A) Late two-cell stage. (B) Early three-cell stage. (C) Late four-ccll stage. (D) Five-cell stage. (E) Six-cell stage. (F) Eight-cell stage; next cleavage beginning.

Fig. 147. (See facing page for legend.)

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303

f) Early Blastocyst. A few hours later or about 70 to 75 hours after mating, a well-defined cleft within the cells of the trophoblast pole becomes evident (fig. 144G). (Cf. fig. 145D, J.) This cleft or cavity enlarges, and the surrounding trophoblast cells lose their rounded shape and become considerably flattened. As the blastocoel gradually increases in size, the formative tissue or inner cell mass becomes displaced toward one end of the early blastocyst, as indicated in fig. 144G, H. The blastocoelic space at this time is filled with fluid, and the blastocyst as a whole completely fills the area within the zona pellucida (fig. 144G, H). The pig embryo reaches a similar condition in about 100 hours after fertilization, and that of the guinea pig in 140 hours.

During its passage down the Fallopian tube, the developing mass of cells continues to be encased by the zona pellucida. The general increase in size is slight. In the rabbit and in the opossum, as the cleaving egg passes down the Fallopian tube, an albuminous coating is deposited around the outside of the zona pellucida (figs. 144G, H; 147A). This albuminous layer forms an accessory egg membrane or covering similar to the albuminous layers deposited around the egg by the oviducal cells in prototherian mammals, birds, and reptiles. At about 80 to 96 hours after mating, the rabbit blastocyst enters the uterus and gradually increases in size. Implantation of the mammalian blastocyst upon the uterine wall will be considered later. (See Chap. 22.)

3) Types of Mammalian Blastocysts (Blastulae). The early blastocyst of the rabbit described above is representative of the early condition of the developing blastula of the eutherian (placental) mammal. However, in the metatherian or marsupial mammals the early blastocyst does not possess a prominent inner cell mass similar to that found in the eutherian mammals. Comparing the early blastocysts of the higher mammals, we find, in general, that there are three main types as follows:

( 1 ; In most of the Eutheria or placental mammals the inner cell mass (embryonic knob) is a prominent mass of cells located at one pole of the blastocyst during the earlier stages of blastocyst formation. (See

Fig. 147. Early development of the opossum egg. (A-H after Hartman, ’16; I-N after McCrady, ’38.) (A) Unfertilized uterine egg, showing the first polar body; yolk

spherules (in black) within the cytoplasm; zona pellucida; albuminous layer; and the outer shell membrane. (B) Two-cell stage. Observe yolk spherules discharged into the cavity of the zona pellucida. (C) Section through three blastomeres of four-cell stage. Observe yolk within and without the blastomeres. (D) Section through 16-celI cleavage stage. Observe yolk within blastomeres and also in cavity of the zona between the blastomeres. (E) Section through early blastocyst showing yolk and cytoplasmic fragments and an included nucleated cell within the blastocoel. (F-H) Early and later blastocyst of the opossum, showing the formative tissue at one pole of the blastocyst. (1) Surface view, fertilized egg. (J) Two-blastomere stage. (K) Cell A has divided meridionally into A, and Aj. (L) Cell B has divided into B, and B.. (M) A, and Aj have divided as indicated. (N) B, and B, divide next as indicated.

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

figs. 144G, H; 145J-L.) This condition is found in the monkey, human, pig, rabbit, etc.

(2) On the other hand, in certain marsupials, such as the American opossum, Didelphys virginiana, and the Brazilian opossum, Didelphys aurita, the inner cell mass is much less prominent during earlier stages of the blastocyst. In these species it is indicated merely by a thickened aggregation of cells at one pole of the blastocyst (fig. 147E— G).

(3) In the marsupial or native cat of Australia, Dasyurus viverrinus, cleavage results in an early blastocyst in the form of a hollow sphere of rounded cells. As the blastocyst expands, the cells increase in number and become flattened to form a thin layer of cells apposed against the shell membrane without an apparent inner cell mass or embryonic knob (fig. 148A-C).

A conspicuous feature of cleavage and early blastocyst formation in the marsupials should be emphasized. For in this group, the early blastomeres apparently use the framework of the zona pellucida as a support upon which they arrange themselves. As a result, the blastocoelic space of the blastocyst

Fig. 148. Early blastular conditions of the marsupial cat of Australia, Dasyurus viverrinus. (After Hill, ’10.) (A) Early blastula. (B) External view of blastocyst, 0.6

mm. in diameter. The cells are becoming flattened and finally reach the condition shown in (C). (C) Section of wall of blastocyst, 2.4 mm. in diameter.

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forms directly by cell arrangement and not by the development of a cleft within the trophoblast cells, as in the eutherian mammals. (See fig. 147C-E; compare with figs. 144G; 145J.)

The descriptions of the mammalian blastocysts presented above pertain only to the primary condition of the blastocyst. The changes involved in later development, resulting in the formation of the secondary blastocyst, will be described in the next chapter which deals specifically with blastulation.

(For more detailed descriptions of early cleavage in the metatherian and eutherian mammals see: Hartman (T6) on the American opossum; Hill (T8) on the opossum from Brazil; Hill (TO) on the Australian native cat, Dasyurus viverrinus; Heuser and Streeter (’29), and Patten (’48) on the pig; Lewis and Gregory (’29), Gregory (’30), and Pincus (’39) on the rabbit; Huber (T5) on the rat; Lewis and Wright (’35) and Snell (’41 ) on the mouse; Lewis and Hartman (’41) and Heuser and Streeter (’41) on the Rhesus monkey.)

b. Holoblastic Cleavage of the Transitional or Intermediate Type

Contrary to the conditions where small amounts of yolk or deutoplasm are present in the egg of the higher mammal or in Amphioxus, the eggs of the vertebrate species described below are heavily laden with yolk. As the quantity of yolk present increases, the cleavage phenomena become less and less typically holoblastic and begin to assume meroblastic characteristics. Hence the designation transitional or intermediate cleavage.

1) Amhy stoma maculatum ( punctatum ), The newly spawned egg of Ambystoma maculatum is nearly spherical and measures about 2 mm. in diameter, although the egg size is somewhat variable. The animal pole contains within its median area a small depression, the “light spot” or “fovea.” Within the fovea is a small pit harboring the first polar body. (A comparable pit is shown in the frog’s egg, fig. 1 1 9C. ) After the second polar body is formed, this pit may appear somewhat elongated, and the light spot disappears. Just before the first cleavage, the animal pole appears flattened similar to the condition in the frog’s egg. The flattened area soon changes to an elongated furrow which progresses gradually downward toward the opposite pole (fig. 149 A, B). This cleavage furrow is meridional, dividing the egg into two, nearly equal blastomeres. The second cleavage furrow is similar to the first but at right angles to the first furrow (fig. 149C). However, considerable variation may exist, and the second furrow may arise at various angles to the first, dividing each of the first two blastomeres into two, slightly unequal, daughter blastomeres. The third set of cleavages is latitudinal, and each blastomere is divided into a smaller animal pole micromere, and a larger vegetal pole macromere (fig. 149D). Later cleavages may not be synchronous.

The first three cleavages described above conform generally to the rules of typical holoblastic cleavage. However, from this time on cleavage digresses from the holoblastic pattern and begins to assume certain characteristics oj

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

Fig. 149, Early cleavage in Arnbystoma maculatum (punctatum). (After Eycleshymer, J. Morphol., 10, and eggs in the laboratory.) (A, B) First cleavage furrow, meridional plane. (C) Second cleavage furrow at right angles to first furrow, meridional plane.

(D) Third cleavage furrow, latitudinal, forming four micromeres and four macromeres.

(E) Fourth cleavage furrow; mixture of meridional and vertical planes of cleavage. (F) Fifth cleavage furrows; mixture of latitudinal and vertical planes of cleavage. Observe equatorial plane cutting the large macromeres. (G-I) Later cleavage stages.

meroblastic cleavage. For example, the fourth set of cleavages may be a mixture of vertical and meridional furrows, as shown in figure 149E. The fifth cleavages are a mixture of horizontal (i.e., latitudinal and equatorial, fig. 149F), vertical and meridional furrows. The sixth set of cleavages is made up of vertical and horizontal cleavage planes of considerable variableness (fig. 149G). From this time on cleavage becomes most variable, with the animal pole micromeres dividing much more rapidly than the yolk-laden macromeres at the vegetal pole (figs. 149H, I).

The blastocoel makes its appearance at the eight-cell stage and appears as a small space between the micromeres and the macromeres, the latter forming the floor of the blastocoelic space. At the late blastula stage, the blastocoel is roofed over by the smaller micromeres, and floored by the yolk

TYPES OF CLEAVAGE

307

VERTICAL FURROW

Fig. 150. Cleavage in the egg of Lepidosiren paradoxa. (After Kerr, ’09.) (A) Be ginning of first cleavage, meridional in position. (B) Second cleavage planes, approximately meridional in position. (C) Third cleavage planes vertical in position, demonstrating a typical meroblastic pattern. (D) Early blastula. (E) Late blastula.

laden macromeres. The blastocoel is small in relation to the size of the egg (Eycleshymer, 1895).

2 ) Lepidosiren paradoxa. The egg of the South American lungfish, Lepidosiren paradoxa, measures about 6,5 to 7 mm. in diameter. Cleavage of the egg is complete (i.e., holoblastic), and a relatively large blastocoel is formed. As in Arnbystoma, the blastocoel is displaced toward the animal pole. The floor of the blastocoel is formed by the large, yolk-laden macromeres.

The first two cleavage furrows are approximately meridional (fig. 150A, B). These two furrows are followed by four vertical furrows, which, when completed, form eight blastomeres (fig. 150C). The latter cleavages are subject to much variation. Although cleavage of the egg is complete, a distinct meroblastic pattern of cleavage is found, composed of two meridional furrows followed by vertical furrowing (see Kerr, ’09).

3) ISecturus maculosus. In this species of amphibia the egg is large and its contained yolk is greater than that of Arnbystoma. It measures about 5 to 6 mm. in diameter. The egg and its envelopes are attached individually by the female beneath the flattened surface of a stone (Bishop, ’26).

Cleavage in this egg proceeds slowly. The first two cleavage furrows tend to be meridional, but variations may occur in different eggs. Sometimes they are more vertical than meridional (fig. 151A). (See Eycleshymer, ’04). The

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

Fig. 151. Cleavage in the egg of Necturus maculosus. (After Eycleshymer and Wilson, ’10.) (A) First two cleavage planes are meridional. (B) Third cleavage planes tend to be vertical and meridional. (C) Fourth cleavage planes are vertical, meridional, and irregular. (D~H) Following cleavage planes become irregular, offering a mixture of modified latitudinal, vertical, and meridional varieties.

third cleavage furrows are irregularly vertical (fig. 15 IB), while the fourth are latitudinal, cutting off four very irregular micromeres at the animal pole. Segmentation then becomes exceedingly irregular. (See Eycleshymer and Wilson,

• 10). One characteristic of cleavage in Necturus is a torsion and twisting of

the cleavage grooves due to a shifting in the position of the blastomeres.

As shown in the figures, the first three cleavage planes assume a distinct meroblastic pattern of two meridional furrows followed by vertical furrows. The yolk material evidently impedes the progress of the furrows considerably.

4 ) Acipenser sturio. In the genus Acipenser are placed the cartilaginous ganoid fishes. Cleavage in Acipenser sturio, the sturgeon, resembles that of

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309

Fig. 152. Cleavage in the egg of the sturgeon, Acipenser sturio, (After Dean, 1895.) (A, B) First and second cleavage planes are approximately meridional. (C) Third cleavage planes are vertical, usually parallel to first cleavage plane. (D) Fourth cleavage planes are vertical, cutting off four central cells from the 12 marginal cells. (E, F) Later cleavage stages.

Necturus, although the furrows in the yolk pole area are retarded more and are definitely superficial. The third and fourth sets of cleavage furrows are vertical and succeed in cutting off four central cells from twelve larger marginal cells (fig. 152). Cleavage in this form is more holoblastic in its essential behavior than that in the egg of Amia and Lepisosteus described below (Dean, 1895).

5) Amia calva. Amia calva is a species of bony ganoid fishes, and it represents one of the oldest living species among the fishes. Its early embryology follows the ganoid habit, namely, its cleavages adhere to the meroblastic pattern of the teleost fishes, with the added feature that the furrows eventually pass distally toward the vegetal pole of the egg. A few yolk nuclei appear to be formed during cleavage. These nuclei aid in dividing the yolk-filled cytoplasm into distinct cells. The latter gradually are added to the early blastomeres and to the later entoderm cells of the developing embryo. In other words, cleavage in this species is holoblastic, but it represents a transitional condition between meroblastic and holoblastic types of cleavage.

The egg of Amia assumes an elongated form, averaging 2.2 by 2.8 mm. The germinal disc is. a whitish cap in the freshly laid egg, reaching down over the animal pole to about one third of the distance along the egg’s longer axis.

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

The vegetal pole is gray in color. The egg membrane is well developed, having a zona radiata and a villous layer. Strands of the villous layer may attach the egg to the stem of a water weed or other structure (fig. 153A).

The first cleavage plane is meridional and partly cleaves the protoplasmic disc into two parts (fig. 153B). This cleavage furrow passes slowly toward the vegetal pole of the egg. The second cleavage is similar to the first furrow and at right angles to it (fig. 153C). The third cleavage is variable but, in general, consists of two furrows passing in a vertical plane at right angles to the first cleavage furrow (fig. 153D). The fourth set of cleavages is hori

SYNr. YTIAL NUCLEI SYNCYTIAL NUCLEI INVADE THE YOLK

Fig. 153. Cleavage in the egg of Amia calva. (After Dean, 1896.) (A) Egg mem

branes of Amia, showing the filamentous (villous) layer attaching the egg to the stem of a water weed. (B) Second cleavage plane shown cutting through the protoplasmic disc at one pole of the egg. Section made parallel to the first cleavage plane. (C) First and second cleavage planes seen from above. (D) Third cleavage planes are vertical in position as indicated. (E) Fourth cleavage, sectioned in a plane approximately parallel to first (or second) cleavage. (F) Section through protoplasmic disc at eighth cleavage. (G) Blastular stage. Blastocoel is indistinct and scattered between (?) blastomeres of blastoderm. The description given by Whitman and Eycleshymer (1897) does not agree in certain features with the above.

TYPES OF CLEAVAGE

311

Fig. 154. Early development of Lepisosteiis osseus. (After Dean, 1895.) (A) Un cleaved egg, showing germinal disc. (B) First cleavage is trench-like, extending beyond (i.e., laterally) to the margin of the germinal disc. (C) Transverse section of cleavage furrows shown in (B). (D) Four-cell stage. (E) Third cleavage planes are vertical

as indicated. (F) Fourth cleavage planes also are vertical. (G) Germinal disc, set tioned 25 hours after fertilization. Blastocoelic spaces dispersed.

zontal. While the latter is in progress the fifth cleavages, which are vertical, begin. As a result of the fourth and fifth sets of cleavages, a mass of eight central cells and twenty or more marginal cells arises. Horizontal (i.e., latitudinal) cleavages begin among the central cells at this time, and other cells (see cell A, fig. 153F) appear to be budded off from the yolk floor from this period on. The latter are contributed to the growing disc of cells above.

Four types of cleavage furrows now appear in the growing blastoderm as follows :

( 1 ) cleavage among the central cells, increasing their number,

(2) cleavage among the marginal cells, contributing cells to the central cells,

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

( 3 ) cleavage of the marginal cells, increasing the number of marginal cells and contributing syncytial nuclei to the yolk floor,

(4) cleavage within the syncytial mass of the yolk floor, contributing cells to the central cells, such as cell A, figure 153F.

Eventually a blastular condition is reached as a result of the foregoing cleavages which does not possess an enlarged blastocoelic space; rather the blastocoel is in the form of scattered spaces within a loosely aggregated cap of cells (fig. 153G). This blastula might be regarded as a stereoblastula, i.e., solid blastula (Dean, 1896; Whitman and Eycleshymer, 1897).

6) Lepisosteus (Lepidosteus) osseiis. The early development of the gar pike, Lepisosteus osseus, another bony ganoid fish, resembles that of Amia described above. The disc of protoplasm which takes part in the early cleavages is a prominent mass located at one pole of the egg (fig. 154A). The first two cleavage furrows appear to be meridional and partly cleave the protoplasmic cap of the egg, as indicated in figure 154B-D. The next cleavages are vertical and somewhat parallel to one of the meridional furrows (fig. 152E). The fourth cleavages are vertical, cutting off four central cells from the peripherally located marginal cells (fig. 152F). As in Amia, the marginal cells contribute syncytial nuclei to the yolk bed below the protoplasmic cap, and these in turn contribute definite cells to the growing blastodisc. The blastula of Lepisosteus consists of a loosely aggregated cap of cells among which are to be found indefinite blastocoelic spaces (fig. 152G). (See Dean, 1895.)

7) Gymnophionan Amphibia. Cleavage presumably is holoblastic, resulting in a disc of small micromeres at the animal pole, with large, irregular macromeres, heavily yolk laden, located toward the vegetal pole (fig. 182A). (See Svensson, ’38.) The latter cells become surrounded during gastrulation by the smaller micromeres (Brauer, 1897). The blastula of the gymnophionan amphibia essentially is solid and may be regarded as a stereoblastula.

3. Meroblastic Cleavage

The word meroblastic is an adjective which refers to a part of the germ; that is, a part of the egg. In meroblastic cleavage only a small portion of the egg becomes segmented and thus gives origin to the blastoderm. Most of the yolk material remains in an uncleaved state and is encompassed eventually by the growing tissues of the embryo. A large number of vertebrate eggs utilize the meroblastic type of cleavage. Some examples of meroblastic cleavage are listed below.

a. Egg of the Common Fowl

{Note: As cleavage in reptiles resembles that of birds, a description of reptilian cleavage will not be given. The reader is referred to figure 231, con

TYPES OF CLEAVAGE

313

cerning the cleavage phenomena in the turtle. The information given below is to be correlated also with the developing pigeon's egg.)

The germinal disc (blastodisc) of the hen’s egg at the time that cleavage begins measures about 3 mm. in diameter. Its general relationship to the egg as a whole is shown in figure 157A.

1) Early Cleavages. The first cleavage furrow makes its appearance at about four and one-half to five hours after fertilization at the time when the egg reaches the isthmus of the oviduct (figs. 155A; 157C, D). The first

Fig. 155. Cleavage in the chick blastoderm, surface views. (C after Olsen, ’42; the rest after Patterson, ’10.) (A) First cleavage is approximately meridional. (B) Second

cleavage is at right angles to first. (C) Third cleavage planes are vertical as indicated and approximately parallel to one of the other cleavage planes. Considerable inequality may exist at this time. (This figure slightly modified from original.) (D) Seventeencell stage. Observe central and marginal cells. (E) Stage approximating 32-cell condition. (F) Surface view of 64-cell stage; 41 central and 23 marginal cells. (G) Surface view of blastoderm in lower portions of oviduct; 31 marginal and 123 central cells. (H) Later blastoderm, showing 34 marginal and 312 central cells.

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315

NUCLEUS OF PAND E R BLASTODISC CELLS

ZONE OF JUNCTURE

(SYNCYTIAL GERM WALL)

CENTRAL PERIBLAST

PRIMITIVE 8LAST0C0EL

CELLULAR GERM WALL '

MARGINAL PERIBLAST

MARGIN OF OVERGROWTH

Fig. 156. Cleavage in chick blastoderm, sectional views. (After Patterson, ’10.) (A)

Median section through blastoderm approximately at right angles to furrow shown in fig. 155A. (B) Section through blastoderm of about eight-cell stage. (C) Section

through blastoderm, showing 32 cells, also showing horizontal cytoplasmic cleft (segmentation cavity). (D) Median section through blastoderm similar to that shown in fig. 155E, (E) Median section through blastoderm similar to that of fig. 155G. (F, G)

Diagrammatic views of developing avian blastoderms. (F) Diagrammatic section and surface view of chick blastoderm shown in fig. 155G and fig. 156E. (G) Section of

chick blastoderm about time that egg is laid, depicting the primary blastocoel below the blastoderm and syncytial tissue at the margins. Observe that the syncytial tissue serves to implant the blastoderm upon the yolk substance.

furrow consists of a slight meridional incision near the center of the blastodisc, cutting across the disc to an extent of about one half of the diameter of the latter (fig. 155A). This furrow passes yolkward but does not reach the lower portion of the disc where the cytoplasm is filled with coarse yolk granules (fig. 156A). The second cleavage occurs about 20 minutes later and consists of two furrows, one on either side of the first furrow and approximately at right angles to the first furrow. These furrows may be regarded as meridional (fig. 155B). Though both of the second furrows tend to meet the first furrow at its midpoint, one of the second furrows may be displaced and, hence, may not contact the corresponding furrow of the other side. The third set of furrows is vertical, cutting across the second set of meridional furrows, and, consequently, tends to parallel the first cleavage furrow (fig. 155C). The fourth set of furrows is also vertical and, although not synchronous, it proceeds gradually to form eight central cells which are surrounded by twelve

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

marginal cells. l|[n figure 155D, five central cells are shown, while in figure 157E, eight central cells are present) The central cells do not have boundaries below and, thus, are open toward the yolk. As a result, their protoplasm is continuous with the protoplasm in the deeper-lying portions of the disc. The marginal cells have boundaries only on two sides, and the cleavage furrows which form the sides of the marginal cells continue slowly to extend in a peripheral direction toward the margins of the disc (fig. 155D). The egg is in this stage of development when it leaves the isthmus and enters the uterus (fig. 157A, F).

Cleavage from this point on becomes very irregular, but three sets of furrows are evident:

(a) There are vertical furrows which extend peripherad toward the margin of the blastodisc^^i'hese furrows meet at various angles the previously established furrows which radiate toward the periphery of the blastodisc (in fig. 155E, see a., b., c.). A branching effect of the radiating furrows, previously established, in this manner may be produced (in fig. 155E, see c.)^

(b) Another set of vertical furrows is found which cut across the median (inner) ends of the radiating furrows. The latter produce peripheral boundaries for the centrally located cells (see fig. 155E, d., e., f. ). The central cells thus increase in number as the blastodisc extends peripherally. As a result of this set of cleavage furrows, a condition of the blastodisc is established in which there is a mass of central cells, having peripheral boundaries, and an area of marginal cells which lies more distally between the radiating furrows. It is to be observed that the marginal cells lack peripheral boundaries (fig. 155E, F).

(c) A third and new kind of cleavage, cytoplasmic but not mitotic, now occurs below the centrally placed cells, namely, a latitudinal or horizontal cleft which establishes a lower boundary for the centrally located cells with the subsequent appearance of a blastocoelic space filled with fluid (fig. 156B, C).

Thus, at the 16- to 32-cell stages (fig. 155D, E) some of the more centrally located central cells have complete cellular boundaries (fig. 156C), but central cells, located more peripherally, may not have the lower boundary. The marginal cells also lack a lower boundary.

A little later, at the 60- to 100-cell stages (fig. 155F), the chick blastoderm presents the following characteristics:

(a) There is a mass of centrally located cells. These cells lie immediately above the horizontal cleft mentioned above (fig. 156C, D). They are completely bounded by a surface membrane and represent distinct cells. These cells continue to increase by mitotic division and, as early as the 64-cell stage (fig. 155F), the centrally located cells are in the

Fig. 157. Chart showing ovary, oviducal, and pituitary relationships in passage of egg from the ovary down the oviduct. Developing blastodisc shown in (B-G) in relation to the oviducal journey. This chart shows an egg which has just been ovulated. Ordinarily, however, this egg would not be ovulated until sometime after the egg shown in the uterus has been laid.

318 CLEAVAGE (SEGMENTATION) AND BLASTULATION

BLASTODERM PERIBLAST

Fig. 158. Early cleavage phenomena in elasmobranch fishes. (A, B, E, F, G after Ziegler, ’02, from Ruchert; C, D after Ziegler.) (A) Germ disc of Torpedo ocellata, showing four cleavage nuclei, sperm nuclei, and beginning of first cleavage furrow. (B) Stage of cleavage, possessing 16 cleavage nuclei. Four central cells and ten marginal cells are evident from surface view. (C) Surface view of blastoderm of Scy Ilium canicula with 64 cleavage nuclei. Twenty-nine central cells and seventeen marginal cells are evident from surface view. (D) Later cleavage stage of 5. canicula with 145 cells showing. (E) Transverse section of (B). (F) Transverse section of blastoderm of

T. ocellata with 64 cells. (G) Median section through blastoderm of T. ocellata at the end of the cleavage period.

form of two layers situated immediately above the horizontal cleft or segmentation cavity (fig. 156D).

(b) The horizontal cleft or segmentation cavity gradually widens and enlarges. It separates the central cells above from the uncleaved germinal disc or central periblast below.

(c) At the margins of the central cells, these cleavages may be found: ( 1 ) Vertical cleavages occur which cut off more central cells from inner ends of the marginal cells. As a result, there is an increase in the number of central cells around the periphery of the already-established cen

TYPES OF CLEAVAGE

319

tral mass of cells. (2) Vertical cleavages arise whose furrows extend peripherad toward the margin of the disc. These furrows and previously formed, similar furrows now approach the outer edge of the blastodisc (germinal disc). (See figs. 155H; 156D). (3) True latitudinal or horizontal cleavages occur which serve to provide lower cell boundaries for the more peripherally located, central cells (see cell A, fig. 156E), and which also contribute nuclei without cell boundaries to the disc substance in this immediate area (see cell B, fig. 156E). As a result, the marginal or peripheral areas of the blastodisc around the mass of completely formed, central cells are composed of: («) marginal cells which appear near the surface of the blastodisc, having partial boundaries at the blastodisc surface, and {b) a deeper-lying protoplasm, possessing nuclei without cell boundaries. This deeperlying, multinucleated, marginal protoplasm constitutes a syncytium (fig. 156F).

2) Formation of the Periblast Tissue. As indicated above, the activities of the blastoderm extend its margins peripherad. In so doing, some of the mitotic divisions in the peripheral areas contribute nuclei which come to lie in the deeper portions of the blastodisc. Some of these nuclei wander distally and yolkward into the more peripherally located, uncleaved portions of the protoplasm below the enlarging primary segmentation cavity or blastocoel. A syncytial protoplasm containing isolated nuclei thus arises around the peripheral margin of the blastoderm in its deeper areas. This entire syncytial protoplasm, composed of a continuous cytoplasm with many nuclei, is known as periblast tissue. It is made up of two general areas: (1) the peripheral periblast around the margin of the blastodisc and (2) a central periblast below the primitive blastocoel (fig. 156G). This periblast tissue is a liaison tissue which brings the yolk and the growing mass of cells of the blastodisc into nutritive contact.

When this condition is reached, two kinds of embryonic tissues exist:

(a) the formative or embryonic tissue proper, composed of an aggregation of distinct cells. These cells constitute the cellular portion of the blastoderm (see blastodisc cells, fig. 156G), and

(b) the peripheral and central periblast tissue (see fig. 156G). The latter functions as a trophoblast tissue, and it is continuous with the segmented portilHi of the blastoderm around the peripheral areas of the blastodisc. Centrally, however, it is separated from the segmented area of the blastoderm by the primary blastocoelic cavity. The developmental condition at this time may be regarded as having reached the primary blastular stage.

3) Morphological Characteristics of the Primary Blastula. This condition of development is reached while the egg continues in the uterus (fig. 157G).

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

A transverse section through one of the diameteis of the primary blastula presents the following features (fig. 156G):

(a) A central mass of cells of two or several cells in depth overlies the blastocoelic space. This is the central or cellular portion of the

blastoderm.

(b) Underneath this central blastoderm is the primary segmentation cavity or primary blastocoel.

(c) Below the primary blastocoel is the central syncytial periblast, which continues downward to the yolk material; many yolk granules are present in the layer of the central periblast near the yolk. Nuclei are not present in the central area of the central periblast, but may be present in its more peripheral portions.

(d) Around the peripheral areas of the central periblast and the cellular portion of the blastoderm is the marginal periblast tissue which now is called the germ wall. The germ-wall tissue contains much yolk material in the process of digestion and assimilation.

The central mass of cells or cellular blastoderm increases in cell number and in size by the multiplication of its own cells and by the contribution of marginal periblast tissue which gradually forms cells with boundaries from its substance. The germ wall thus may be divided into two main zones: ( 1 ) an inner zone of distinct cells, which are dividing rapidly and, in consequence, contribute cells to the peripheral portions of the growing cellular blastoderm and (2) an outer peripheral zone, the syncytial germ wall (zone of junction). The latter is in intimate contact with the yolk (fig. 156G). The central periblast tissue gradually disappears. At the outer boundary of the peripheral periblast, there is an edge of blastodermic cells overlying the yolk. These cells have complete boundaries and are known as the margin of overgrowth (fig. 156G). A resume of the early development of the hen’s egg in relation to the parts of the oviduct, pituitary control, laying, etc., is shown in figure 157.

4) Polyspermy and Fate of the Accessory Sperm Nuclei. The bird’s egg is polyspermic and several sperm make their entrance at the time of fertilization (see fig. 157B). The supernumerary sperm stimulate abortive cleavage phenomena in the peripheral area of the early blastodisc (fig. 155D). However, these cleavage furrows together with the extra sperm nuclei soon disappear.

(References: Blount (’09); Lillie (’30); Olsen (’42); and Patterson (’10).)

For later stages in the development of the hen’s egg, see chapter 7.

b. Elasmobranch Fishes

1) Cleavage and Formation of the Early Blastula. Like the egg of the bird, the egg of the elasmobranch fishes is strongly telolecithal, and a small disc of protoplasm at one pole of the egg alone takes part in the cleavage phe

TYPES OF CLEAVAGE

321

nomena. Cleavage in the majority of these fishes simulates that of the bird, but certain exceptional features are present. In some, as in Torpedo ocellata, meroblastic cleavage is present in an extreme form. The zygotic nucleus divides and the two daughter nuclei divide again forming a syncytial state before the appearance of the first cleavage furrow. The tendency of retardation or suppression of the cytoplasmic mechanism of cleavage which occurs in the bird blastoderm thus is carried to an extreme form in the early development of some elasmobranch fishes.

The first cleavage furrow is meridional or nearly so (fig. 158A), and the second furrow is similar and at right angles to the first furrow. The third set of furrows is vertical and meets the previous furrows at various angles. The fourth set of cleavages is vertical and synchronous, as is the preceding, and gives origin to three or four central cells, which, on surface viewing, have complete cell boundaries but below their cytoplasms are confluent with the cytoplasm of the blastodisc (fig. 158B and E ). Around the periphery of these central cells, are on the average ten marginal cells which have their cytoplasms confluent below and peripherally with the general cytoplasm of the disc. The fifth cleavage furrows are mixed. That is, in the central part of the disc the cleavage furrows are latitudinal, as the mitotic spindles in this area form perpendicular to the surface. As a result, distinct daughter cells are cut off above, while the daughter cells below have cytoplasms confluent with the general cytoplasm of the disc. A blastocoelic cavity appears between these two sets of central cells. In the marginal areas the fifth set of cleavages is vertical, cutting off more central cells and giving origin to more marginal cells. The sixth set of cleavages is a mixture of vertical cleavages at the periphery and latitudinal cleavages centrally; it produces a condition shown in figure 158F. In surface view, the blastoderm appears as in figure 158C, D.

From this time on cleavage becomes very irregular and a developmental condition soon is produced which possesses a central blastoderm of many cells with an enlarged blastocoelic cavity below (fig. 158G). A syncytial periblast tissue is present at the margins of the blastoderm which also extends centrally below the blastocoelic space where it forms a central periblast (fig. 158G). In this manner, two kinds of cells are produced:

(a) a blastoderm of distinct cells which ultimately produces the embryo and

(b) a surrounding trophoblast or periblast tissue which borders the yolk substance peripherally and centrally. As in the chick, the periblast tissue has nutritive (i.c., trophoblast) functions.

2) Problem of the Periblast Tissue in Elasmobranch Fishes. Two views have been maintained, regarding the origin of the periblast nuclei in the elasmobranch fishes. One view maintains that they arise from the accessory sperm nuclei derived from polyspermy, for polyspermy is the rule here as it

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

is in reptiles and birds. In the latter groups, these accessory nuclei may divide for a time but ultimately degenerate, playing no real part in ontogeny. In the case of the elasmobranch fishes, the accessory nuclei tend to persist somewhat longer, and accordingly, it is upon this evidence that some have maintained that the periblast nuclei arise from them. Others hold that the sperm nuclei degenerate as they do in reptiles and birds, and the periblast nuclei arise as a result of the regular embryonic process. A third view concedes that both these sources contribute nuclei.

In view of the origin of the periblast nuclei in teleost fishes, in the ganoid fishes, Amia and Lepisosteus, and in reptiles and birds, and of the syncytial tissue of the later mammalian trophoblast, it is probable that embryonic cells and tissues and not accessory sperm nuclei are the progenitors of the periblast tissue. This probability is suggested by figure 158F, G. Furthermore, later on in the development of the elasmobranch fishes, the entoderm appears to contribute nuclei which wander into the periblast tissue which lies between the entoderm and the yolk material (fig. 21 3K, L). In later stages the periblast tissue is referred to as the yolk syncytium. In the yolk syncytium the periblast nuclei gradually assume a much larger size.

For further details of the early development of the elasmobranch fishes, consult Ziegler (’02) and Kerr (’19) and Chapter 7.

c. Teleost Fishes

1) Cleavage and Early Blastula Formation. During the fertilization process of the egg in teleost fishes, the superficial cytoplasm of the egg migrates toward the point of sperm entrance and hence a mound-like disc of protoplasm forms at the pole of the egg where the sperm enters (figs. 122C; 123B, C). It is this protoplasmic mass which takes part in cleavage (fig. 123E). The cleavage planes in the teleost fishes manifest great regularity. The early cleavage furrows almost cut through the entire protoplasmic disc in most teleost eggs, and a mere strand of cytoplasm is left near the yolk which is not cleaved (fig. 159E).

In the sea bass, Serranus atrarius, the first two cleavage planes are meridional and at right angles to each other (fig. 159A); the third planes are vertical and parallel to the first plane. The result is a group of eight cells in two rows (fig. 159B). The fourth cleavage furrows are vertical and parallel to the long axis of the eight cells previously established. These furrows divide each of the eight blastomeres into inner and outer daughter cells. The result is 16 cells, arranged in parallel rows of four cells each (fig. 159C, D).

As the 16-cell condition is converted into 32 cells, the four inner cells divide latitudinally, that is, the cleavage spindle forms perpendicular to the surface, while the twelve surrounding cells divide vertically (fig. 159D, F, G). From this time on latitudinal and vertical cleavages become mixed, and the

Fig. 159. Early development of the sea bass, Serraniis atrarius, and the trout, Salmo fario. (A-M after Wilson, 1889 and 1891; N R after Kopsch, ’ll.) (A) Two-blastomere

stage, showing anaphase of next division. (B) Eight-blastomere stage (slightly modified). (C) Sixteen-cell blastoderm. (D) Sixteen-cell stage, showing anaphase nuclei of next division. In the four centrally placed cells, the spindles are at right angles to the surface, thus forming a latitudinal cleavage furrow in these cells. (E) Section through center of four-blastomore stage. (F) Section through center of (D). Observe periblast tissue. (G) Section showing change from 16-cell stage into 32 cells; see (D). (H)

Thirty-two to 64 cells. (I) Fate cleavage blastoderm. Observe marginal and central periblast. (J) Multiplication of periblast nuclei around the margin of the blastoderm. (K~M) Late blastoderm, showing marginal and central periblast tissue. (N-R) Cleavage of the blastodisc of the trout. Observe that periblast tissue is derived from the blastodisc cytoplasm directly.

323

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

Fig. 160. Cleaving eggs of Platypus and Echidna. (After Flynn and Hill, ’39.) (A)

Egg, shell, and early cleavage in Ornithorhynchus. (B) Early cleavage in Echidna. See fig. 16 ID.

synchronization of mitotic division is lost. In certain other teleost fishes, latitudinal cleavages begin as early as the 8-cell stage.

At the 32- to 64-cell stages in Serranus atrarius, the blastoderm presents a cap-like mass of dividing cells overlying a forming blastocoel (fig. 159H, I). Between the blastocoel and the yolk, there is a thin layer of protoplasm connecting the edges of the cap. This thin protoplasmic layer is the forerunner of the central periblast tissue; at this stage it contains no nuclei (fig. 159F, H).

2) Origin of the Periblast Tissue in Teleost Fishes. In the sea bass and many other teleost fishes, some of the surrounding cells at the edge of the blastoderm lose their cell boundaries and fuse together to form a common syncytial tissue. The nuclei in this tissue continue to divide (fig. 159J) and eventually migrate into the periblast tissue below the blastocoel (see arrow, fig. 159L). The latter then becomes the central periblast, while the syncytial tissue around the edges of the growing blastodisc forms the peripheral or marginal periblast (fig. 159K-M).

In the trout, the early cleavage furrows of the blastodisc are incomplete, and the periblast arises from the syncytial tissue established directly below and at the sides of the protoplasmic cap (fig. 159N-R), This condition resembles the cleavage process in the elasmobranch fishes.

See Kerr (T9); Kopsch (Tl); and H. V. Wilson (1889).

d. Protolherian Mammalia

The Prototheria normally are placed in the class Mammalia along with the Metatheria (marsupials) and Eutheria (true placental mammals). How

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

ever, the prototherian mammals are aberrant, highly specialized animals, whose general anatomy and embryology delineates a group quite distinct from the higher mammals. The duckbill or Platypus (Ornithorhynchus) is found only in Australia. The other species belonging to this group is the spiny anteater Echidna aculeata found in New Guinea, Tasmania, and Australia. The duckbill lays from one to three heavily yolk-laden eggs in an underground chamber on a nest of weeds and grasses. The eggs have a leathery shell. The young are hatched naked, and the mother holds them against her abdomen with her tail, where they feed upon a milk-like substance which exudes from the milk glands by means of pore-like openings. The Echidna lays two white, leathery eggs about the size of the eggs of a sparrow which she places in a temporary pouch or fold of skin on the ventral abdominal wall. They feed similarly to the duckbill young.

The early cleavages of Echidna and Ornithorhynchus follow different cleavage patterns. (See Flynn and Hill, ’39, ’42.) The cleavage planes of the Platypus are more regular and symmetrical and resemble to a degree the pattern of early cleavage in teleost fishes (fig. 160A), whereas the early cleavage planes in Echidna simulate to some degree those found in reptiles (fig. 160B). In both species cleavage is meroblastic.

In Echidna the cleavage furrows cut almost all the way through the protoplasmic disc (fig. 161 E). The second cleavage in this species is at right angles to the first, and divides the blastodisc into two larger and two smaller cells (fig. 16 lA). The third cleavage furrows tend to parallel the first furrow, forming eight cells (fig. 16 IB), while the fourth cleavages run parallel to the second furrow, and 16 cells are formed (fig. 161C). The fifth cleavages lack the constancy of the first four sets although they continue to be synchronous; they result in the formation of 32 cells (fig. 16 ID).

In transverse section, the cells of the 32-cell blastoderm appear as rounded masses, each cell in its upper portion being free from the surrounding cells but in its lower extremity intimately attached to the yolk substance (fig. 161F) . Another feature of the early cleavages in Echidna is the tendency of the cells to separate from each other; wide spaces consequently appear between the blastomeres (fig. 161G). This tendency toward independence and isolationism of the early blastomeres is characteristic of the higher mammals, as previously observed. After the 32-cell stage, synchronization is lost and cleavage becomes very irregular. A central mass of blastodermic cells eventually is formed, surrounded by marginal cells, known as vitellocytes (fig. 175A).

As cleavage and development proceeds, the central blastomeres become free from the underlying yolk, expand, and form a layer about two cells in thickness (fig. 175B). The vitellocytes around the periphery of the blastoderm eventually fuse to form a syncytium or multinucleated cytoplasmic mass intimately associated with the yolk (fig. 175B, C). This marginal mass of syncytial tissue forms the marginal periblast. Within the central portion of the blasto

FORCE WHICH CAUSES THE BLASTOMERES TO ADHERE

327

derm itself two types of cells may be observed, namely, a superficial ectodermal cell and a more deeply situated, somewhat vacuolated, smaller entodermal cell (fig. 175B). (For later stages of blastulation, see chapter 7.)

e. Cleavage in the California Hagfish, Polistotrema (Bdellostoma) stouti

The California hagfish spawns an egg which is strongly telolecithal. The germinal disc (blastodisc) is situated immediately below the egg membrane at one end of the egg, adjacent to the micropyle and the anchor filaments (fig. 162A). Cleavage begins in this disc, and the enlarging blastoderm slowly creeps downward to envelop the massive yolk material. The freshly laid egg measures about 29 mm. by 14 mm., including the shell. Without the shell, the egg is about 22 mm, by 10 mm. and is rounded at each end (Dean, 1899).

The first two cleavage planes may be regarded as meridional (or vertical) (fig. 162B). The third cleavage appears to be a mixture of vertical and horizontal (latitudinal) cleavages, with the former predominating (fig. 162D, E). Cleavage from this time on becomes irregular, and a typical meroblastic blastoderm soon is attained with central and marginal cells (fig. 162F).

C. What is the Force Which Causes the Blastomeres to Adhere Together During Early Cleavage?

A question naturally arises concerning the force which makes the blastomeres of most chordates adhere to one another during the early cleavage

Fig. 162. Egg and cleavage in the marine lamprey, Polistotrema (Bdellostoma) stouti. After Dean, 1899.) (A) Animal pole end of the egg. (B) Surface view of blasto dermic hillock, showing first cleavage furrow. (C) Same, second cleavage. (D) Third cleavages. (E, F) Later cleavages, strongly irregular. (G) Egg with shell removed.

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period. This subject was investigated in the amphibian blastula by Holtfreter, ’39. According to this investigator, blastomeres, when isolated by mechanical means, appear to wander aimlessly about. When contact is made with other blastomeres during this wandering process the cells stick or adhere together. As a result, the mass of adhering cells gradually is formed which becomes rounded into a ball-shaped structure. The results of this work suggest that the force which draws the cells together is one of thigmotaxis or contact affinity, aided by a surface stickiness of the cells. This force only becomes influential when an isolated cell has made contact with another cell or cells.

On the other hand, the early blastomeres of the cleaving mammalian egg are evidently held together also by the binding influence of the egg membrane or zona pellucida. An adhering influence is not prominent until later cleavage stages.

However, one must not be too ready to espouse a single, mechanical factor as the main binding force which causes the blastomeres to adhere together, to move in relation to each other, and to form a definite configuration. Factors tending toward organization are at work during early and late cleavage as well as in subsequent development. Relative to these matters, it is well to cogitate upon the statement of Whitman (1893). “Comparative embryology reminds us at every turn that the organism dominates cell-formation, using for the same purpose one, several, or many cells, massing its material and directing its movements, and shaping its organs, as if cells did not exist, or as if they existed only in complete subordination to its will” (p. 653).

D. Progressive Cytoplasmic Inequality and Nuclear Equality of the Cleavage Blastomeres

1. Cytoplasmic Inequality of the Early Blastomeres

In harmony with the differences in the location and activities of the various blastomeres of the cleaving egg, it is apparent that a difference exists in the ooplasmic substance within the various cells in many species. In the frog, for example, the quantity of yolk substance present in the cells of the yolk pole is much greater than that of the animal pole. Similarly in the four-cell stage the substance of the gray crescent is located in two of the blastomeres, while the other two blastomeres have little or none of this substance. Two of these four cells, therefore, are qualitatively different from the other two. In the ascidian, Styela partita, the presence of the yellow crescent, yolk substance, and gray crescent materials demonstrates that in the four- or eightcell stages there are qualitative differences in the ooplasmic substances which enter into the composition of the respective blastomeres (Conklin, ’05, a and b). Similar conditions may be demonstrated for Amphioxus although pigmented materials are not present in the egg (fig. 167). (See Conklin, ’32, ’33.) As cleavage continues in the eggs of Styela and Amphioxus, a progres

Fig. 163. Developmental potencies (cell lineage) of isolated blastomeres of the cleaving sea-urchin egg, representing different levels along the egg axis (from Huxley and DeBcer, ’34, after Horstadius). Observe the following: (1) Progressing from the animal pole to the vegetative pole, the potency for developing the sensory cilia decreases from animal pole cells 1 to animal pole cells 11. (2) The potency for developing motile cilia increases from animal pole cell II to vegetative pole cell I. (3) The potency for gastrulation becomes greater from vegetative pole cell I to vegetative pole cell II. (4) In the development of vegetative pole cell I, shown at the right of vegetative I, if the third (equatorial) cleavage plane happens to be displaced near the animal pole, an isolated vegetative cell I has more animal pole potencies and will develop apical cilia; if the cleavage plane is displaced toward the vegetative pole, the vegetative pole cell I will attempt to gastrulate. (5) The disc of vegetative cells II plus the micromeres produce a gut so large it will not invaginate and hence forms an exogastrula.

329

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

sive difference in the cytoplasmic substances which enter into the various blastomeres becomes evident.

That the presence or absence of a specific ooplasmic substance within the blastomeres determines a difference in the developmental history of the cell or cells has been shown experimentally for many animal species. For example, in the amphibian embryo it has been demonstrated both by constriction of the developing egg and its membranes with hair loops (Spemann, ’02, ’03) and by placing a small glass rod in the cleavage furrow after the egg membranes have been removed (Ruud, ’25) that each of the blastomeres of the two-cell stage will develop a complete embryo if the first cleavage plane bisects the gray crescent. If, on the other hand, the first cleavage plane is at right angles to the median plane of the embryo, the blastomere which contains the substance of the gray crescent will develop a complete embryo, whereas the other one will give origin to a very imperfect form which does not gastrulate normally or produce a semblance of a normal embryo.

Similar experiments upon the egg of the newt, Triton palrnatus, indicate that a marked difference in the “developmental potencies exists between the dorsal and ventral sides of the egg within a few minutes from fertilization. The formation of the gray crescent seems to be a secondary phenomenon which makes this difference clearly visible in the eggs of some species” (Fankhauser, ’48, p. 694).

In Amphioxus, similar evidence is obtained after the blastomeres have been mechanically isolated. Typical embryos are developed always from the first two blastomeres, for unlike the frog or newt, the first cleavage plane consistently furrows the median axis of the embryo. These twin embryos are half the normal size (Wilson, E. B., 1893; Conklin, ’33). Right and left halves of the four-cell stage also give rise to normal larvae. Moreover, blastulae also develop from isolated blastomeres of the eight-cell stage, but the blastulae which develop from the micromeres are smaller and have only one type of cell, namely, ectoderm, and they never go further than the blastular stage. On the other hand, those from the macromeres are larger and have entoderm, mesoderm, as well as ectoderm, but they never progress further than the gastrular stage of development (Conklin, ’33). Reference should be made to figure 167B in this connection. It is to be observed that the macromeres contain potential mesodermal, entodermal, and ectodermal ooplasm, whereas the micromeres lack the mesodermal and entodermal substances and contain only ectodermal material.

In the protochordate, Styela, a somewhat different condition is found. If the cleaving egg of this species, is separated at the two-cell stage into two separate blastomeres, each blastomere develops only one half of an embryo (Conklin, ’05b, ’06). That is, the right blastomere develops an embryo minus the left half, while the left blastomere produces the opposite condition. There is some tendency to develop or regulate into a complete embryo in that the

Fig. 164. Distribution of presumptive organ regions (cell lineage) during cleavage in Ascaris. (After Durken: Experimental Analysis of Development, New York, W, W. Norton, based upon figures by Boveri and zur Strassen.) (A) Two-cell stage, showing primordial soma cell and first stem cell. (B) Two ectodermal cells, A and B. Soma cell, Sj, is a mixture of mesoderm, stomodaeum, and entoderm; second stem cell, Pj, is a mixture of mesoderm and germ-cell material. The symbolism used to designate the various organ-forming substances is shown in (G). The progressive segregation into separate cells of the substances shown in cells S* and P 2 is given in (C-G). Cf. also fig. 6 IE.

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POSITrON OF 2ND. MATURATION SPINDLE I POINT OF SPERM PENETRATION

CLEAVAGE FURROW OF FIRST CLEAVAGE

ON PART OF NUCLEUS WHICH ENTERED "BRIDGE** IN C

Fig. 165. Drawings of cleavage of a partially constricted egg of Triturus viridescens, illustrating delayed nucleation. (Slightly modified from Fankhauser, ’48.) (A) Shows

constricting loop, point of sperm entrance, and second maturation spindle. The constricted portion to the right will contain the fusion nucleus. ^B) First cleavage furrow in right half of egg. (C) Second cleavage. The nucleus in the “bridge” area has migrated into the “bridge.” (D) Third cleavage. The nucleus in the bridge area has divided and produced cleavage furrow through the bridge cytoplasm as indicated. One of the daughter nuclei of this cleavage is now in the constricted part of the egg at the left. (E) Fourth cleavage — first division of left half. (F) Blastular stage — late blastula at right, middle blastula at left.

ectoderm grows over the half of the embryo which failed to develop. Also, the notochord rounds up into^ normally shaped notochord but is only half the normal size. Essentially, however, these separated blastomeres develop into “half embryos in which some cells have grown over from the uninjured to the injured side, but in which absolutely no change has taken place in the potency of the individual cells or of the different ooplasmic substances” (Conklin, ’06). Similarly, at the four-cell stage isolation of anterior and posterior blastomeres gives origin to anterior and posterior half embryos respectively.

The developing sea-urchin egg has been used extensively for experimental work in the study of isolated blastomeres. In figures 163 and 166A-D are shown the different developmental possibilities which arise from isolated blastomeres of the early cleavage stages. Also, in cell-lineage studies on the developing egg of A scans, a difference in the developmental potencies of the blastomeres is evident (fig. 164). (See also fig. 145A-D in respect to the early development of the pig.)

The foregoing experiments and observations and others of a similar nature suggest that, during the early cleavage stages of many different animal species, a sorting-out process is at work which segregates into different blastomeres

CYTOPLASMIC INEQUALITY AND NUCLEAR EQUALITY

333

distinct ooplasmic substances which possess different developmental potencies. This segregation of different substances into separate blastomeric channels is one of the functions of cleavage. .

2. Nuclear Equality of the Early Blastomeres

Another question next arises: Is there a similar sorting out of nuclear substances during the cleavage period and do the nuclei in certain cells become different from those of other cells? Or, do all of the nuclei retain an equality during cleavage and development? Experimental evidence indicates a negative answer to the former question and a positive one to the latter.

A precise and illuminating experiment demonstrating nuclear equality of the early blastomeres may be performed by the hair-loop constriction method (Spemann, ’28; Fankhauser, ’48). For example, the fertilized egg of the newt, Triturus viridescens, may be constricted partially by a hair loop so that the zygotic nucleus is confined to one side (fig. 165 A, B). The side possessing the nucleus divides, but the other side does not divide (fig. 165B, C). By releasing the ligature between the two sides at various stages of development of the cleaving side, i.e., 2-, 4-, 8-, 16-, and 32-cell stages, a nucleus is permitted to “escape” into the cytoplasm of the uncleaved side (fig. 165C, E; in D the escaped nucleus is seen in the blastomere to the left). By tightening the loop again after the escaping nucleus has entered the uncleaved cytoplasm, further nuclear “invasion” of the uncleaved part is blocked. If the original constriction was made so that the plane of constriction coincides with the plane of bilateral symmetry, i.e., if it constricts the gray crescent into two halves, the result is two normal embryos. This occurs after the 2-, 4-, 8- and 16-cell stages of the cleaving half of the egg. Nuclei permitted to escape when the cleaving side has reached the 32-cell stage do not produce normal embryos in the uncleaved side, probably because of the changes which have occurred in the meantime in the cytoplasm of the uncleaved side and not to the qualitative differences in the nuclei at this stage.

Another type of experiment upon the early cleaving blastomeres which demonstrates nuclear equality may be performed. It has been shown by Pfliiger, Roux, and Driesch (Wilson, E. B., ’25, p. 1059) that a cleaving egg pressed between two glass surfaces will divide parallel to the pressure surfaces. That is, the mitotic spindle is moved into a position parallel to the pressure surfaces. Under these circumstances, the spindle obeys the second law of Hertwig, namely, that the mitotic spindle tends to coincide with the long axis of the protoplasmic mass. Cleavage under pressure so applied, therefore, will result in a series of vertical cleavage planes. In the sea urchin (fig. 166) if pressure is applied in the four-cell stage, the mitotic spindles will form in a horizontal position, as shown in figure 166E, instead of in the vertical position, as indicated in figure 166B, C, where no pressure is applied. In other words, all of the nuclei shown in white in the upper blastomeres of

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figure 166C will be displaced horizontally by the applied pressure, as shown in figure 165F. If pressure is released at this stage, the mitotic spindle again obeys Hertwig’s rule and forms in the long axis of the cytoplasm which is now vertical in position. As a result, upper and lower cells are formed, as in figure 166G. The original destiny of the nuclei in the cells producing ectoderm is shown in white circles; that for the cells destined to produce mesenchyme, entoderm, and ectoderm is shown in black (figs. 163, mesomeres; 166C, D). As shown in figure 166G, there is a mixture of these nuclei after the pressure is released. Regardless of this redistribution of nuclei, development proceeds almost normally. Development thus appears to be governed by the presence of special ooplasmic substances contained within the respective blastomeres (figs. 163; 166A-D).

The evidence from the foregoing experiments suggests the conclusion that the nuclei in the early blastomeres are qualitatively equal. Consequently, this body of experimental evidence is antagonistic to the older view of Weismann, who held that differences in the various parts of the developing organism are to be attributed to “differential nuclear divisions” whereby different hereditary qualities (i.e., biophors) are dispersed to different cells. To quote from Weismann (1893, p. 76):

Ontogeny depends on a gradual process of disintegration of the id of germplasm, which splits into smaller and smaller groups of determinants in the development of each individual, so that in place of a million different determinants, of which we may suppose the id of the germ-plasm to be composed, each daughter-cell in the next ontogenetic stage would only possess half a million, and each cell of the following stage only a quarter of a million and so on. Finally, if we neglect possible complications, only one kind of determinant remains in each cell, viz., that which has to control that particular cell or group of cells.

£. Quantitative and Qualitative Cleavages and Their Influence upon Later Development

One of the earliest students of the problem of the developmental possibilities of isolated blastomeres was Hans Driesch (1891 and 1892). In these publications, Driesch offered the results of experiments in which he shook apart the early blastomeres of the sea urchin and studied their development. Driesch found that the two blastomeres resulting from the first division continued to divide, and as though the other blastomeres were present. The first division of the isolated blastomere was meridional, as if it had retained contact with its mate of the two-cell stage. The next division was latitudinal, also, as if it had retained contact with its original mate. Ultimately each isolated blastomere developed into swimming blastulae of half the normal size. The four blastomeres of the four-cell stage were similarly isolated. Here, also, each divides as if it were part of the whole, and free-swimming blastulae develop. However, later development is imperfect or definitely abnormal.

QUANTITATIVE AND QUALITATIVE CLEAVAGES

335

Isolation of blastomeres in the eight-cell stage of development, in most cases, results in abnormal development.

In Amphioxus, as mentioned previously, isolation of the first two blastomeres results in the production of twin embryos of half the normal size. In the eight-cell stage in Amphioxus, the isolated smaller micromeres will develop blastulae of ectoderm only, whereas the macromeres will develop blastulae with developed entoderm, mesoderm, and ectoderm. In the four-cell stage, if the two posterior blastomeres are separated from the two anterior blastomeres, the former develop early embryos which have entoderm and mesoderm together with ectoderm; the latter have notochord and neural plate together with ectoderm and possibly a little of the mesoderm (Conklin, ’33). Similarly, in the frog or in the newt, when the first cleavage plane bisects the gray crescent, the isolation of the first two blastomeres results in the

Fig. 166. Nuclear equality in the sea-urchin egg. (A-D) Normal cleavage. White nuclei and black nuclei theoretically so designed to show nuclei in animal and vegetal pole cells respectively. (E) Four-cell stage flattened by pressure, showing position of spindles for the third cleavage parallel to pressure surface. (F) Eight-cell stage under pressure. Compare with (C), normal. (G) Horizontal cleavage resulting from release of pressure after eight-cell stage. Note mixed distribution of nuclei. Later development normal, with cytoplasmic, organ-forming substances determining development as in fig. 163. Thus it appears that the nuclei are equal within the blastomeres, whereas the cytoplasm is unequally (i.e., qualitatively) distributed to the respective blastomeres, the particular type of development of the blastomeres being dependent upon the cytoplasmic substance present.

Black cytoplasm ~ micromeres which form primary mesenchyme. Coarse dotting = entoderm, secondary mesenchyme and coelomic material. White, light stipple, and vertical lines = ectodermal cells.

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CLEAVAGE (SEGMENTATION) AND BLASTULATION

formation of two normal embryos. However, if the first cleavage is at right angles to the plane of bilateral symmetry of the egg, the blastomere containing the gray crescent material will develop a normal embryo, but the other blastomere will not do so.

The above results from isolated blastomeres suggest the following; When the division of the early egg is purely quantitative, so that the resulting blastomeres contain all of the cytoplasmic substances equally, as in the first one or two cleavage planes in the sea urchin (fig. 166A, B) or the first cleavage in the frog when it bisects the gray crescent, the isolation of the resulting blastomeres tends to produce complete embryos. Such blastomeres are known as totipotent blastomeres. (See Chap. 8.) However, when cleavage is qualitative, such as the second cleavage of Amphioxus, the third cleavage of the sea urchin (fig. 166C), or the first cleavage of the frog when it occurs at right angles to the median axis of the embryo, the resulting development depends upon the qualities (that is, ooplasmic substances) resident in the isolated blastomeres.

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7

Tlie CkorJate Blastula and Its Significance

A. Introduction

1. Blastulae without auxiliary tissue

2. Blastulae with auxiliary or trophoblast tissue

3. Comparison of the two main blastular types

B. History of the concept of specific, organ-forming areas

C. Theory of epigenesis and the germ-layer concept of development

D. Introduction of the words ectoderm, mesoderm, endoderm

E. Importance of the blastular stage in Haeckel’s theory of “The Biogenetic Law of Embryonic Recapitulation”

F. Importance of the blastular stage in embryonic development

G. Description of the various types of chordate blastulae with an outline of their organforming areas

1. Protochordate blastula

2. Amphibian blastula

3. Mature blastula in birds

4. Primary and secondary reptilian blastulae

5. Formation of the late mammalian blastocyst (blastula)

a. Prototherian mammal, Echidna

b. Metatherian mammal, Didelphys

c. Eutherian mammals

6. Blastulae of teleost and elasmobranch fishes

7. Blastulae of gymnophionan amphibia

A. Introduction

In the previous chapter it was observed thatftwo main types of blastulae are formed in the chordate group: ^

(1) those blastulae without accessory or trophoblast tissue, e.g., Amphioxus, frog, etc. and

(2) those possessing such auxiliary tissue, e.g., elasmobranch and teleost fishes, reptiles, birds, and mammals.

1. Blastulae Without Auxiliary Tissue

The blastulae which do not have the auxiliary tissues are rounded affairs composed of a layer. of blastomeres surrounding a blastocoelic cavity (figs.

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140T; 143C). The layer of blastomeres forms the blastoderm. The latter may be one cell in thickness, as in Amphioxus (fig. MOT), or several cells in thickness, as in the frog (fig. M3C). This hollow type of blastula often is referred to as a coeloblastula or blastosphere. However, in the gymnophionan amphibia, the blastula departs from this vesicular condition and appears quite solid. The latter condition may be regarded as a stereoblastula, i.e., a solid blastula. A somewhat comparable condition is present in the bony ganoid fishes, Amia and Lepisosteus,

The main characteristic of the blastula which does not possess auxiliary tissue is that the entire blastula is composed of formative cells, i.e., all the cells enter directly into the formation of the embryo’s body.

2. Blastulae with Auxiliary or Trophoblast Tissue^

examination of those blastulae which possess auxiliary or trophoblast tissues shows a less simple condition than the round blastulae mentioned above. In the first plac^fi two types of cells are present, namely, formative cells which enter into the "exposition of the embryonic body and auxiliary cells concerned mainly with trophoblast, or nutritional, functions. In the second place, in the blastula which possesses auxiliary tissue, the latter often develops precociously, that is, in advance of the formative cells of the blastul^ As a result, the arrangement of the formative cells into a configuration comparable to that of those blastulae without trophoblast cells may be much retarded in certain instances. This condition is true particularly of the mammalian blastula (blastocyst).

Generally speak ingj(^the blastulae which possess auxiliary tissue consist in their earlier stages of a disc or a mass of formative cells at the peripheral margins of which are attached the non-formative, auxiliary cells (fig. 159, blastoderm-formative cells, periblast-non-formative; also figs. M5K, L; M7G, H). The blastocoelic space lies below this disc of cells. However, in mammals the auxiliary or nourishment-getting tissue tends to circumscribe the blastocoel, whereas the formative cells occupy a polar area (fig. MSG, H). Blastulae, composed of a disc-shaped mass of cells overlying a blastocoelic space, have been described in classical terms as discoblastulae.')

3. Comparison of the Two Main Blastular Types

If we compare these two types of blastulae in terms of structure, it is evident that a comparison is not logical unless the essential or formative cells and their arrangement are made the sole basis for the comparison, for only the formative cells are common to both types of blastulae. To make the foregoing statement more obvious, let us examine the essential structure of a typical coeloblastula, such as found in Amphioxus, as it is defined by the presentday embryologist.

The studies by Conklin, ’32 and ’33, demonstrated that the fertilized egg

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of Amphioxus possesses five major, presumptive, organ-forming areas (fig. 167A). These areas ultimately give origin to the ectodermal, mesodermal, entodermal, notochordal, and neural tissues. In the eight-cell stage of cleavage, the cytoplasmic substances concerned with these areas are distributed in such a way that the blastomeres have different substances and, consequently, differ qualitatively (fig. 167B). Specifically, the entoderm forms the ventral part of the four ventral blastomeres; the ectoderm forms the upper or dorsal portion of the four micromeres, while the mesodermal, notochordal, and neural substances lie in an intermediate zone between these two organ-forming areas, particularly so in the blastomeres shown at the left in figure 167B. In figure 167C and D is shown a later arrangement of the presumptive, organ-forming areas in the middle and late stages of blastular development. These figures represent sections of the blastulae. Consequently, the organ-forming areas are contained within cells which occupy definite regions of the blastula. In figure 167E-G are presented lateral, vegetal pole, and dorso-posterior pole views of the mature blastula (fig. 167D), representing the organ-forming areas as viewed from the outside of the blastula.

It is evident from this study by Conklin that the organization of the fertilized egg of Amphioxus passes gradually but directly through the cleavage stages into the organization of the mature blastula; also, that the latter, like the egg, is composed of five, major, presumptive, organ-forming areas. It is evident further that one of the important tasks of cleavage and blastulation is to develop and arrange these major, organ-forming areas into a particular pattern. (Note: Later the mesodermal area divides in two, forming a total of six, presumptive, organ-forming areas.)

If we analyze the arrangement of these presumptive, organ-forming areas, we see that the mature blastula is composed of a floor or hypoblast, made up of potential, entoderm-forming substance, and a roof of potential ectoderm with a zone of mesoderm and chordoneural cells which lie in the area between these two general regions. In fact, the mesodermal and chordoneural materials form the lower margins of the roof of the mature blastula (fig. 167D). Consequently, the mature blastula of Amphioxus may be pictured as a bilaminar affair composed essentially of a hypoblast or lower layer of presumptive entoderm, and an upper concave roof or epiblast containing presumptive ectoderm, neural plate, notochord, and mesodermal cells. It is to be observed further that the blastocoel is interposed between these two layers. This is the basic structure of a typical coeloblastula. Furthermore, this blastula is composed entirely of formative tissue made up of certain definite, potential, organforming areas which later enter into the formation of the body of the embryo; auxiliary or non-formative tissue has no part in its composition. All coeloblastulae conform to this general structure.

If we pass to the blastula of the early chick embryo, a striking similarity may be observed in reference to the presumptive, organ-forming areas (fig.

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173). An upper, epiblast layer is present, composed of presumptive ectodermal, neural, notochordal, and mesodermal cells, while a hypoblast layer of entodermal potency lies below. Between these two layers the blastocoelic space is located. However, in the chick blastoderm, in addition to the formative cells, a peripheral area of auxiliary or trophoblast (periblast) tissue is present.

B. History of the Concept of Specific, Organ-forming Areas

The idea that the mature egg or the early developing embryo possesses certain definite areas having different qualities, each of which contributes to the formation of a particular organic structure or of several structures, finds its roots in the writings of Karl Ernst von Baer, 1828-1837. Von Baer’s comparative thinking and comprehensive insight into embryology and its processes established the foundation for many of the results and conclusions that have been achieved in this field during the past one hundred years.

Some forty years later, in 1874, Wilhelm His in his book, Unsere Korperform, definitely put forth the organ-forming concept relative to the germ layers of the chick, stating that “the germ-disc contains the organ-germs spread out in a flat plate,” and he called this the principle of the organ-forming germregions (Wilson, ’25, p. 1041). Ray Lankester, in 1877, advanced views supporting an early segregation from the fertilized egg of “already formed and individualized” substances, as did C. O. Whitman (1878) in his classical work on the leech, Clepsine. In this work. Whitman concludes that there is definite evidence in favor of the preformation of organ-forming stuffs within the egg. Other workers in embryology, such as Rabl, Van Beneden, etc., began to formulate similar views (Wilson, ’25, pp. 1041-1042).

The ideology embodied within the statement of Ray Lankester referred to above was the incentive for considerable research in that branch of embryological investigation known as “cell lineage.” To quote more fully from Lankester’s statement in this connection, p. 410:

Though the substance of a cell may appear homogeneous under the most powerful microscope, excepting for the fine granular matter suspended in it, it is quite possible, indeed certain, that it may contain, already formed and individualized, various kinds of physiological molecules. The visible process of segregation is only the sequel of a differentiation already established, and not visible.

The studies on cell lineage in many invertebrate forms, such as that of Whitman (1878) on Clepsine, of Wilson (1892) on Nereis, of Boveri (1892) and zur Strassen (1896; fig. 163B) on Ascaris, or the work of Horstadius (’28, ’37; fig. 163A) on the sea urchin, serve to emphasize more forcefully the implications of this statement. In these studies the developmental prospective fates of the various early cleavage blastomeres were carefully observed and followed.

Much of the earlier work on cell lineage was devoted to invertebrate forms. One of the first students to study the matter in the phylum Chordata was

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E. G. Conklin who published in 1905 a classical contribution to chordate embryology relative to cell lineage in the ascidian, Styela (Cynthia) partita. This monumental work extended the principle of organ-forming, germinal areas to the chordate embryo. However, the significance of the latter observations, relative to the chordate phylum as a whole, was not fully appreciated until many years later when it was brought into prominence by the German investigator, W. Vogt (’25, ’29).

Vogt began a series of studies which involved the staining of different parts of the amphibian blastula with vital dyes and published his results in 1925 and 1929. The method employed by Vogt is as follows:

Various parts of the late amphibian blastula are stained with such vital dyes as Nile-blue sulfate, Bismarck brown, or neutral red (fig. 168A). These stains color the cells but do not kill them. When a certain area of the blastula is stained in this manner, its behavior during later stages of development can be observed by the following procedure: After staining a particular area, the embryo is observed at various later periods, and the history of the stained area is noted. When the embryo reaches a condition in which body form is fully established, it is killed, fixed in suitable fluids, embedded in paraffin, and sectioned. Or, the embryo may be dissected after fixation in a suitable fluid. The cellular area of the embryo containing the stain thus may be detected and correlated with its original position in the blastula (cf. fig. 168A, B). This procedure then is repeated for other areas of the blastula (fig. 168C-E). Vogt thus was able to mark definite areas of the late blastula, to follow their migration during gastrulation, and observe their later contribution to the formation of the embryonic body. Definite maps of the amphibian blastula in relation to the future history of the respective blastular areas were in this way established (fig. 169C).

This method has been used by other investigators in the study of similar phenomena in other amphibian blastulae and in the blastulae and gastrulae of other chordate embryos. Consequently, the principle of presumptive, organforming areas of the blastula has been established for all of the major chordate groups other than the mammals. The latter group presents special technical difficulties. However, due to the similarity of early mammalian development with the development of other Chordata, it is quite safe to conclude that they also possess similar, organ-forming areas in the late blastular and early gastrular stages.

The major, presumptive, organ-forming areas of the late chordate blastula are as follows (figs. 167, 169, 1-73, 174, 179, 180, 181):

(1) There is an ectodermal area which forms normally the epidermal layer of the skin;

(2) also, there is an ectodermal region which contributes to the formation of the neural tube and nervous system;

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(3) a notochordal area is present which later gives origin to the primitive axis;

(4) the future mesodermal tissue is represented by two areas, one on either side of the notochordal area. In Amphioxus, however, this mesodermal area is present as a single area, the ventral crescent, which divides during gastrulation into two areas;

(5) the entodermal area, which gives origin to the future lining tissue of the gut, occupies a position in the blastula either at or toward the vegetative pole;

(6) there is a possibility that another potential area, containing germinal plasm, may be present and integrated with the presumptive entoderm or mesoderm. This eventually may give origin to the primitive germ cells;

(7) the pre-chordal plate region is associated with the notochordal area in all chordates in which it has been identified and lies at the caudal margin of the latter. In gastrulation it maintains this association. The pre-chordal plate material is an area which gives origin to some of the head mesoderm and possibly also to a portion of the roof of the foregut. It acts potently in the organization of the head region. Accordingly, it may be regarded as a complex of entomesodermal cells, at least in lower vertebrates.

C. Theory of Epigenesis and the Germ-layer Concept of Development

As the three classical germ layers take their origin from the blastular state (see Chap. 9), it is well to pause momentarily to survey briefly the germ-layer concept.

That the embryonic body is derived from definite tissue layers is an old concept in embryology. Casper Friedrich Wolff (1733-94) recognized that the early embryonic condition of the chick blastoderm possessed certain layers of tissue. This fact was set forth in his Theoria Generationis, published in 1759, and in De forniatione intestinorum praecipue, published in 1769, devoted to the description of the intestinal tract and other parts of the chick embryo. In these works Wolff presented the thesis that embryonic development of both plants and animals occurred by “a host of minute and always visible elements that assimilated food, grew and multiplied, and thus gradually in associated masses” produced the various structures which eventually become recognizable as “the heart, blood vessels, limbs, alimentary canal, kidneys, etc.” (The foregoing quotations are from Wheeler, 1898.) These statements contain the essence of Wolff’s theory of epigenesis. That is, that development is not a process of unfolding and growth in size of preformed structures; rather, it is an indirect one, in which certain elements increase in number and gradually become molded into the form of layers which later give rise to the organ structures of the organism.

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Two Other men contributed much to the layer theory of development, namely, Heinrich Christian Pander (1794-1865) and Karl Ernst von Baer (1792-1876). In 1817, Pander described the trilaminar or triploblast condition of the chick blastoderm, and von Baer, in his first volume (1828) and second volume (1837) on comparative embryology of animals, delineated four body layers. The four layers of von Baer’s scheme are derived from Pander’s three layers by dividing the middle layer into two separate layers of tissue. Von Baer is often referred to as the founder of comparative embryology for various reasons, one of which was that he recognized that the layer concept described by Pander held true for many types of embryos, vertebrate and invertebrate. The layer concept of development thus became an accepted embryological principle.

While Pander and von Baer, especially the latter, formulated the germlayer concept as a structural fact for vertebrate embryology, to Kowalewski (1846-1901) probably belongs the credit for setting forth the idea, in his paper devoted to the early development of Amphioxus (1867), that a primary, single-layered condition changes gradually into a double-layered condition. The concept of a single-layered condition transforming into a double-layered condition by an invaginative procedure soon became regarded as a fundamental embryological sequence of development.

Gradually a series of developmental steps eventually became crystallized from the fact and speculation present during the latter half of the nineteenth century as follows:

( 1 ) The blastula, typically a single-layered, hollow structure, becomes converted into

(2) the two-layered gastrula by a process of invagination of one wall or delamination of cells from one wall of the blastula; then,

(3) by an outpouching of a part of the inner layer of the gastrula, or by an ingression of cells from this layer, or from the outside ectoderm, a third layer of cells, the mesoderm, comes to lie between the entoderm and ectoderm; and finally,

(4) the inner layer of mesoderm eventually develops into a two-layered structure with a coelomic cavity between the layers.

This developmental progression became accepted as the basic procedure in the development of most Metazoa.

The original concept of the germ layers maintained that the layers were specific. That is, entodermal tissue came only from entoderm, ectodermal tissue from ectoderm, etc. However, experimental work on the early embryo in which cells are transplanted from one potential layer to another has overthrown this concept ( Oppenheimer, ’40). The work on cell lineage and the demonstration of the early presence of the presumptive, organ-forming areas

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also have done much to overthrow the concept concerning the rigid specificity of the three primary germ layers of entoderm, mesoderm, and ectoderm.

D. Introduction of the Words Ectoderm^ Mesoderm^ Endoderm

Various students of the Coelenterata, such as Huxley (1849), Haeckel (1866) and Kleinenberg (1872), early recognized that the coelenterate body was constructed of two layers, an outer and an inner layer. Soon the terms ectoderm (outside skin) and endoderm (inside skin) were applied to the outer and inner layers or membranes of the coelenterate body, and the word mesoderm (middle skin) was used to refer to the middle layer which appeared in those embryos having three body layers. The more dynamic embryological words epiblast, mesoblast, and hypoblast (entoblast) soon came to be used in England by Balfour, Lankester, and others for the words ectoderm, mesoderm, and endoderm, respectively. The word entoderm is used in this text in preference to endoderm.

E. Importance of the Blastular Stage in Haeckel’s Theory of ^^The Biogenetic Law of Embryonic Recapitulation”

In 1859, Charles Darwin (1809-82) published his work On the Origin of Species by Means of Natural Selection, This theory set the scientific world aflame with discussions for or against it.

In 1872 and 1874, E. Haeckel (1834-1919), an enthusiast of Darwin’s evolutionary concept, associated the findings of Kowalewski regarding the early, two-layered condition of invertebrate and vertebrate embryos together with the adult, two-layered structure of the Coelenterata and published the blastaea-gastraea theory and biogenetic principle of recapitulation. In these publications he applied the term gastrula to the two-layered condition of the embryo which Kowalewski has described as the next developmental step succeeding the blastula and put forward the idea that the gastrula was an embryonic form common to all metazoan animals.

In his reasoning (1874, translation, ’10, Chap. 8, Vol. I), Haeckel applied the word blastaea to a “long-extinct common stem form of substantially the same structure as the blastula.” This form, he concluded, resembled the “permanent blastospheres” of primitive multicellular animals, such as the colonial Protozoa. The body of the blastaea was a “simple hollow ball, filled with fluid or structureless jelly with a wall composed of a single stratum of homogeneous ciliated cells.”

The next phylogenetic stage, according to Haeckel, was the gastraea, a permanent, free-swimming form which resembled the embryonic, two-layered, gastrular stage described by Kowalewski. This was the simple stock form for all of the Metazoa above the Protozoa and other Protista. Moreover, he postulated that the gastrula represented an embryonic recapitulation of the adult stage of the gastraea or the progenitor of all Metazoa.

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The assumed importance of the blastula and gastrula thus became the foundation for Haeckel’s biogenetic principle of recapitulation. Starting with the postulation that the hypothetical blastaea and gastraea represented the adult phylogenetic stages comparable to the embryonic blastula and gastrula, respectively, Haeckel proceeded, step by step, to compress into the embryological stages of all higher forms the adult stages of the lower forms through which the higher forms supposedly passed in reaching their present state through evolutionary change. The two-chambered condition of the developing mammalian heart thus became a representation of the two-chambered, adult heart of the fish, while the three-chambered condition recapitulated the adult amphibian heart, etc. Again, the visceral arches of the embryonic pharyngeal regions of the mammal represented the gill-slit condition of the fish. Ontogeny thus recapitulates phytogeny y and phytogeny of a higher species is the result of the modification of the adult stages of lower species in the phylogenetic scale. The various steps in the embryological development of any particular species, according to this reasoning, were caused by the evolutionary history of the species; the conditions present in the adult stage of an earlier phylogenetic ancestor became at once the cause for its existence in the embryological development of all higher forms. Embryology in this way became chained to a repetition of phylogenetic links!

Many have been the supporters of the biogenetic law, and for a long time it was one of the most popular theories of biology. A surprising supporter of the recapitulation doctrine was Thomas Henry Huxley (1825-95). To quote from Oppenheimer (’40): “One wonders how the promulgator of such a distorted doctrine of cause and effect could have been championed by the same Huxley who wrote: Tact I know and Law I know; but what is this Necessity save an empty Shadow of my own mind’s throwing?’.”

The Haeckelian dogma that ontogeny recapitulates phytogeny fell into error because it was formulated upon three false premises due to the fragmentary knowledge of the period. These premises were:

( 1 ) That in evolution or phytogeny, recently acquired, hereditary characters were added to the hereditary characters already present in the species;

(2) that the hereditary traits revealed themselves during embryonic development in the same sequence in which they were acquired in phytogeny; and

(3) that Darwin’s concept of heredity, namely, pangenesis, essentially was correct.

The theory of pangenesis assumed that the germ cells with their hereditary factors were produced by the parental body or soma and that the contained hereditary factors within the germ cells were produced by gemmules which

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migrated from the various soma cells into the germ cells. This theory further postulated the inheritance of acquired characters.

If these three assumptions are granted, then it is easy to understand Haeckel’s contention that embryological development consists in the repetition of previous stages in phylogeny. For example, if we assume that the blastaea changed into the gastraea by the addition of the features pertaining to the primitive gut with its enteric lining, then the gastraea possessed the hereditary factors of the blastaea plus the new enteric factors. These enteric features could easily be added to the deric (outer-skin) factors of the blastaea, according to Darwin’s theory of pangenesis. Furthermore, according to assumption (2) above, in the embryonic development of the gastraea, the hereditary factors of the blastaea would reveal themselves during development first and would produce the blastaea form, to be followed by the appearance of the specific enteric features of the gastraea. And so it proceeded in the phylogeny and embryology of later forms. In this way the preceding stage in phylogeny became at once the cause of its appearance in the development of the next phylogenetic stage.

•These assumptions, relative to heredity and its mechanism of transference, were shown to be untenable by the birth of the Nageli-Roux-Weismann concept of the germ plasm (see Chaps. 3 and 5) and by the rebirth or rediscovery of Mendelism during the latter part of the nineteenth century. Studies in embryology since the days of Weismann have demonstrated in many animal species the essential correctness of Weismann’s assumption that the germ plasm produces the soma during development, as well as the future germ plasm, and thus have overthrown the pangenesis theory of Darwin. The assiduous study of Mendelian principles during the first twenty-five years of the twentieth century have demonstrated that a fixed relation does not exist between the original character and the appearance of a new character as implied in the Haeckelian law (Morgan, ’34, p. 148). Furthermore, that “in many cases, perhaps in most, a new end character simply replaces the original one. The embryo does not pass through the last stage of the original character and then develop the new one — although this may happen at times — but the new character takes the place of the original one” (Morgan, ’34, p. 148).

How then does one explain the resemblances of structure to be found among the embryos at various stages of development in a large group of animals such as the Chordata? Let us endeavor to seek an explanation.

In development, nature always proceeds from the general to the specific, both in embryological development and in the development of phylogeny or a variety of forms. The hereditary factors which determine these generalized states or structural conditions apparently are retained, and specialized factors come into play after the generalized pattern is established. Generalized or basic conditions, therefore, appear before the specialized ones. An example of this generalized type of development is shown in the formation of the

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blastula in chordate animals. Although many different specific types and shapes of blastulae are present in the group as a whole, all of them can be resolved into two basic groups. These groups, as mentioned in the beginning of this chapter, are:

( 1 ) blastulae without auxiliary, nutritive tissue and

(2) blastulae with auxiliary tissue.

Moreover, if the auxiliary tissue of those blastulae which possess this tissue is not considered, all mature chordate blastulae can be reduced to a fundamental condition which contains two basic layers, namely, hypoblast and epiblast layers. The epiblast possesses presumptive epidermal, neural, notochordal, and mesodermal, organ-forming areas, while the hypoblast cells form the presumptive entodermal area. The shapes and sizes of these blastulae will, of course, vary greatly. Moreover, the hypoblast cells may be present in various positions, such as a mass of cells at the caudal end of a disc-shaped epiblast (teleost and elasmobranch fishes), an enlarged, thickened area or pole of a hollow sphere (many Amphibia) y a single, relatively thin layer of cells, forming part of the wall of a hollow sphere (Arnphioxus), a rounded, disc-shaped mass of cells overlain by the thin, cup-shaped epiblast (Clavelina), a thickened mass attached to the underside of the caudal end of the disc-shaped epiblast (chick; certain reptiles), a thin layer of cells situated below the epiblast layer (mammals), or a solid mass of cells, lying below a covering of epiblast cells (gymnophionan Amphibia). Although many different morphological shapes are to be found in the blastulae of the chordate group, the essential, presumptive, organ-forming areas always are present, and all are organized around the presumptive notochordal area.

But the question arises: Why is a generalized blastular pattern developed instead of a series of separate, distinct patterns? For instance, why should the notochordal area appear to occupy the center of the presumptive, organforming areas of all the chordate blastulae when this area persists as a prominent morphological entity only in the adult condition of lower chordates? The answer appears to be this: The notochordal area at this particular stage of development is not alone a morphological area, but it is also a physiological instrument, an instrument which plays a part in a method or procedure of development. The point of importance, therefore, in the late blastular stage of development is not that the notochordal area is going to contribute to the skeletal axis in the adult of the shark, but rather that it forms an integral part of the biolgical mechanism which organizes the chordate embryo during the period immediately following the blastular stage. Thus, if the notochordal material can play an important role in the organization of the embryo and in the induction of the neural tube in the fish or in the frog, it also can fulfill a similar function in the developing chick or human embryo. Whatever it does later in development depends upon the requirements of the species. To use

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a naive analogy, nature does not build ten tracks to send ten trains with different destinies out of a station when she can use one track for all for at least part of the way. So it is in development. A simple tubular heart appears in all vertebrate embryos, followed by a simple, two-chambered* condition, not because the two-chambered heart represents the recapitulated, two-chambered, fish heart but rather because it, like the notochord, is a stage in a dynamic developmental procedure of heart development in all vertebrates. As far as the fish is concerned, when the common, two-chambered, rudimentary stage of the heart is reached, nature shunts it off on a special track which develops this simple, two-chambered condition into the highly muscular and efficient two-chambered, adult heart adapted to the fish level of existence in its watery environment. The three-chambered,* amphibian heart follows a similar pattern, and it specializes at the three-chambered level because it fits into the amphibian way of life. So it is with the embryonic pharyngeal area with its visceral and aortal arches which resemble one another throughout the vertebrate group during early embryonic development. The elaboration of a common, pharyngeal area with striking resemblances throughout the vertebrate group can be explained more easily and rationally on the assumption that it represents a common, physiologically important step in a developmental procedure.

This general view suggests the conclusion that ontogeny tends to use common developmental methods wherever and whenever these methods can be utilized in the development of a large group of animals. Development or ontogeny, therefore, recapitulates phylogenetic procedures and not adult morphological stages. One explanation for this conservation of effort may be that, physiologically speaking, the number of essential methods, whereby a specific end may be produced, probably is limited. Another explanation suggests that an efficient method never is discarded.

F. Importance of the Blastular Stage in Embryonic Development

Superficially in many forms, chordate and non-chordate, the blastula is a hollow, rounded structure containing the blastocoelic space within. It is tempting to visualize this form as the basic, essential form of the blastula. However, the so-called blastular stage in reality presents many forms throughout the animal kingdom, some solid, some round and hollow, and others in the form of a flattened disc or even an elongated band. Regardless of their shape, all blastulae have this in common/ they represent an association of presumptive organ-forming areas, areas which later move to new positions in the forming body, increase in cellular mass, and eventually become molded into definite structures. One of the main purposes of blastulation, therefore, may be stated as the elaboration (or establishment) of the major, presumptive organ-forming areas of the particular species and their arrangement in a particular pattern which permits their ready manipulation during the next

• Exclusive of the sinus venosus.

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step of development or gastrulation. jThc particular shape of the blastula has its importance. However, this importance does not lie in the supposition that it conforms to a primitive spherical type but rather that the various, presumptive, organ-forming areas are so arranged and so poised that the cell movements so necessary to the next phase of development or gastrulation may be properly executed for the particular species. In most species, the formation of a blastocoelic space also is a necessary function of blastulation. In some species, however, this space actually is not formed until the next stage of development or gastrulation is in progress.

In summary, therefore, it may be stated that the importance of the blastula does not reside in the supposed fact that it is a one-layered structure or blastoderm having a particular shape. Rather, its importance emerges from the fact that the blastoderm has certain, well-defined areas segregated within it — areas which will give origin to future organ structures. Moreover, these areas foreshadow the future germ layers of the body. In diploblastic Metazoa, two germ layers are foreshadowed, while in triploblastic forms, three germ layers are outlined. As far as the Chordata are concerned, the hypoblast is the forerunner of the entoderm or the internal germ layer; whereas the epiblast is composed potentially of two germ layers, namely, the epidermal, neural plate areas which form the ectodermal layer and the chordamesodermal or marginal zone cells which give origin to the middle germ layer.

In the following pages, the chordate blastula is described as a two-layered structure composed of various, potential, organ-forming areas. This twolayered configuration, composed of a lower hypoblast and an upper epiblast, is used to describe the chordate blastula for the dual purpose of comparison and analysis of the essential structure of the various blastulae. The bilaminar picture, it is believed, will enable the student to understand better the changes which the embryo experiences during the gastrulative period.

G. Description of the Various Types of Chordate Blastulae with an Outline of Their Organ-forming Areas

1. Protochordate Blastula

The following description pertains particularly to Amphioxus. With slight modification it may be applied to other protochordates, such as Clavelina, Ascidiella, Styela, etc.

As noted in the introduction to this chapter, the potential entodermal cells of Amphioxus lie at the vegetal pole and form most of the floor or hypoblast of the blastula (fig. 167D). The upper or animal pole cells form a roof of presumptive epidermal, notochordal, mesodermal, and neural cells arched above and around the entoderm. The latter complex of organ-forming cells forms the epiblast. The blastocoelic cavity is large and insinuated between the hypoblast and epiblast. The presumptive notochordal and mesodermal

Fig. 167. Presumptive organ-forming areas in the uncleaved egg and during cleavage and blastulation in Amphioxus. (Original diagram based upon data obtained from Conklin, ’32, ’33.) (A) Uncleaved egg. (B) Eight-cell stage. (C) Early blastula in

section. (D) Late blastula in section. (E) Late blastula, external view from side. (F) Late blastula, external, vegetal pole view. (G) Late blastula, external, dorsoposterior view. The localization of cytoplasmic materials in Styela partita is similar to that of Amphioxus. Observe that the pointed end of the arrow defines the future cephalic end of the embryo. The position of the polar body denotes the antero-ventral area, while the position of the notochordal and neural plate material represents the antero-dorsal region. The “tail end’’ of the arrow is the postero-ventral area of the embryo.

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354 THE CHORDATE BLASTULA AND ITS SIGNIFICANCE

areas lie at the margins of the entodermal layer and surround it. As such, some of the cells of these two, organ-forming areas may form part of the floor of the bias tula. The presumptive, notochordal and neural plate cells lie at the future dorsal lip of the blastopore and form the dorsal crescent, while the mesodermal area occupies the ventral-lip region as the ventral crescent (fig. 167F). In Amphioxus, the mature blastula is pear shaped, with the body

Fig. 168. Ultimate destiny within the developing body of presumptive organ-forming areas of the late amphibian blastula, stained by means of vital dyes. (After Pastecls: J. Exper. Zool., 89.) (A) Area of blastula, stained. (B) Destiny of cellular area, stained

in (A). (D, E) Ultimate destiny shown by broken lines of cellular areas, stained in

late blastula shown in (C). (E) Anterior trunk segment. (D) Posterior trunk segment.

Fig. 169. Presumptive organ-forming areas in the amphibian late blastula and beginning gastrula. (A, B) General epiblast and hypoblast areas of the early and late blastular conditions, respectively. The hypoblast is composed mainly of entodermal or gut-lining structures, whereas the epiblast is a composite of ectodermal (i.e., epidermal and neural), mesodermal, and notochordal presumptive areas. Observe that the epiblast gradually grows downward over the hypoblast as the late blastula is formed. (C) Beginning gastrula of the urodele, Triton. (Presumptive areas shown according to Vogt, ’29.) (D) Same as above, from vegetative pole. (Slightly modified from Vogt, ’29.)

(E) Lateral view of beginning gastrula of anuran amphibia. (F) Dorsal view of the same. (E, F derived from description by Vogt, ’29, relative to Rami fusca and Bombinator; also Pasteels: J. Exper. Zool., 89, relative to Discogkmus.) Observe that an antero-posterior progression of somites is indicated in C and D.

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THE CHORDATE BLASTULA AND ITS SIGNIFICANCE

of the mesodermal crescent comprising much of the neck portion of the “pear” (fig. 167E).

The blastula of Amphioxus thus may be regarded essentially as a bilaminar structure (i.e., two-layered structure) in which the hypoblast forms the lower layer while the epiblast forms the upper composite layer.

.^ 1 , Amphibian Blastula

In the amphibian type of blastula, a spherical condition exists similar to that in Amphioxus (fig. 169). The future entoderm is located at the vegetative (vegetal) pole, smaller in amount in the frog, Rana pipiens, and larger in such forms as Necturus maculosus (fig. 169 A, B). The presumptive notochordal material occupies an area just anterior to and above the future dorsal lip of the blastopore. The dorsal lip of the gastrula, when it develops, arises within the entodermal area (fig. 169C-F). Extending laterally on either side of the presumptive notochordal region is an area of presumptive mesoderm (fig. 169C-F). Each of these two mesodermal areas tapers to a smaller dimension as it extends outward from the notochordal region. The presumptive notochordal and mesodermal areas thus form a composite area or circular marginal zone which surrounds the upper rim of the entodermal material.

Above the chordamesodermal zone are two areas. The presumptive neural area is a crescent-hke region lying above or anterior to the presumptive notochord-mesoderm complex. Anterior to the neural crescent and occupying the remainder of the blastular surface, is the presumptive epidermal crescent (fig. 169C-F).

In the various kinds of blastulae of this group, the yolk-laden, vegetal pole cells actually form a mass which projects upward into the blastocoelic space (fig. 169A, B). The irregularly rounded, presumptive entodermal, organforming area, therefore, is encapsulated partially by the other potential germinal areas, particularly by the chordamesodermal zone (fig. 169B). In a sense, this is true also of the protochordate group (fig. 167D).

The amphibian type of blastula includes those of the petromyzontoid Cyclostomes, the ganoid fishes with the exception of bony ganoids, the dipnoan fishes, and the Amphibia with the exception of the Gymnophiona, where a kind of solid blastula is present.

It is to be observed that the amphibian and protochordate blastulae differ in several details. In the first place, there is a greater quantity of yolk material in the blastula of the Amphibia; hence the presumptive entodermal area or hypoblast projects considerably into and encroaches upon the blastocoel. Also, in Amphioxus, the presumptive notochordal area forms a distinct dorsal crescent apart from the presumptive mesodermal or ventral crescent (fig. 167F), whereas, in the Amphibia, the notochordal material is sandwiched in between the two wings of mesoderm, so that these two areas form one composite marginal zone crescent (fig. 169D, E).

TYPES OF CHORDATE BLASTULAE

357

As in Amphioxus, the amphibian blastula may be resolved into a twolayered structure composed of a presumptive entodermal or hypoblast layer and an upper, epiblast layer of presumptive epidermal, notochordal, mesodermal, and neural tissues. Each of these layers, unlike that of Amphioxus, is several cells in thickness.

^3. Mature Blastula in Birds

Development of the hen’s egg proceeds rapidly in the oviduct (fig. 157B-G), and at the time that the egg is laid, the blastodisc (blastula) presents the following cellular conditions:

( 1 ) a central, cellular blastoderm above the primary blastocoel and

(2) a more peripheral portion, associated with the yolk material forming the germ-wall tissue (fig. 156G).

The central blastoderm is free from the yolk substance and is known as the area pellucida, whereas the germ-wall area with its adhering yolk material forms the area opaca (fig. 170). Around its peripheral margin the area pellucida is somewhat thicker, particularly so in that region which will form the posterior end of the future embryo. In the latter area, the pellucid margin may consist of a layer of three or even four cells in thickness (fig. 172A). This thickened posterior portion of the early pellucid area forms the embryonic shield (fig. 170). Anterior to the embryonic shield, the pellucid area is one or two cells in thickness (figs. 171A; 172B).

Eventually the pellucid area becomes converted into a two-layered structure with an upper or overlying layer, the primitive ectoderm or epiblast and a lower underlying sheet of cells, the primitive entoderm or hypoblast (figs. 171 A; 172A). The space between these two layers forms the true or secondary blastocoel. The cavity below the hypoblast is the primitive archenteric space. At the caudal and lateral edges of the pellucid area, cells from the inner zone of the germ wall appear to contribute to both hypoblast and epiblast.

The two-layered condition of the avian blastula shown in figure 171 A may be regarded as a secondary or late blastula. At about the time that the secondary blastula is formed (or almost completely formed), the hen’s egg is laid, and further development depends upon proper incubational conditions outside the body of the hen. Shortly after the latter incubation period is initiated, the primitive streak begins to make its appearance in the midcaudal region of the blastoderm, as described in Chapter 9.

Much controversy has prevailed concerning the method of formation of the entoderm and the two-layered condition in the avian blastoderm. Greatest attention has been given to the origin of the entoderm in the eggs of the pigeon, hen, and duck. The second layer is formed in the pigeon’s egg as it passes down the oviduct, in the hen’s egg at about the time of laying, and in the duck’s egg during the first hours of the external incubation period. The

Fig. 171. Origin of the hypoblast (entoderm) in the avian blastoderm. (A) Median, antero-posterior section of chick blastoderm. Entoderm arises by delamination from upper or epiblast layer; possibly also by cells that grow anteriad from thickened posterior area. (Based upon data supplied by Peter, ’34, ’38, and Jacobson, ’38.) (B-D) For mation of the hypoblast (entoderm) from epiblast by a process of delamination in the duck embryo. (Based upon data supplied by Pasteels, ’45.)

unincubated chick blastoderm is about 3 mm. in diameter, that of the duck, about 2 to 3 mm.

The most recent observations, relative to the formation of the second or hypoblast layer, have been made upon the duck’s egg (Pasteels, ’45). In this egg, Pasteels found that, at about nine hours after incubation is initiated, a two-layered condition is definitely formed and that “the primary entoblast of the duck is the result of a progressive delamination of the segmenting blastodisc

TYPES OF CHORDATE BLASTULAE

359

separating the superficial cells from the deeper ones” (fig. 171B-D). He further suggests that “the bilaminar embryo of birds is to be homologized with the blastula of the Amphibia, the cleft separating the two layers being equivalent to the blastocoele” (p. 13). The formation of the hypoblast (primary entoderm ) by a process of delamination from the upper layer or epiblast agrees with the observations by Peter (’38) on the developing chick and pigeon blastoderm (fig. 172) and of Spratt (’46) on the chick. It also agrees with some of the oldest observations, concerning the matter of entoderm formation, going back to Ollacher in 1869, Kionka, 1894, and Assheton, 1896. Others, such as Duval (1884, 1888) in the chick, and Patterson (’09) in the pigeon, have ascribed the formation of the primary entoderm to a process of invagination and involution at the caudal margin of the blastoderm, while Jacobson (’38) came to the conclusion that the entoderm of the pellucid area arose in chick and sparrow embryos through a process of outgrowth of cells from the primitive plate and from an archenteric canal produced by an inward bending of the epiblast and primitive plate tissue. The latter author believed that the entoderm of the area opaca arose by delamination.

The hypoblast of the chick gives origin to most of the tissue which lines the future gut, and, therefore, may be regarded as the potential entodermal area. As in the amphibia and Amphioxus, the epiblast is composed of several, presumptive organ-forming areas (fig. 173A). (See Pasteels, ’36c; Spratt, ’42, ’46.) At the caudal part of the epiblast is an extensive region of presumptive mesoderm bisected by the midplane of the future embryonic axis. Just anterior to this region and in the midplane is the relatively small, presumptive notochordal area. Between the latter and the mesodermal area is located the presumptive prechordal plate of mesodermal cells. Immediately in front of the notochordal region lies the presumptive neural area in the form of a crescent with its crescentic arms extending in a lateral direc

Fig. 172. Delamination of hypoblast (entoderm) cells from upper or epiblast layer in the chick blastoderm. (A) Posterior end of blastoderm (cf. fig. 17 lA). (B) Anterior end of blastoderm.

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THE CHORDATE BLASTULA AND ITS SIGNIFICANCE

Fig. 173. Presumptive organ-forming areas in the chick blastoderm. (A) Slightly modified from Spratt, ’46. (B) Schematic section of early chick blastoderm passing

through antero-posterior median axis.

tion from the midline of the future embryonic axis. Anterior to the neural crescent is the presumptive epidermal crescent. Within the area opaca is found potential blood-vessel and blood-cell-forming tissue, as well as the extensive extra-embryonic-tissue materials.

The above description of the presumptive organ-forming areas pertains to the avian blastula just previous to the inward migrations of the notochordal, pre-chordal plate, and mesodermal areas; that is, just previous to the appearance of the primitive streak and the gastrulative process.

4. Primary and Secondary Reptilian Blastulae

The primary blastula of turtle, snake, and lizard embryos is akin in essential features to that of birds. It consists of a central blastoderm or area pellucida, overlying a primary blastocoelic cavity, and a more distally situated opaque blastoderm, together with an indefinite periblast syncytium. A localized region of the central blastoderm, situated along the midline of the future embryonic axis and eccentrically placed toward the caudal end, is known as the embryonic shield.

A specialized, posterior portion of the embryonic shield, in which the upper layer (epiblast) is not separated from the underlying cells (hypoblast), is known as the primitive plate (fig. 174A-D). (Consult also Will, 1892, for

Fig. 174. Formation of hypoblast (entoderm) layer in certain reptiles; major presumptive organ-forming areas of reptilian blastoderm. (A) Section through blastoderm of the turtle, Clemmys leprosa. This section passes through the primitive plate in the region where the entoderm cells are rapidly budded off (invaginated?) from the surface layer. It presumably passes through (E) in the area marked entoblast. It is difficult to determine whether the entoderm cells are actually invaginated, according to the view of Pasteels, or whether this area represents a region where cells are delaminated or budded off in a rapid fashion from the overlying cells. (B) Similar to (A), diagrammatized to show hypoblast cells in black. (C) Section through early blastoderm of the gecko, Platydactylus. Epiblast cells are shown above, primitive entoderm cells below. (D) A later stage showing primitive plate area with the appearance of a delamination or proliferation of entoderm (hypoblast) cells from the upper layer of cells. (E) Presumptive, organ-forming areas of the turtle, Clemmys leprosa, before gastrulation. (F) Presumptive, organ-forming areas of the epiblast of turtle and other reptiles if the hypoblast is budded off or separated from the underside of the epiblast without invagination. It is to be observed that B and D represent modifications by the author.

361

362

THE CHORDATE BLASTULA AND ITS SIGNIFICANCE

accurate diagrams of the reptilian blastoderm.) Surrounding the primitive plate, the central blastoderm is thinner and is but one (occasionally two cells) cell in thickness (see margins of figs. 174A, C). As development proceeds, a layer of cells appears to be delaminated or proliferated off from the undersurface of the primitive plate area (fig. 174C, D). This delamination gives origin to a second layer of cells, the entoderm or hypoblast (Peter, ’34). Some of these entodermal cells may arise by delamination from more peripheral areas of the central blastoderm outside the primitive plate area. In the case of the turtle, Clemmys leprosa, Pasteels (’37a) believes that there is an actual invagination of entodermal cells (fig. 174A-B). More study is needed to substantiate this view.

Eventually, therefore, a secondary blastula arises which is composed of a floor of entodermal cells, the hypoblast, closely associated with the yolk, and an overlying layer or epiblast. The epiblast layer is formed of presumptive epidermal, mesodermal, neural, and notochordal, organ-forming areas. The essential arrangement of the presumptive organ-forming areas in the reptiles is very similar to that described for the secondary avian blastula. The space between the epiblast and hypoblast layers is the secondary blastocoelic space.

Fig. 175. Early blastoderms of the prototherian mammal, Echidna. (A) Early blastoderm showing central mass of cells: with peripherally placed vitellocytes. (B) Later blastoderm. Central cells are expanding and the blastoderm is thinning out. Smaller cells (in black) are migrating into surface layer. Vitellocytes have fused to form a peripheral syncytial tissue. (C) Later blastoderm composed of a single layer of cells of two kinds. The smaller cells in black represent potential entoderm cells. (D) Increase of hypoblast cells and their migration into the archenteric space below to form a second or hypoblast layer.

TYPES OF CHORDATE BLASTULAE

363

Fig. 176. Early development of blastoderm of the opossum. (Modified from Hartman,

• 16.) (A) Blastocyst wall composed of one layer of cells from which entoderm cells

are migrating inward, (B-D) Later development of the formative portion of the blastoderm. Two layers of cells are present in the formative area, viz., an upper epiblast layer and a lower hypoblast. Trophoblast cells are shown at the margins of the epiblast and hypoblast layers.

Both hypoblast and epiblast are connected peripherally with the periblast tissue.

5. Formation of the Late Mammalian Blastocyst (Blastula) a. Prototherian Mammal, Echidna

In Echidna, according to Flynn and Hill (’39, ’42), a blastoderm somewhat comparable to that of reptiles and birds is produced. An early primary blastular condition is first established, consisting of a mass of central cells with specialized vitellocytes at its margin (fig. 175A). A little later, an extension of this blastoderm occurs, and a definite primary blastocoelic space is formed below the blastoderm (fig. 175B). During this transformation, small, deeper lying cells (shown in black, fig. 175B) move up to the surface and become associated with the thinning blastoderm which essentially becomes a single layer of cells (fig. 175C). The marginal vitellocytes in the meantime fuse to form a germ-wall syncytium. This state of development may be regarded as the fully developed primary blastula. A little later, this primary condition becomes converted into a two-layered, secondary blastula, as shown in figure 175D by the secondary multiplication and migration inward of the small cells to form a lower layer or hypoblast. The latter process may be

364

THE CHORDATE BLASTULA AND ITS SIGNIFICANCE

regarded as a kind of polyinvagination. In this manner the secondary blastula is formed. It is composed of two layers of cells, the epiblast above and the hypoblast below with the secondary blastocoelic space insinuated between these two layers.

b. Metatherian Mammal, Didelphys

The opossum, Didelphys virginiana, possesses a hollow blastocyst akin to the eutherian variety. (See Hartman, T6, T9; McCrady, ’38.) As observed in the previous chapter, it is produced by a peculiar method. The early blastomeres do not adhere together to form a typical morula as in most other forms; rather, they move outward and adhere to the zona pellucida and come to line the inner aspect of this membrane. As cleavage continues, they eventually form a primary blastula with an enlarged blastocoel.

Following this primary phase of development, one pole of the blastocyst begins to show increased mitotic activity, and this polar area gradually thickens (fig. 176A). At this time certain cells detach themselves from the thickened polar area of the blastocyst and move inward into the blastocoel (fig. 176A, B) .

Fig. 177. Schematic drawings of early pig development. (A) Early developing blastocyst. (B) Later blastocyst, showing two kinds of cells in the inner cell mass. (C) Later blastocyst, showing disappearance of trophoblast cells overlying the inner cell mass. (D) Later blastocyst. Two layers of formative cells are present as indicated with trophoblast tissue attached at the margins.

TYPES OF CHORDATE BLASTULAE

365

Fig. 178. Schematic drawings of the developing blastocyst of the monkey. (After Hcuser and Streeter: Carnegie Inst,, Washington, Publ. 538. Contrib. to Embryol. No. 181.) (A, B) Early blastocysts showing formative and non-formative cells in the inner

cell mass. (C-E) Later arrangement of the formative cells into an upper epiblast and lower hypoblast layer.

These cells form the mother entoderm cells, and by mitotic activity they give origin to an entodermal layer which adheres to the underside of the thickened polar area (fig. 176B, C). The polar area then thins out to form the expansive condition shown in figure 176D. A bilaminar, disc-shaped area thus is formed in this immediate region of the blastocyst, and it represents the area occupied

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THE CHORDATE BLASTULA AND ITS SIGNIFICANCE

by the formative cells of the blastula. The edge of this disc of formative cells is attached to the trophoblast or auxiliary cells (fig. 176D). Only the formative cells give origin to the future embryonic body.

c. Eutherian Mammals

The eutherian mammals as a whole present a slightly different picture of blastocyst development from that described above for marsupial species. These differences may be outlined as follows:

( 1 ) During the earliest phases of blastocyst development in most eutherian mammals, a distinct, inner cell mass is elaborated at the formative or animal pole (fig. 177 A, B). This characteristic is marked in some species (pig, rabbit, man, and monkey) and weaker in others (mink and armadillo). It may be entirely absent in the early blastula of the Madagascan insectivore, Hemicentetes semispinosus; however, in the latter, a thickening corresponding to the inner cell mass later

Fig. 179. Presumptive organ-forming areas in the blastoderm of the shark embryo. (A) Median section of the blastoderm of Torpedo ocellata. Hypoblast cells are shown in black. Caudal portion of the blastoderm is shown at the right. Cf. (B). (This figure partly modified from Ziegler, ’02 — see Chap. 6 for complete reference.) (B) Map of the presumptive organ-forming areas of the blastoderm of the shark, Scyllium canicula.

TYPES OF CHORDATE BLASTULAE

367

E P I BLAST

N TO DERM OR PRIMARY ' YPOBLAST

NEURAL ECTOOE RM

NOTOCHORD

ENTO DE RM DORSAL BLASTOPORAL LIP

Fig. 180. Presumptive organ-forming areas of the teleost fish blastoderm. (A) Median section through the late blastoderm of Fundulus heteroclitus just previous to gastrulation. Somewhat schematized from the author’s sections. Presumptive entoderm or hypoblast is shown exposed to the surface at the caudal end of the blastoderm and, therefore, follows the conditions shown in (B). (B) Presumptive organ-forming areas

of the blastoderm of Fundulus heteroclitus. Arrows show the direction of cell movements during gastrulation. (Modified from diagram by Oppenheimer, ’36.)

appears. Within the inner cell mass, two types of cells are present, namely, formative and trophoblast (figs. 177B; 178A).

(2) Unlike that of the marsupial mammal, an overlying layer of trophoblast cells, covering the layer of formative cells, always is present (fig. 177B). In some cases (rabbit, pig, and cat) they degenerate (the cells of Rauber, fig. 177C), while in others (man, rat, and monkey) the overlying cells remain and increase in number (fig. 178A-E).

(3) The entodermal cells arise by a separation (delamination) of cells from the lower aspect of the inner cell mass (figs. 177C; 178A), with the exception of the armadillo where their origin is similar to that of marsupials. With these differences, the same essential goal arrived at in the marsupial mammals is achieved, namely, a bilaminar, formative area, the embryonic disc, composed of epiblast and hypoblast layers (figs. 177D; 178D, E), which ultimately gives origin to the embryonic body. A bilaminar, extra-embryonic, trophoblast area, consisting of extra-embryonic entoderm and ectoderm, also is formed (figs. 177D; 178D, E). The secondary blastocoel originates between the epiblast and hypoblast of the embryonic disc, while below the hypoblast layer is the archenteric space (fig. 178E).

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THE CHORDATE BLASTULA AND ITS SIGNIFICANCE

6. Blastulae of Teleost and Elasmobranch Fishes

In the teleost and elasmobranch fishes, the primary blastula is a flattened, disc-shaped structure constructed during its earlier stages of an upper blastoderm layer of cells, the formative or strictly embryonic tissue, and a peripheral and lower layer of trophoblast or periblast tissues; the latter is closely associated with the yolk substance (figs. 179A; 180A; 181 A). The primary blastocoelic space lies between the blastoderm and the periblast tissue.

That margin of the formative portion of the blastoderm which lies at the future caudal end of the embryo is thickened considerably, and presumptive entodermal material or primary hypoblast is associated with this area. Its relationship is variable, however. In some teleost fishes, such as the trout, the entodermal cells are not exposed to the surface at the caudal portion of the blastodisc (fig. 181 A; Pasteels, ’36a). In other teleosts, a considerable portion of the entodermal cells may lie at the surface along the caudal margin of the blastoderm (fig. 180A; Oppenheimer, ’36). In the elasmobranch fishes the disposition of the entodermal material is not clear. A portion undoubtedly lies exposed to the surface at the caudal margin of the disc (fig. 179 A, B; Vandebroek, ’36), but some entodermal cells lie in the deeper regions of the blastoderm (fig. 179A).

Turning now to a consideration of the other presumptive organ-forming areas of the fish blastoderm, we find that the presumptive pre-chordal plate material lies exposed on the surface in the median plane of the future embryo immediately in front of the entoderm and near the caudal edge of the blastoderm. (It is to be observed that, in comparison, the pre-chordal plate lies well forward within the area pellucida of the bird blastoderm.) This condition is found in the shark, Scyllium, in Fundulus, and in the trout, Salrno (figs. 179B; 180B). However, in the trout it lies a little more posteriorly at the caudal margin of the disc (fig. 181B). Anterior to the pre-chordal plate is the presumptive notochordal material, and anterior to the latter is a rather expansive region of presumptive neural cells. These three areas thus lie along the future median plane of the embryo, but they exhibit a considerable variation in size and in the extent of area covered in Scyllium, Fundulus, and Salrno (figs. 179, 180, 181).

Extending on either side of these presumptive organ-forming areas, is an indefinite region of potential mesoderm. In Salrno, presumptive mesodermal cells lie along the lateral and anterior portions of the blastoderm edge (fig. 18 IB). However, in Scyllium and in Fundulus, it is not as extensive (figs. 179B; 180B). In front of the presumptive neural organ-forming area is a circular region, the presumptive epidermal area.

In their development thus far the three blastulae described above represent a primary blastuiar condition, and the cavity between the blastodisc and the underlying trophoblast or periblast tissue forms a primary blastocoel. This condition presents certain resemblances to the early blastocyst in the higher

PERI BLA ST ^

ENTODERM OR PRIMARY . . . HYPOBLAST

— MESODERM

1 . •• ■ >;.'.\ rr E P I OE R M AL

ECTODERM

-NEURAL ECTO DERM

-NOTO CHORD

— -—P RE-CHORDAL PLATE

DORSAL BLASTOPORAL LIP

Fig. 181. Presumptive organ-forming areas of the blastoderm of the trout, Salma irideus. (A) Schematized section through blastoderm just previous to gastrulation. Presumptive entoderm (hypoblast) shown in black at caudal end of the blastoderm. Observe that entoderm is not exposed to surface. Cf. (B). (B) Surface view of presumptive

organ-forming areas of the blastoderm just before gastrulation.

Fig. 182. Late blastoderms of Gymnophiona. (Modified from Brauer, 1897.) (A)

Late blastoderm of Hypogeophis alternans. Entoderm cells in black lie below. (B) Beginning gastrula of H. rostratus. Observe blastocoelic spaces in white between the entoderm cells.

369

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THE CHORDATE BLASTULA AND ITS SIGNIFICANCE

mammals and the late blastula of birds. In both groups the trophoblast tissue is attached to the edges of the formative tissue and extends below in such a way that the formative cells and trophoblast tissue tend to form a hollow vesicle. In both, the formative portion of the blastula is present as a disc or mass of cells composed of presumptive, organ-forming cells closely associated at its lateral margins with the trophoblast or food-getting tissue. A marked distinction between the two groups, however, is present in that the future entodermal cells in fishes are localized at the caudal margin of the disc, whereas in mammals and birds they may be extensively spread along the under margin of the disc. In reptiles the condition appears to be somewhat similar to that in birds and mammals, with the exception possibly of the turtles, where the future entoderm appears more localized and possibly may be superficially exposed. Therefore, while great differences in particular features exist between the fishes and the higher vertebrates, the essential fundamental conditions of the early blastulae in teleost and in elasmobranch fishes show striking resemblances to the early blastulae of reptiles, birds, and mammals.

The blastulae of teleost fishes remain in this generalized condition until about the time when the gastrulative processes begin. At that time the notochordal and mesodermal, cellular areas begin their migrations over the caudal edge of the blastodisc to the blastococlic space below, where they ultimately come to lie beneath the epidermal and neural areas. Associated with the migration of notochordal and mesodermal cells, an entodermal floor or secondary hypoblast is established below the notochordal and mesodermal cells by the active migration of primary hypoblast cells in an antero-lateral direction. In the elasmobranch fishes there is a similar cell movement from the caudal disc margin, as found in teleost fishes, but, in addition, a delamination of entodermal (and possibly mesodermal cells) occurs from the deeper lying parts of the blastodisc.

7. Blastulae of Gymnophionan Amphibia

In the Gymnophiona, nature has consummated a blastular condition different from that in other Amphibia. It represents an intermediate condition between the blastula of the frog and the blastodiscs of the teleost and elasmobranch fishes and of higher vertebrates (fig. 182). In harmony with the frog blastula, for example, a specialized periblast or food-getting group of cells is absent. On the other hand, the presumptive entoderm and the presumptive notochordal, mesodermal, neural, and epidermal cells form a compact mass at one pole of the egg, as in teleosts, the ohick, and mammal. Similar to the condition in the chick and mammal, the entodermal cells delaminate (see Chap. 9) from the under surface of the blastodisc (Brauer, 1897).

Bibliography

Assheton, R. 1896. An experimental examination into the growth of the blastoderm of the chick. Proc. Roy. Soc., London, s.B. 60:349.

Baer, K. E., von. 1828-1837. Tiber Entwickelungsgeschichte der Thiere. Beobachtung und Reflexion. Borntrager, Konigsberg.

Boveri, T. 1892. Tiber die Entstehung des gegensatzes zwischen den Geschlcchtszellen und den somatischen Zellen bei ^scaris megalocephala, etc., in: Sitz. d. gesellsch. d. Morph, u. Physiol. Miinchen. vol. 8.

Brauer, A. 1897. Beitriige zur Kenntniss der Entwicklungsgeschichte und der Aiiatomie der Gy mnophionen. Zool. Jahrb. 10:389.

Conklin, E. G. 1905. The organization and cell-lineage of the ascidian egg. J. Acad. Nat. Sc., Philadelphia. 13:5.

. 1932. The embryology of Aniphi oxus. J. Morphol. 54:69.

. 1933. The development of isolated

and partially separated blastomeres of Amphioxus. J. Exper. Zool. 64:303.

Duval, M. 1884. De la formation du blastoderme dans I’oeuf d’oiseau. Ann. d. Sc. Nat., Serie. 18:1.

. 1889. Atlas d’embryologic. G.

Masson, editeur. Librairie de I’academie de medicine, Paris.

Flynn, T. T. and Hill, J. P. 1939. The development of the Monotrematci. IV. Growth of the ovarian ovum, maturation, fertilization and early cleavage. Trans. Zool. Soc., London, s.A. 24: Part 6, 445.

and . 1942. The later stages

of cleavage and the formation of the primary germ layers in the Monotremata (preliminary communication ) . Proc. Zool. Soc., London, s.A. 111:233.

Haeckel, E. 1866. Generelle Morphologic. Reimer, Berlin.

. 1872. Die Kalkschwamme. Eine

Monographie. Reimer, Berlin.

. 1874. Vols. 1 and 2 in the English translation, 1910. The Evolution of Man, translated by J. McCabe. G. P. Putnam’s Sons, New York.

Hartman, C. G. 1916. Studies in the development of the opossum, Didelphys virginiana. 1. History of early cleavage. II. Formation of the blastocyst. J. Morphol. 27:1.

. 1919. III. Description of new material on maturation, cleavage and entoderm formation. IV. The bilaminar blastocyst. J. Morphol. 32:1.

Horstadiiis, S. 1928. Tjber die determination des Keimes bei Echinodermen. Acta Zool. Stockholm. 9:1.

. 1937. Investigations as to the localization of the micromere-, the skeleton-, and the entoderm-forming material in the unfertilized egg of Arbacia punctiilata. Biol. Bull. 73:295.

Huxley, T. H. 1849. On the anatomy and affinities of the family of the Medusae, Philos. Tr. Roy. Soc., London, s.B. 139:413.

. 1888. Anatomy of Invertebrated

Animals. D. Appleton & Co., New York.

Jacobson, W. 1938* The early development of the avian embryo. 1. Entoderm formation. J. Morphol. 62:415.

Kionka, H. 1894. Die Furchung des Huhnereies. Anat. Hefte. 3:428.

Kleinenberg, N. 1872. Hydra. Eine Monographic. Engelmann, Leipzig.

Kowalewski, A. 1867. Entwicklungsgeschichte des Amphioxus lanceolatus. Mem. Acad, imp d. sc. de St. Petersburg, VIE Serie. 11: No. 4.

Lankester, R. 1877. Notes on the embryology and classification of the animal kingdom. Quart. J. M. Sc. 17:399.

McCrady, E., Jr. 1938. The embryology of the opossum. Am. Anat. Memoirs, 16, The Wistar Institute of Anatomy and Biology, Philadelphia.

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Morgan, T. H. 1934. Embryology and Genetics. Columbia University Press, New York.

Gllacher, J. 1869. Untersuchungen iiber die Furchung und Bliitterbildung im Huhnerei. Inst. f. Exper. Path., Wien. 1:54.

Oppenheimer, J. M. 1936. Processes of localization in developing Fundulus. J. Exper. Zool. 73:405.

. 1940. The non-specificity of the

germ layers. Quart. Rev. Biol. 15:1.

Pander. H. C. 1817. Beitrage zur Entwickelungsgeschichte des Huhnchcns im Eye. Wurzburg.

Pasteels, J. 1936a. Etude sur la gastrulation des vertebres meroblastiques. 1. Teleosteens. Arch, biol., Paris. 47:205.

. 1937a. Etudes sur la gastrulation

des vertebres meroblastiques. II. Reptiles. Arch, biol., Paris. 48:105.

. 1937b. III. Oiseaux. Arch, biol.,

Paris. 48:381.

. 1945. On the formation of the

primary entoderm of the duck (Anas domestica) and on the significance of the bilaminar embryo in birds. Anat. Rcc. 93:5.

Patterson, J. T. 1909. Gastrulation in the pigeon’s egg — a morphological and experimental study. J. Morphol. 20:65.

Peter, K. 1934. Die erste Entwicklung des Chamiileons (Chamaeleon vulgaris) vergleichen mit der Eidechse (Ei, Keimbildung, Furchung, Entodermobildung). Zeit. f. anat. u. Entwicklngesch. Abteil. 2, 102-103:11.

. 1938. Untersuchungen fiber die

Entwicklung des Dotter entoderms. 1. Die Entwicklung des Entoderms beim Hfihnchen. 2. Die Entwicklung des Entoderms bei der Taube. Zeit. mikr.-anat. Forsch. 43:362 and 416.

Spratt, N. T., Jr. 1942, Location of organspecific regions and their relationship to the development of the primitive .streak in the early chick blastoderm. J. Exper. Zool. 89:69.

. 1946. Formation of the primitive

streak in the explanted chick blastoderm marked with carbon particles. J. Exper. Zool. 103:259.

Vandebroek, G. 1936. Les mouvements morphogenetiques au cours de la gastrulation chez Scyllium canicula Cuv. Arch, biol., Paris. 47:499.

Vogt, W. 1925. Gestaltungsanalyse am Amphibienkeim mit ortlicher Vitalfarbung. Vorwort fiber Wege und Ziele. I. Methodik und Wirkungsweise der ortlichen Vitalfarbung mit Agar als Farbtriiger. Arch. f. Entwicklngsmech. d. Organ. 106:542.

. 1929. Gestaltungsanalyse, etc. II.

Teil. Gastrulation und Mesodermbildung bei Urodelen und Anuren. Arch. f. Entwicklngsmech. d. Organ. 120:384.

Wheeler, W. M. 1898. Caspar Friedrich Wolff and the Theoria Generationis. Biological Lectures, Marine Biol. Lab., Woods Hole, Mass. Ginn & Co., Boston.

Whitman, C. O. 1878. The embryology of Clepsine. Quart. J. M. Sc. 18:215.

Will, L. 1892. Beitrage zur Entwicklungsgeschichte der Reptilien. I. Die Anlage der Keimblatter beim Gecko (Platydactylus facetanus Schreib). Zool. Jahrb. 6 : 1 .

Wilson, E. B. 1892. The cell lineage of Nereis. J. Morphol. 6:361.

. 1898. Cell-Lineage and ancestral

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. 1925. The Cell in Development

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Wolff, C. F. 1759. Theoria Generationis. Halle.

. 1812. De formatione intestinorum

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Zur Strassen, O. 1896. Embryonalentwickelung der Ascaris me galocephala. Arch. f. Entwicklngsmech. 3:27, 133.

8

Tlie Late Blastiila in Relation to Certain Innate Pliysiolo^ical Conditions: Twinning

A. Introduction

B. Problem of differentiation

1. Definition of differentiation; kinds of differentiation

2. Self-differentiation and dependent differentiation

C. Concept of potency in relation to differentiation . 1. Definition of potency

2. Some terms used to describe different states of potency

a. Totipotency and harmonious totipotency

b. Determination and potency limitation

c. Prospective potency and prospective fate

d. Autonomous potency c. Competence

D. The blastula in relation to twinning

1. Some definitions

a. Dizygotic or fraternal twins

b. Monozygotic or identical twins

c. Polyembryony •

2. Basis of true or identical twinning

3. Some experimentally produced, twinning conditions

E. Importance of the organization center of the late blastula

A. Introduction

In the preceding two chapters the blastula is defined as a morphological entity composed of six, presumptive, organ-forming areas — areas which are poised and ready for the next phase of development or gastrulation. However, the attainment of this morphological condition with its presumptive, organ-forming areas is valid and fruitful in a developmental way only if it has developed within certain physiological conditions which serve as a spark to initiate gastrulation and carry it through to its completion.

The physiological conditions of the blastula are attained, as are its morphological characteristics, through a process of differentiation. Moreover, during the development of the blastula, different areas acquire different abilities to undergo physiological change and, hence, possess different abilities or

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powers of differentiation. To state the matter differently, the various, presumptive, organ-forming areas of the blastula have acquired different abilities not only in their power to produce specific organs of the future body of the embryo, but also in that some presumptive areas possess this propensity in a greater degree than do other areas. However, at this point, certain terms in common usage relating to the problem of differentiation are defined in order that a better understanding may be obtained concerning the ability to differentiate on the part of the presumptive, organ-forming areas of the late blastula.

B. Problem of Differentiation

1. Definition of Differentiation; Kinds of Differentiation

The word differentiation is applied to that phase of development when a cell, a group of cells, cell product experiences a change which results in a persistent alteration of its activities. Under ordinary conditions an alteration in structure or function is the only visible evidence that such a change has occurred.

To illustrate these matters, let us recall the conditions involved in the maturation of the egg. A subtle change occurs within the primitive oogonium which causes it to enlarge and to grow. This growth results in an increase in size and change in structure of both the cytoplasm and the nucleus. A little later, as the egg approaches that condition which is called maturity, observable morphological changes of the nucleus occur which accompany or initiate an invisible change in behavior. These latter changes make the egg fertilizable. Here we have illustrated, first of all, a subtle, invisible, biochemical change in the oogonium which arouses the formation of visible morphological changes in the oocyte and, secondly, a morphological change (i.e., nuclear maturation) which accompanies an invisible physiological transformation.

Another illustration will prove profitable. Let us recall the development of the mammary-gland tissue (fig. 58). Through the action of the lactogenic (luteotrophic) hormone, LTH, the cells of the various acini of the fully developed gland begin to secrete milk. The acini, it will be recalled, were caused to differentiate as a result of the presence of progesterone. Similarly, the various parts of the complicated duct system were stimulated to differentiate from a very rudimentary condition by the presence of estrogenic hormone. Earlier in development, however, the particular area of the body from which the duct rudiments ultimately arose was conditioned by a change which dictated the origin of the duct rudiments from the cells of this area and restricted their origin from other areas.

In the foregoing history of the mammary gland, various types of differentiation are exemplified. The final elaboration of milk from the acinous cells is effected by a change in the activity of the cells under the influence of LTH. The type of change which brings about the functional activities of a structure is called physiological differentiation. The morphological changes in the cells

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which result in the formation of the duct system and the acini are examples of morphological differentiation. On the other hand, the invisible, subtle change or changes which originally altered the respective cells of the nipple area and, thereby, ordained or determined that the cells in this particular locale should produce duct and nipple tissue is an example of biochemical differentiation or chemodifferentiation. Chemodifterentiation, morphological differentiation, and physiological differentiation, therefore, represent the three types or levels of differentiation. Moreover, all of these differentiations stem from a persistent change in the fundamental activities of cells or cell parts.

It should be observed further that chemodifferentiation represents the initial step in the entire differentiation process, for it is this change which determines or restricts the future possible activities and changes which the cell or cells in a particular area may experience. Also, in many cases, differentiation appears to arise as a result of stimuli which are applied to the cell or cells externally. That is, internal changes within a cell may be called forth by an environmental change applied to the cell from without.

In embryological thinking, therefore, the word differentiation implies a process of becoming something new and different from an antecedent, lessdifferentiated condition. But beyond this, differentiation also connotes a certain suitableness or purposefulness of the structure which is differentiated. Such a connotation, however, applies only to normal embryonic differentiation; abnormal growths and monstrosities of many kinds may fulfill the first phase (i.e., of producing something new) of differentiation as defined in the first sentence of this paragraph, but they do not satisfy the criteria of purpose and of suitableness within the organized economy of the developing body as a whole. It is important to keep the latter implications in mind, for various structures may appear to be vestigial or aberrant during embryonic development, nevertheless their presence may assume an important, purposeful status in the ultimate scheme which constructs the organization of the developing body.

2. Self-differentiation and Dependent Differentiation

In the amphibian, very late blastula and beginning gastrula, the presumptive, chordamesodermal area, when undisturbed and in its normal position in the embryo, eventually differentiates into notochordal and mesodermal tissues. This is true also when it is transplanted to other positions. That is, at this period in the history of the chordamesodermal cells the ability resides within the cells to differentiate into notochordal and mesodermal structures. Consequently, these cells are not dependent upon surrounding or external factors to induce or call forth differentiation in these specific directions. Embryonic cells in this condition are described as self-differentiating (Roux). Similarly, the entodermal area with its potential subareas of liver, foregut, and intestine develops by itself and this area does not rely upon stimuli from other con

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THE BLASTULA IN RELATION TO INNATE CONDITIONS

tiguous cells to realize a specific potency. On the other hand, the presumptive, neural plate region at this time is dependent upon the inducing influence of the chordamesodermal cells during the process of gastrulation for its future realization as neural tissue. This area has little inherent ability to differentiate neural tissue and is described, therefore, as being in a state of dependent differentiation (Roux). Furthermore, the presumptive skin ectoderm (i.e., epidermis), if left alone, will proceed to epidermize during gastrulation, but foreign influences, such as transplantation, into the future neural plate area may induce neural plate cells to form from the presumptive skin ectoderm (fig. 183). The differentiation of neural cells from any of the ectodermal cells of the late blastula thus is dependent upon special influencing factors applied to the cells from without.

C. Concept of Potency in Relation to Differentiation

1. Definition of Potency

The word potency, as used in the field of embryology, refers to that property of a cell which enables it to undergo differentiation. From this viewpoint, potency may be defined as the power or ability of a cell to give origin to a specific kind of cell or structure or to various kinds of cells and structures.

It is questionable, in a fundamental sense, whether potency actually is gained or lost during development. It may be that the expression of a given kind of potency, resulting in the formation of a specific type of cell, is merely the result of a restriction imposed upon other potentialities by certain modifying factors, while the total or latent potency remains relatively constant. All types of differentiated cells, from this point of view, basically are totipotent; that is, they possess the latent power to give origin to all the kinds of cells and tissues of the particular animal species to which they belong.

The specific potencies which denote the normal development of particular organs undoubtedly have their respective, although often quite devious, connections with the fertilized egg. However, one must concede the origin of abnormal or acquired potency values due to the insinuation of special inductive or modifying factors which disturb the expression of normal potency value. For example, tumors and other abnormal growths and tissue distortions may be examples of such special potencies induced by special conditions which upset the mechanism controlling normal potency expression.

2. Some Terms Used to Describe Different States of Potency a. Totipotency and Harmonious Totipotency

The word totipotent, as applied to embryonic development, was introduced into embryological theory by Wilhelm Roux, and it refers to the power or ability of an early blastomere or blastomeres of a particular animal species to give origin to the many different types of cells and structures characteristic of the individual species. Speculation concerning the meaning of totipotency

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377

of a single blastomere received encouragement from the discovery by Hans Driesch, in 1891, that an isolated blastomere of the tWo- or four-cell stage of the cleaving, sea-urchin’s egg could give origin to a “perfect larva.” Driesch described this condition as constituting an equipotential state, while Roux referred to it as a totipotential condition. As the word totipotential seems more fitting and better suited to describe the condition than the word equipotential, which simply means equal potency, the word lotipotency is used herein. The word omnipotent is sometimes used to describe the totipotent condition; as it has connotations of supreme power, it will not be used.

The totipotent state is a concept which may be considered in different ways. In many instances it has been used as described above, namely, as a potency condition that has within it the ability to produce a perfect embryo or individual. The word also has been used, however, to describe a condition which is capable of giving origin to all or nearly all the cells and tissues of the body in a haphazard way but which are not necessarily organized to produce a normally formed body of the particular species. Therefore, as a basis for clear thinking, it is well to define two kinds of totipotency, namely, totipotency and harmonious totipotency. The former term is used to describe the ability of a cell or cell group to give origin to all or nearly all the different cells and tissues of the particular species to which it belongs, but it is lacking in the ability to organize them into an harmonious organism. Harmonious totipotency, on the other hand, is used to denote a condition which has the above ability to produce the various types of tissues of the species, but possesses, in addition, the power to develop a perfectly organized body.

The fertilized egg or the naturally parthenogenetic egg constitutes an harmonious totipotential system. This condition is true also of isolated blastomeres of the two- or iour-blastomere stage of the sea-urchin development, as mentioned above, of the two-cell state of Amphioxus, or of the first twoblastomere stage of the frog’s egg when the first cleavage plane bisects the gray crescent. However, in the eight-cell stage in these forms, potency becomes more limited in the respective cells of the embryo. Restriction of potency, therefore, is indicated by a restriction of power to develop into a variety of cells and tissues, and potency restriction is a characteristic of cleavage and the blastulative process (figs. 61; 163 A; 163B). When a stage is reached in which the cells of a particular area are limited in potency value to the expression of one type of cell or tissue, the condition is spoken of as one of unipotency. A pluripotent state, on the other hand, is a condition in which the potency is not so limited, and two or more types of tissues may be derived from the cell or cells.

b. Determination and Potency Limitation

The limitation or restriction of potency, therefore, may form a part of the process of differentiation; as such, it is a characteristic feature of embryonic

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development. Potency limitation, however, is not always the result of the differentiation process. For instance, in the development of the oocyte in the ovary, the building up of the various conditions, characteristic of the totipotent state, is a feature of the differentiation of the oocyte.

The word determination is applied to those unknown and invisible changes occurring within a cell or cells which effect a limitation or restriction of potency. As a result of this potency limitation, differentiation becomes restricted to a specific channel of development, denoting a particular kind of cell or structure. Ultimately, by the activities of limiting influences upon the resulting blastomeres during cleavage, the totipotent condition of the mature egg becomes dismembered and segregated into a patchwork or mosaic of general areas of the blastula, each area having a generalized, presumptive, organ-forming potency. As we have already observed, in the mature chordate blastula there are six of these major, presumptive organ-forming areas (five if we regard the two mesodermal areas as one). By the application of other limiting influences during gastrulation or the next phase of development, each of these general areas becomes divided into minor areas which are limited to a potency value of a particular organ or part of an organ. The process which brings about the determination of individual organs or parts of organs is called individuation.

When potency limitation has reduced generalized and greater potency value to the status of a general organ system (e.g., nervous system or digestive system) with the determination (i.e., individuation) of particular organs within such a system, the condition is described as one of rigid or irrevocable determination. Such tissues, transplanted to other parts of the embryo favorable for their development, tend to remain limited to an expression of one inherent potency value and do not give origin to different kinds of tissues or organs. Thus, determined liver rudiment will differentiate into liver tissue, stomach rudiment into stomach tissue, forebrain material into forebrain tissue, etc.

In many instances determination within a group of cells is brought about because of their position in the developing organism and not because of intrinsic, self-differentiating conditions within the cells. Because their position foreordains their determination in the future, the condition is spoken of as positional or presumptive determination. For example, in the late amphibian blastula, the composite ectodermal area of the epiblast will become divided, during the next phase of development, into epidermal and neural areas as a result of the influences at work during gastrulation, especially the activities of the chordamesodermal area. Therefore, one may regard these areas as already determined, in a presumptive sense, even in the late blastula, although their actual determination as definite epidermal and neural tissue will not occur until later.

As stated in the preceding paragraphs, determination is the result of potency

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limitation or inhibition. However, there is another aspect to determination, namely, potency expression, which simply means potency release or development. Potency expression, probably, is due to an activating stimulus (Spemann, ’38). Consequently, the individuation of a particular organ structure within a larger system of organs is the result of two synchronous processes:

( 1 ) inhibition of potency or potencies and

(2) release or calling forth of a specific kind of potency (Wiggles worth, ’48).

Associated with the phenomenon of potency inhibition or limitation is the loss of power for regulation. Consequently, individuation and the loss of regulative power appear to proceed synchronously in any group of cells.

c. Prospective Potency and Prospective Fate

Prospective fate is the end or destiny that a group of cells normally reaches in its differentiation during its normal course of development in the embryo. The presumptive epidermal area of the late blastula differentiates normally into skin epidermis. This is its prospective fate. Its prospective potency, however, is greater, for under certain circumstances it may be induced, by transplantation to other areas of the late blastula, to form other tissue, e.g., neural plate cells or mesodermal tissues.

d. Autonomous Potency

Autonomous potency is the inherent ability which a group of cells possesses to differentiate into a definite structure or structures, e.g., notochord, stomach, or liver rudiments of the late blastula of the frog.

Versatility of autonomous potency is the inherent ability which a group of cells possesses to differentiate, when isolated under cultural conditions outside the embryo, into tissues not normally developed from the particular cell group in normal development. In the amphibian late blastula this is true of the notochordal and somitic areas of the chordamesodermal area, which may give origin to skin or neural plate tissue under these artificially imposed conditions.

e. Competence

Certain areas of the late amphibian blastula have the ability to differentiate into diverse structures under the stimulus of varied influence. Consequently, we say that these areas have competence for the production of this or that structure. The word competence is used to denote all of the possible reactions which a group of cells may produce under various sorts of stimulations. The entodermal area of the late amphibian blastula and early gastrula has great power for self-differentiation but no competence, whereas the general, neural plate-epidermal area has competence but little power of self

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differentiation (see p. 375). On the other hand, the notochord, mesodermal area possesses both competence and the ability for self-differentiation.

Competence appears to be a function of a developmental time sequence. That is, the time or period of development is all important, for a particular area may possess competence only at a single, optimum period of development. The word competence is sometimes used to supersede the other terms of potency or potentiality (Needham, ’42, p. 112).

D. The Blastula in Relation to Twinning

1. Some Definitions

a. Dizygotic or Fraternal Twins

Fraternal twins arise from the fertilization of two separate eggs in a species which normally produces one egg in the reproductive cycle, as, for example, in the human species. Essentially, fraternal twins are much the same as the “siblings” of a human family (i.e., the members born as a result of separate pregnancies) or the members of a litter of several young produced during a single pregnancy in animals, such as cats, dogs, pigs, etc. Fraternal twins are often called “false twins.”

b. Monozygotic or Identical Twins

This condition is known as “true twinning,” and it results from the development of two embryos from a single egg. Such twins presumably have an identical genetic composition.

c. Polyembryony

Polyembrony is a condition in which several embryos normally arise from one egg. It occurs regularly in armadillos (Dasypopidae) where one ovum gives origin normally to four identical embryos (fig. 186).

2. Basis of True or Identical Twinning

The work of Driesch (1891) on the cleaving, sea-urchin egg and that of Wilson (1893) on the isolated blastomeres of Amphioxus mentioned above initiated the approach to a scientific understanding of monozygotic or identical twinning. Numerous studies have been made in the intervening years on the developing eggs of various animal species, vertebrate and invertebrate, and from these studies has emerged the present concept concerning the matter of twinning. True twinning appears to arise from four, requisite, fundamental, morphological and physiological conditions. These conditions are as follows:

( 1 ) there must be a sufficient protoplasmic substrate;

(2) the substrate must contain all the organ-forming stuffs necessary to assure totipotency, that is, to produce all the necessary organs;

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381

(3) an organization center or the ability to develop such a center must be present in order that the various organs may be integrated into an harmonious whole; and

(4) the ability or faculty for regulation, that is, the power to rearrange materials as well as to reproduce and compensate for the loss of substance, must be present.

3. Some Experimentally Produced, Twinning Conditions

The isolation of the first two blastomeres in the sea-urchin egg and in Amphioxus with the production of complete embryos from each blastomere

Fig. 183. Early gastrula of darkly pigmented Triton taeniatus with a small piece of presumptive ectoderm of T. cristatus lightly pigmented inserted into the presumptive, neural plate area shown in (A). (B) Later stage of development. (C) Cross section of the later embryo. I hc lighter eye region shown to the right was derived from the original implant from T. cristatus, (After Spemann, ’38.)

Fig. 184. Demonstration that the presence of the organizer region or organization center is necessary for development. (Redrawn from Spemann, ’38.) (A) Hair-loop

constriction isolates the organizer areas in the dorsal portion of the early gastrula. (B) Later development of the dorsal portion isolated in (A). (C) Later development of

ventral portion of gastrula isolated in (A). (D) Constriction of organizer area of early

gastrula into two halves. (E) Result of constriction made in (D). Constrictions were made at 2-cell stage.

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THE BLASTULA IN RELATION TO INNATE CONDITIONS

CENTER OF ORGANIZATION

Fig. 185. Twinning in teleost fishes. (After Morgan, ’34; Embryology and Genetics, Columbia University Press, pp. 102-104. A, B, C from Rauber; D from Stockard.) In certain teleost fishes, especially in the trout, under certain environmental conditions, two or more organization centers arise in the early gastrula. (A-C) These represent such conditions. If they lie opposite each other as in (A), the resulting embryos often appear as in (D). If they lie nearer each other as in (B) or (C), a two-headed monster may be produced.

has been described in Chapter 6, In these cases all the conditions mentioned above are fulfilled. However, in the case of the isolation of the first two blastomeres in Stye la described in Chapter 6, evidently conditions (1), (2), and (3) are present in each blastomere when the two blastomeres are separated, but (4) is absent and only half embryos result. That is, each blastomere has been determined as either a right or left blastomere; with this determination of potency, the power for regulation is lost. In the frog, if the first two blastomeres are separated when the first cleavage plane bisects the gray crescent, all four conditions are present and two tadpoles result. If, however, the first cleavage plane separates the gray-crescent material mainly into one blastomere while the other gets little or none, the blastomere containing the gray-crescent material will be able to satisfy all the requirements above, and it, consequently, develops a normal embryo. However, the other blastomere lacks (2), (3), and (4) and, as a result, forms a mere mass of cells. Again, animal pole blastomeres, even when they contain the gray-crescent material, when separated entirely from the yolk blastomeres, fail to go beyond the late blastular or beginning gastrular state (Vintemberger, ’36). Such animal pole blastomeres appear to lack requirements (I), (2), and possibly (3) above. Many other illustrations of embryological experiments could be given, establishing

Fig. 186. Polyembryony or the development of multiple embryos in the armadillo, Tatusia novemcincta. (After Patterson, ’13.) (A) Separate centers of organization in

the early blastocyst. (B) Later stage in development of multiple embryos. Each embryo is connected with a common amniotic vesicle. (C) Section through organization centers a and b in (A). The two centers of organization are indicated by thickenings at right and left. (D) Later development of four embryos, the normal procedure from one fertilized egg in this species.

383

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THE BLASTULA IN RELATION TO INNATE CONDITIONS

the necessity for the presence of all the above conditions. Successful whole embryos have resulted in the amphibia when the two-cell stage and beginning gastrula is bisected in such a manner that each half contains half of the chordamesodermal field and yolk substance; that is, each will contain half of the organization center (fig. 184).

Monozygotic twinning occurs occasionally under normal conditions in the teleost fishes. In these cases, separate centers of organization arise in the blastoderm, as shown in figure 185. When they arise on opposite sides of the blastoderm, as shown in figure 185 A, twins arise which may later become fused ventrally (fig. 185D). When the centers of organization arise as shown in figure I85B, C, the embryos become fused laterally. Stockard (’21) found that by arresting development in the trout or in the blastoderm of Fundutus for a period of time during the late blastula, either by exposure to low temperatures or a lack of oxygen, twinning conditions were produced. The arrest of development probably allows separate centers of organfzation to arise. Normally, one center of organization makes its appearance in the late blastula of these fishes, becomes dominant, and thus suppresses the tendency toward totipotency in other parts of the blastoderm. However, in the cases of arrested development, a physiological isolation of different areas of the blastoderm evidently occurs, and two organization centers arise which forthwith proceed to organize separate embryos in the single blastoderm. Conditions appear more favorable for twinning in the trout blastoderm than in Fundulus. After the late blastular period is past and gastrulation begins, i.e., after one organization center definitely has been established, Stockard found that twinning could not be produced.

In the Texas armadillo, Tatusia novemcincta, Patterson (’13) found that, in the relatively late blastocyst (blastula), two centers of organization arise, and that, a little later, each of these buds into two separate organization centers, producing four organization centers in the blastula (fig. 186A-C). Each of these centers organizes a separate embryo; hence, under normal conditions, four embryos (polyembryony) are developed from each fertilized egg (fig. 186D).

It is interesting in connection with the experiments mentioned by Stockard above, that the blastocyst (blastula) in Tatusia normally lies free in the uterus for about three weeks before becoming implanted upon the uterus. It may be that this free period of blastocystic existence results in a slowing down of development, permitting the origin of separate organization centers. In harmony with this concept, Patterson (’13) failed to find mitotic conditions in the blastoderms of the blastocysts during this period.

In the chick it is possible to produce twinning conditions by separating the anterior end (Hensen’s node) of the early primitive streak into two parts along the median axis of the developing embryo. Twins fused at the caudal end may be produced under these conditions. In the duck egg, Wolff and Lutz (’47) found that if the early blastoderm is cut through the primitive node

THE BLASTULA IN RELATION TO TWINNING

385

area (fig. 187A), two embryos are produced as in figure 187 A'. However, if the primitive node and primitive streak are split antero-posteriorly, as indicated in figure 1876, two embryos, placed as in figure 187B', are produced.

It is evident, therefore, that in the production of monozygotic twins, condition (3) or the presence of the ability to produce an organization center is of greatest importance. In the case of the separation of the two blastomeres of the two-cell stage in Amphioxus or of the division of the dorsal lip of the early gastrula of the amphibian by a hair loop, as shown in figure 184, a mechanical division and separation of the ability to produce an organization center in each blastomere (Amphioxus) or of the separation into two centers of the organization center already produced (Amphibia) is achieved. Once these centers are isolated, they act independently, producing twin conditions, providing the substrate is competent. Similar conditions evidently are produced in the duck-embryo experiments of Wolff and Lutz referred to above.

In some teleost blastulae, e.g., Fundidus and Salmo, during the earlier period of development, it has been found possible to separate the early blastoderm into various groups of cells (Oppenheimer, ’47) or into quadrants (Luther, ’36), .and a condition of totipotency is established in each part. Totipotency appears thus to be a generalized characteristic in certain teleost blastoderms during the earlier phases of blastular development. Harmonious totipotency, however, appears not to be achieved in any one part of the blastodisc of these species during the early conditions of blastular formation. During the

Fig. 187. Isolation of the organization center in the early duck embryo. (From Dalcq, ’49, after Wolff and Lutz.) (A') Derived from blastoderm cut as in (A). (B') Derived

from blastoderm cut as in (B).

386

THE BLASTULA IN RELATION TO INNATE CONDITIONS

development of the late blastula, however, the posterior quadrant normally acquires a dominant condition together with a faculty for producing harmonious totipotency. The other totipotent areas then become suppressed. These basic conditions, therefore, serve to explain the experiments by Stockard (’21) referred to above, where two organization centers tend to become dominant as a result of isolating physiological conditions which tend to interfere with the processes working toward the development of but one center of organization. This probable explanation of the twinning conditions in the teleost blastoderm suggests strongly that the separation and isolation of separate organization centers is a fundamental condition necessary for the production of monozygotic or true twinning.

It becomes apparent, therefore, that, in the development of the trout blastoderm (blastula), the development oj an area which possesses a dominant organization center is an important aspect of blastulation. In other blastulae, the seat or area of the organization center apparently is established at an earlier period, as, for example, the gray crescent in the amphibian egg which appears to be associated with the organization center during the late blastula state. Similarly, in the teleost fish, Carassius, totipotency appears to be limited to one part of the early blastula (Tung and Tung, ’43).

It also follows from the analysis in the foregoing paragraphs that in the production of polyembryony in the armadillo or of spontaneous twinning in forms, such as the trout (Salmo), a generalized totipotency throughout the early blastoderm is a prerequisite condition. When a single dominant area once assumes totipotency, it tends to suppress and control the surrounding areas, probably because it succeeds in “monopolizing” certain, substrate, “food” substances (Dalcq, ’49).

£. Importance of the Organization Center of the Late Blastula

It is also evident that one of the main functions of cleavage and blastulation is the formation of a physiological, or organization, center which must be present to dominate and direct the course of development during the next stage of development. Consequently, the elaboration of a blastocoel with the various, presumptive, organ-forming areas properly oriented in relation to it is not enough. A definite physiological condition entrenched within the so-called organization center must be present to arouse and direct the movement of the major, organ-forming areas during gastrulation.

Bibliography

Butler, E. 1935. The developmental capacities of regions of the unincubated chick blastoderm as tested in chorioallantoic grafts. J. Exper. Zool. 70:357.

Daicq, A. M. 1949. The concept of physiological competition (Spiegelman) and the interpretation of vertebrate morphogenesis. Experimental Cell Research, Supplement 1 : p. 483. Academic Press, Inc., New York.

Driesch, H. 1891. Entwicklungsmechanische Studien I-II. Zeit. Wiss. Zool. 53:160.

Holtfreter, J. 1938. Differenzierungspotenzen isolierter teile der Anuren-gastrula. Roux’ Arch. f. Entwick. d. Organ. 138.657.

Luther, W. 1936. Potenzpriifungen an isolierten Teilstiicken der Forellenkeimscheibe. Arch. f. Entwicklngsmech. d. Organ. 135:359.

Morgan, T. H. 1934. Chap. IX in Embryology and Genetics. Columbia University Press, New York.

Needham, J. 1942. Biochemistry and Morphogenesis. Cambridge University Press, London.

Oppenheimer, J. M. 1947. ‘Organization of the teleost blastoderm. Quart. Rev. Biol. 22:105.

Patterson, J. T. 1913. Polyembryonic development in Tatusia novemcincta. J. Morphol. 24:559.

Spemann, H. 1938. Embryonic Development and Induction. Yale University Press, New Haven.

Stockard, C. R. 1921. Developmental rate and structural expression: an experimental study of twins, “double monsters” and single deformities, and the interaction among embyronic organs during their origin and development. Am. J. Anat. 28: 1 15.

Tung, T. C. and Tung, Y. F. Y. 1943. Experimental studies on the development of the goldfish. (Cited from Oppenheimer, ’47.) Proc. Clin. Physiol. Soc. 2 : 11 .

Vintemberger, P. 1936. Sur le developpement compare dcs micromeres dc I’oeuf de Rana fusca divise enhuit (a) Apres isolement (b) Apres transplantation sur un socle de cellules vitellines. Compt. rend. Soc. de Biol. 122:927.

Wigglesworth, V. B. 1948. The role of the cell in determination. Symposia of the Soc. for Exper. Biol. No. II. Academic Press, Inc., New York.

Wilson. E. B. 1893. Amphioxiis and the mosaic theory of development. J. Morphol. 8:579.

Wolff, E. and Lutz, H. 1947. Embryologie experimentale — sur la production experimcntale dc jumeaux chez I’embryon d’oiseau. Compt. rend. Acad. d. Sc. 224:1301.

387

9

Gastrulation

A. Some definitions and concepts

1. Gastrulation

2. Primitive vertebrate body plan in relation to the process of gastrulation

a. Fundamental body plan of the vertebrate animal

b. The gastrula in relation to the primitive body plan

c. Chart of blastula, gastrula, and primitive, body-form relationships (fig. 188)

B. General processes involved in gastrulation

C. Morphogenetic movement of cells

1. Importance of cell movements during development and in gastrulation

2. Types of cell movement during gastrulation

a. Epiboly

b. Emboly

3. Description of the processes concerned with epiboly

4. Description of the processes involved in emboly

a. Involution and convergence

b. Invagination

c. Concrescence

d. Cell proliferation

e. Polyinvagination

f. Ingression

g. Delamination

h. Divergence

i. Extension

D. The organization center and its relation to the gastrulative process

1. The organization center and the primary organizer

2. Divisions of the primary organizer

E. Chemodifferentiation and the gastrulative process

F. Gastrulation in various Chordata 1. Amphioxus

a. Orientation

b. Gastrulative movements

1 ) Emboly

2) Epiboly

3) Antero-posterior extension of the gastrula and dorsal convergence of the mesodermal cells

4) Closure of the blastopore

c. Resume of cell movements and processes involved in gastrulation of Amphioxus

1 ) Emboly

2) Epiboly

388

GASTRULATION

389

2. Gastrulation in Amphibia with particular reference to the frog

a. Introduction

1) Orientation

2) Physiological changes which occur in the presumptive, organ-forming areas of the late blastula and early gastrula as gastrulation progresses

b. Gastrulation

1) Emboly

2) Epiboly

3) Embryo produced by the gastrulative processes

4) Position occupied by the pre -chordal plate material

c. Closure of the blastopore and formation of the neurenteric canal

d. Summary of morphogenetic movements of cells during gastrulation in the frog and other Amphibia

1) Emboly

2) Epiboly

3. Gastrulation in reptiles

a. Orientation

b. Gastrulation

4. Gastrulation in the chick

a. Orientation

b. Gastrulative changes

1) Development of primitive streak as viewed from the surface of stained blastoderms

2) Cell movements in the epiblast involved in primitive-streak formation as indicated by carbon-particle marking and vital-staining experiments

3) Cell movements in the hypoblast and the importance of these movements in primitive-streak formation

4) Primitive pit notochordal canal

5) Resume of morphogenetic movements of cells during gastrulation in the chick

5. Gastrulation in mammals

a. Orientation

b. Gastrulation in the pig embryo

c. Gastrulation in other mammals

6. Gastrulation in teleost and elasmobranch fishes

a. Orientation

b. Gastrulation in teleost fishes

1) Emboly

2) Epiboly

3) Summary of the gastrulative processes in teleost fishes

a) Emboly

b) Epiboly

4) Developmental potencies of the germ ring of teleost fishes

c. Gastrulation in elasmobranch fishes

7. Intermediate types of gastrulative behavior

G. The late gastrula as a mosaic of specific, organ-forming territories

H. Autonomous theory of gastrulative movements

I. Exogastrulation

J. Pre-chordal plate and cephalic projection in various chordates

K. Blastoporal and primitive-streak comparisons

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GASTRULATION

A. Some Definitions and Concepts

1 . Gastrulation

(According to Haeckel, the word gastrula is the name given to “the important embryonic form” having “the two primary germ-layers,” and the word gastrulation is applied to the process which produces the gastrula. Furthermore, “this ontogenetic process has a very great significance, and is the real starting-point of the construction of the multicellular animal body” (1874, see translation, ’10, p. 1 23 ji).^thers such as Lankester (1875) and Hubrecht (’06) did much to establish the idea that gastrulation is a process during which the. monolayered blastula is converted into a bilaminar or didermic gastrulajjHaeckel emphasized invagination or the infolding of one portion of the blastula as the primitive and essential process in this conversion, while Lankester proposed delamination or the mass separation of cells as the primitive process^ While it was granted that invagination was the main process of gastrulation in Amphioxus, in the Vertebrata, especially in reptiles, birds, and mammals, delajpination was considered to be an essential process by many embryologists. Some, however, maintained that the process of invagination held true for all the Chordata other than the Mammalia, It may be mentioned in passing that Lankester conferred the name “blastopore” upon the opening into the interior of the blastoderm which results during gastrulation. The words “blastopore” and “primitive mouth” soon were regarded as synonymous, for in the Coelenterata, the blastopore eventually becomes the oral opening.

The definition of the gastrula as a didermic stage, following the monolayered blastula, is a simple concept, easy to visualize, and, hence, may have some pedagogical value. However, it is not in accord with the facts unearthed by many careful studies relative to cell lineage and it does not agree with the results obtained by the Vogt method (see Chap. 7) applied to the process of gastrulation in the vertebrate group.

One of the first to define gastrulation in a way which is more consonant with the studies mentioned in the previous paragraph was Keibel (’01). He defined gastrulation in the vertebrates (’01, p. 1111) as “the process by which the entodermal, mesodermal and notochordal cells find their way into the interior of the embryo.” It is to be observed that this definition embodies the concept of migration of specific, organ-forming areas. We may restate the concept involved in this definitioq^ in a way which includes invertebrates as well as vertebrates as Gastrulation is the dynamic process during

which the major, presumptive \rgan-forming areas of the blastula (Chaps. 6 and 7) become rearranged and reorganized in a way which permits their ready conversion into the body plan of the particular species. That is to say, during the process of gastrulation, the presumptive organ-forming areas of the blastula undergo axiation in terms of the body organization of the species. In some animal species, this reorganization of the blastula into the structural

DEFINITIONS AND CONCEPTS

391

pattern of the gastrula results in the production of a two-layered form, for example, as in Amphioxus; in others (actually in most metazoan species) it brings about the formation of a three-layered condition^ It is apparent, therefore, as observed by Pasteels (’37b, p. 464), that “it is impossible to give a general definition of the gastrula stage.” It is obvious, also, that one cannot define gastrulation in terms of simple invagination, delamination, or the production of a two-layered condition. Many processes, involving intricate movements of cell groups, occur as outlined in the succeeding pages of this chapter.

Relative to the process of gastrulation and later development, emphasis should be placed upon the importance of the blastocoel. The latter takes its origin largely by the movement of groups of cells in relation to one another during cleavage and blastulation. Therefore, we may enumerate the following events related to the blastocoel during the early phases of embryonic development:

(1) The blastocoel is associated with those movements in the developing blastula which produce the specific cellular configuration of the mature blastula;

(2) during gastrulation, it enables the various, presumptive organ-forming areas of the blastula to be rearranged and to migrate into the particular areas which permit their ready organization and axiation into the scheme of the body form of the particular species; and

(3) in the period of development immediately following gastrulation, it affords the initial space necessary for the tubulation of the major, organ-forming areas.

The events mentioned in (3) will be described in Chapter 10.

2. Primitive Veiitebrate Body Plan in Relation to the Process of Gastrulation

In the animal kingdom, each of the major animal groupings has a specific body plan. In the phylum, Chordata, the cephalochordate, Amphioxus, and the vertebrates possess such a plan. It is necessary at this point to review briefly the rudiments of this primitive or basic body plan.

a. Fundamental Body Plan of the Vertebrate Animal

The vertebrate body essentially is a cylindrical structure with a head or cephalic end, a middle trunk region, and a tail or caudal end. The dorsum or dorsal region is the uppermost aspect, while the venter or belly lies below. Also, the body as a whole may be slightly compressed laterally. Viewed in transverse section, the body is composed basically of five hollow tubes, particularly in the trunk area. The epidermal tube forms the exterior and within the latter are placed the neural, enteric, and two mesodermal tubes, all oriented around the median skeletal axis or notochord as indicated in figures 188C and 217G and N.

392

GASTRUtATION

b. The Gastrula in Relation to the Primitive Body Plan

If one watches a large transport plane preparing to take off at an airfield, the following events may be observed:

( 1 ) The cargo and passengers are boarded, the engines are warmed, and the plane is taxied toward the runway.

(2) Upon reaching the starting end of the runway, the engines are accelerated, and the plane is turned around and headed in the direction of the take-off.

Fig. 188. Relationship between the presumptive organ-forming areas of the blastula (diagram A) and the primitive tubular condition of the developing vertebrate body (diagram C). The gastrula (diagram B) represents an intermediate stage. Consult chart in text.

GENERAL PROCESSES

393

(3) The engines are further accelerated and the plane is moved down the runway for the take-off into the airy regions.

Similarly, during cleavage and blastulation, the embryonic machine develops a readiness, elaborates the major, organ-forming areas in their correct positions in the blastula, and taxies into position with its engines warming up, as it were. Once in the position of the mature blastula, the various, major, presumptive organ-forming areas are turned around and reoriented by the gastrulafive processes, and thus, each major, organ-forming area of the gastrula is placed in readiness for the final developmental surge which results in primitive body formation. During the latter process the major, presumptive organforming areas in the vertebrate group are molded into the form of elongated tubular structures with the exception of the notochordal area which forms an elongated skeletal axis. (The latter phenomena are described in Chapter 10.)

c. Chart of Blastula, Gastrula, and Primitive Body-form Relationships in the Vertebrate Group (Fig. 188)

The major, presumptive organ-forming areas are designated by separate numerals.

Blastula

Gastrula

Primitive Body Form

1. Epidermal crescent

2. Neural crescent

3. Entodermal area

4. Two «nesodermal areas

5. Notochordal crescent

1. Part of ectodermal layer

2. Elongated neural plate a part of ectoderm layer

3. Primitive archenteron in rounded gastrulae, such as frog; archenteric layer in flattened gastrulae, such as chick

4. Two mesodermal layers on either side of notochord

5. Elongated band of cells lying between mesodermal layers

1. External epidermal tube

2. Dorsally placed neural tube

3. Primitive gut tube

4. Two primitive mesodermal tubes; one along either side of neural tube, notochord, and gut tube; especially true of trunk region

5. Rounded rod of cells lying below neural tube and above entodermal or gut tube; these three structures lie in the meson or median plane of the body

B. General Processes Involved in Gastrulation

Gastrulation is a nicely integrated, dynamic process; one which is controlled largely by intrinsic (i.e., autonomous) forces bound up in the specific, physicochemical conditions of the various, presumptive, organ-forming areas of the late blastula and early gastrula. These internal forces in turn are correlated

394

GASTRULATION

with external conditions. One of the important intrinsic factors involves the so-called organization center referred to in Chapter 7. However, before consideration is given to this center, we shall define some of the major processes involved in gastrulation.

There are two words which have come into use in embryology relative to the process of gastrulation, namely, epiboly and emboly. These words are derived from the Greek, and in the original they denote motion, in fact, two different kinds of motion. The word emboly is derived from a word meaning to throw in or thrust in. In other words, it means insertion. The word epiboly, on the other hand, denotes a throwing on or extending upon. These words, therefore, have quite opposite meanings, but they aptly describe the general movements which occur during gastrulation. If, for example, we consider figure 169, these two words mean the following: All the presumptive organforming areas below line a-b in (C) during the process of gastrulation are moved to the inside by the forces involved in emboly. On the other hand, due to the forces concerned with epiboly, the presumptive organ-forming materials above line a-b are extended upon or around the inwardly moving cells.

Associated with the comprehensive molding processes of epiboly and emboly are a series of subactivities. These activities may be classified under the following headings:

( 1 ) morphogenetic movement of cells,

(2) the organization center and its organizing influences, and

(3) chemodifferentiation.

C. Morphogenetic Movement of Cells

1.^ Importance of Cell Movements During Development AND IN Gastrulation

The movement of cells from one place in the embryo to another to establish a particular form or structure is a common embryological procedure. This type of cell movement is described as a morphogenetic movement because it results in the generation of a particular form or structural arrangement. It is involved not only in the formation of the blastula where the movements are slow, or in gastrulation where the cell migrations are dynamic and rapid, but also in later development. (See Chap. 11.) In consequence, we may say that cell migration is one of the basic procedures involved in tissue and organ formation.

The actual factors — physical, chemical, physiological, and mechanical — which effect cell movements are quite unknown. However, this lack of knowledge is not discouraging. In fact, it makes the problem more interesting, for cells are living entities utilizing physicochemical and mechanical forces peculiar

MOVEMENT OF CELLS

395

to that condition which we call living. The living state is a problem which awaits solution.

At the period when the process of blastulation comes to an end and the process of gastrulation is initiated, there is an urge directed toward cell movement throughout the entire early gastrula. Needham (’42, p. 145) uses the term “inner compulsion” to describe the tendency of the cells of the dorsal-Up area to move inward (invaginate) at this time. Whatever it is called and however it may be described, the important feature to remember is that this tendency to move and the actual movement of the cells represent a living process in which masses of cells move in accordance with the dictates of a precise and guiding center of activity, known as the primary organizer or organization center.

2. Types of Cell Movement During Gastrulation

The following types of cell movement are important aspects of the process of gastrulation.

a. Epiboly

( 1 ) Extension along the antero-posterior axis of the future embryo.

(2) Peripheral expansion or divergence.

b. Emboly

(1) Involution.

(2) Invagination.

(3) Concrescence (probably does not occur).

(4) Convergence.

(5) Polyinvagination.

(6) Delamination.

(7) Divergence or expansion.

(8) Extension or elongation.

(9) Blastoporal constriction.

Note: While cell proliferation is not listed as a specific activity above, it is an important aspect of gastrulation in many forms.

3. Description of the Processes Concerned with Epiboly

Epiboly or ectodermal expansion involves the movements of the presumptive epidermal and neural areas during the gastrulative process. The general migration of these two areas is in the direction of the antero-posterior axis of the future embryonic body in ail chordate embryos. In the rounded blastula (e.g., frog, Amphioxus, etc.), the tendency to extend antero-posteriorly produces an enveloping movement in the antero-posterior direction. As a result, the presumptive epidermal and neural areas actually engulf and surround the inwardly moving presumptive notochordal, mesodermal, and ento

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GASTRULATION

dermal areas. (Study fig. 190A-H.) In flattened blastulae the movements of epiboly are concerned largely with antero-posterior extension, associated with peripheral migration and expansion of the epidermal area. (See fig. 202.) The latter movement of the presumptive epidermal area is pronounced in teleost fishes, where the yolk is engulfed as a result of epidermal growth and expansion (figs. 210B; 21 ID).

The above-mentioned activities, together with cell proliferation, effect spatial changes in the presumptive epidermal and neural areas as shown in figures 189, 190, 191, 198, and the left portion of figure 202A-I. It is to be observed that the epidermal crescent is greatly expanded, and the area covered is increased; also, that the neural crescent is changed into a shield-shaped area, extended in an antero-posterior direction (figs. 192A; 2021).

4. Description of the Processes Involved in Emboly

While forces engaged in epiboly are rearranging the presumptive neural and epidermal areas, the morphogenetic movements concerned with emboly move the presumptive chordamesodermal and entodermal areas inward and extend them along the antero-posterior axis of the forming embryo. This inward movement of cells is due to innate forces within various cell groups; some apparently are autonomous (i.e., they arise from forces within a particular cell group), while others are dependent upon the movement of other cell groups. ) (See p. 447.) We may classify the types of cell behavior during this migration and rearrangement of the chordamcsoderm-entodermal areas as follows:

a. Involution and Convergence

Involution is a process which is dependent largely upon the migration of cells toward the blastoporal lip (e.g., frog, see heavy arrows, fig. 192) or to the primitive streak (e.g., bird, see arrows, fig. 204C-E). The word involution, as used in gastrulation, denotes a “turning in” or inward rotation of cells which have migrated to the blastoporal margin. In doing so, cells located along the external margin of the blastoporal lip move over the lip to the inside edge of the lip (see arrows, figs. 191C-E, H; 192B, C). The inturned or involuted cells thus are deposited on the inside of the embryo along the inner margin of the blastopore. The actual migration of cells from the outside surface of the blastula to the external margin of the blastoporal lip is called convergence. In the case of the primitive streak of the chick, the same essential movements are present, namely, a convergence of cells to the primitive streak and then an inward rotation of cells through the substance of the streak to the inside (arrows, fig. 204; black arrows, fig. 202). If it were not for the process of involution, the converging cells would tend to pile up along the outer edges of the blastoporal lip or along the primitive streak. Involution

MOVEMENT OF CELLS

397

thus represents a small but extremely important step in the migration of cells from the exterior to the interior during gastrulation.

b. Invagination

The phenomenon of invagination, as used in embryological development, implies an infolding or insinking of a layer of cells, resulting in the formation of a cavity surrounded by the infolded cells (figs. 189, 190, the entoderm). Relative to gastrulation, this process has two aspects:

(1 ) mechanical or passive infolding of cells, and

( 2 ) active inward streaming or inpushing of cells into the blastocoelic space.

In lower vertebrates, the dorsal-lip area of the blastopore is prone to exhibit the active form of invagination, whereas the entoderm of the lateral- and ventral-lip regions of the blastopore tends to move in a passive manner. The notochordal-canal, primitive-pit area of the primitive streak of higher vertebrates is concerned especially with the active phase of invagination.

c. Concrescence

This term is used in older descriptions of gastrulation. The word denotes the movement of masses of cells toward each other, particularly in the region of the blastopore, and implies the idea of fusion of cell groups from two bilaterally situated areas. It probably does not occur, (However, see development of the feather in Chap. 12.)

d. Cell Proliferation

An increase in the number of cells is intimately concerned with the process of gastrulation to the extent that gastrulation would be impeded without it, in some species more than in others. Cell proliferation in Amphioxus, for example, is intimately associated with the gastrulative process, whereas in the frog it assumes a lesser importance.

e. Polyinvagination

Polyinvagination is a concept which implies that individual or small groups of cells in different parts of the external layer of the blastula or blastodisc invaginate or ingress into the segmentation (blastocoelic) cavity. That is, there are several different and separate inward migrations of one or more cells. This idea recently was repudiated by Pasteels (’45) relative to the formation of the entodermal layer in the avian blastoderm. It applies, presumably, to the ingression of cells during the formation of the two-layered blastula in the prototherian mammal, Echidna (see p. 364).

/. Ingression

The word ingression is suitable for use in cases where a cell or small groups of cells separate from other layers and migrate into the segmentation

398

GASTRULATION

cavity or into spaces or cavities developed within the developing body. In the primitive-streak area of reptiles, birds, and mammals, for example, mesodermal cells detach themselves from the primitive streak and migrate into the space between the epiblast and hypoblast. Also, in the formation of the twolayered embryo in the prototherian mammal. Echidna, the inward migration of small entodermal cells to form the hypoblast may be regarded as cellular ingrcssion (fig. 175D). Ingression and polyinvagination have similar meanings.

g. Delamination

The word delamination denotes a mass sunderance or separation of groups of cells from other cell groups. The separation of notochordal, mesodermal, and entodermal tissues from each other to form discrete cellular masses in such forms as the teleost fish or the frog, after these materials have moved to the inside during gastrulation, is an example of delamination (fig. 210E, F).

h. Divergence

This phenomenon is the opposite of convergence. For example, after cells have involuted over the blastoporal lips during gastrulation, they migrate and diverge to their future positions within the forming gastrula. This movement particularly is true of the lateral plate and ventral mesoderm in the frog, or of lateral plate and extra-embryonic mesoderm in the reptile, bird, or mammal (fig. 192B, C, small arrows).

/. Extension

The elongation of the presumptive neural and epidermal areas externally and of the notochordal, mesodermal, and entodermal materials after they have moved inward beneath the neural plate and epidermal material are examples of extension. The extension of cellular masses is a prominent factor in gastrulation in all Chordata from Amphioxus to the Mammalia. In fact, as a result of this tendency to extend or elongate on the part of the various cellular groups, the entire gastrula, in many instances, begins to elongate in the anteroposterior axis as gastrulation proceeds. The faculty for elongation and extension is a paramount influence in development of axiation in the gastrula and later on in the development of primitive body form. The presumptive notochordal material possesses great autonomous powers for extension, and hence during gastrulation it becomes extended into an elongated band of cells.l^"^

D. The Organization Center and Its Relation to the Gastrulative Process

1. The Organization Center and the Primary Organizer

Using a transplantation technic on the beginning gastrula of the newt, it was shown by Spemann (T8) and Spemann and Mangold (’24) that the dorsallip region of the blastopore (that is, the chordamesoderm-entoderm cells in this area), when transplanted to the epidermal area of another embryo of the

THE ORGANIZATION CENTER

399

same stage of development, is able to produce a secondary gastrulative process and thus initiate the formation of a secondary embryo (fig. 193). Because the dorsal-lip tissue was able thus to organize the development of a second or twin embryo, Spemann and Mangold described the dorsal-lip region of the beginning gastrula as an “organizer” of the gastrulative process. In its normal position during gastrulation this area of cells has since been regarded as the organization center of amphibian development. It is to be observed in this connection that Lewis ( ’07 ) performed the same type of experiment but failed to use an embryo of the same age as a host. As he used an older embryo, the notochordal and mesodermal cells developed according to their presumptive fate into notochordal and somitic tissue but failed to organize a new embryo.

More recent experiments upon early frog embryos by Vintemberger (’36) and by Dalcq and Pasteels (’37), and upon early teleost fish embryos by other investigators (Oppenheimer, ’36 and ’47) have demonstrated the necessity and importance of yolk substance in the gastrulative process. This fact led Dalcq and Pasteels (’37) to suggest a new concept of the organization center, namely, that this center is dependent upon two factors: “the yolk and something normally bound to the gray crescent” (i.e., chordamesodermal area).

It was thought at first that the transplanted organizer material actually organized and produced the new embryo itself (Spemann, ’18, p. 477). But this idea had to be modified in the light of the following experiment by Spemann and Mangold (’24): Dorsal-lip material of unpigmented Triton cristatus was transplanted to an embryo of T. taeniatus of the same age. The latter species is pigmented. This experiment demonstrated that the neural plate tissue of the secondary embryo was almost entirely derived from the host and not from the transplanted tissue. Consequently, this experiment further suggested that the organizer not only possessed the ability to organize but also to induce host tissue to differentiate. Induction of neural plate cells from cells which ordinarily v'ould not produce neural plate tissue thus became a demonstrated fact.

The concept of an organizer in embryonic development had profound implications and stimulated many studies relating to its nature. Particularly, intensive efforts were made regarding the kinds of cells, tissues, and other substances which would effect induction of secondary neural tubes. The results of these experiments eventually showed that various types of tissues and tissue substances, some alive, some dead, from many animal species, including the invertebrates, were able to induce amphibian neural plate and tube formation. (See Spemann, ’38, Chap. X and XI; also see fig. 196A, B and compare with fig. 193.) Moreover, microcautery, fuller’s earth, calcium carbonate, silica, etc., have on occasion induced neural tube formation. However, the mere induction of neural tube development should not be confused with the organizing action of normal, living, chordamesoderm-entoderm cells of the dorsal-lip region of the beginning gastruIayThe latter’s activities are more comprehensive, for the cells of the dorsal-lip area direct and organize the normal gastrulative

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process as a whole and bring about the organization of the entire dorsal axial system of notochord, neural tube, somites, etc. In this series of activities, neural plate induction and neural tube formation merely are secondary events of a general organization process.

A clear-cut distinction should be drawn, therefore, between the action of the dorsal-lip organizer, in its normal position and capacity,’ and that of an ordinary inductor which induces secondary neural tube development. The characteristics of the primary organizer or organization center of the early gastrula are:

(a) its ability for autonomous or self-differentiation (that is, it possesses the ability to give origin to a considerable portion of the notochord, prechordal plate material, and axial mesoderm of the secondary embryo),

(b) its capacity for self-organizatiotu

(c) its power to induce changes within and to organize surrounding cells, including the induction and early organization of the neural tube.

As a result of its comprehensive powers, it is well to look upon the organization center (primary organizer) as the area which determines the main features of axiation and organization of the vertebrate embryo. In other words, it directs the conversion of the late blast ula into the axiated gastrular condition — a condition from which the primitive vertebrate body is formed. Induction is a tool-like process, utilized by this center of activity, through which it effects changes in surrounding cells and thus influences organization and differentiation. Moreover, these surrounding cells, changed by the process of induction, may in turn act as secondary inductor centers, with abilities to organize specific subareas.

An example of the ability of a group of cells, changed by inductive influence, to act as an inducing agent to cause further inductive processes is shown by the following experiment performed by O. Mangold (’32). The right, presumptive, half brain of a neurula of Ambystoma mexicanum, the axolotl, was removed and inserted into the blastocoel of a midgastrula of Triton taeniatus. Eight days after the implant was made, a secondary anterior end of an embryo was observed protruding from the anterior, ventral aspect of the host larva. An analysis of this secondarily induced anterior portion of an embryo demonstrated the following:

( 1 ) The original implant had developed into a half brain with one eye and one olfactory pit. However,

(2) it also had induced a more or less complete secondary larval head with a complete brain, two eyes, with lenses, two olfactory pits, one ganglion, four auditory vesicles, and one balancer. One of the eyes had become intimately associated with the eye of the implant, both having the same lens.

IHfc. URUAINIZAIION CtlN 1 tK

HUl

The series of inductive processes presumably occurred as follows: The implanted half brain induced from the epidermis of the host a secondary anterior end of a neural plate; the latter developed into a brain which induced the lenses, auditory vesicles, etc. from the host epidermis. Thus, the original implant, through its ability to induce anterior neural plate formation from the overlying epidermis, acted as a “head organizer.”

The transformation of the late blastula into the organized condition of the late gastrula thus appears to be dependent upon a number of separate inductions, all integrated into one coordinated whole by the “formative stimulus” of the primary organizer located in the pre -chordal plate area of entodermalmesodermal cells and adjacent chordamesodermal material of the early gastrula.

2. Divisions of the Primary Organizer

The primary organizer is divisible into two general inductor areas as follows:

(a) the pre-chordal plate of entomesodermal material, and

(b) the chordamesodermal cells which come to lie posterior to the prechordal plate area of the late gastrula.

The pre-chordal plate is a complex of entodermal and mesodermal cells associated at the anterior end of the notochordal cells in the late gastrula. In the beginning gastrula, however, it lies between the notochordal material and the dorsal-lip inpushing of the entoderm in amphibia, and just caudal to the notochordal area in teleosts, elasmobranch fishes, reptiles, and birds (figs. 169; 173 A; 179B; 180B). The chordamesodermal portion of the primary organizer is composed of presumptive notochordal cells and that part of the presumptive mesoderm destined to form the somites. The pre-chordal plate is known as the head organizer, because of its ability to induce brain structures and other activities in the head region. (The use of the phrase head organizer as a synonymous term for pre-chordal plate is correct in part only, for a portion of the anterior notochord and adjacent mesoderm normally is concerned also with the organization of the head.) On the other hand, the presumptive notochord with the adjacent somitic (somite) material is described as the trunk or tail organizer (fig. 19 IG) because of its more limited inductive power. For example, Spemann (’31) demonstrated that the head organizer transplanted to another host embryo of the same age produced a secondary head with eye and ear vesicles when placed at the normal head level of the host. Also when placed at trunk level, it induced a complete secondary embryo including the head structures. However, the trunk organizer is able to induce head and trunk structures at the head level of the host; but in the trunk region it induces only trunk and tail tissues. (See Holtfreter, ’48, pp. 18-19; Needham, ’42, pp. 271-272; Spemann, ’31, ’38. The student is referred also to Huxley and De Beer, ’34, Chaps. 6 and 7; and Lewis, ’07.)

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£. Chemodifferentiation and the Gastrulative Process

In the previous chapter it was observed that certain areas of the amphibian blastula are foreordained to give origin to certain organ rudiments in the future embryo because of their position and not because of their innate physiological condition. This condition is true of the future neural plate ectoderm and epidermal ectoderm. During the conversion of the late blastula into the late gastrula, these areas become changed physiologically, and they no longer are determined in a presumptive sense but have undergone changes which make them self-differentiating. This change from a presumptively determined condition to a self-differentiating, fixed state is called determination and the biochemical change which effects this alteration is known as chemodifferentiation (see Chap. 8).

Chemodifferentiation is an important phenomenon during gastrulation. As a result of the physiological changes involved in chemodifferentiation, restrictive changes in potency are imposed upon many localized cellular areas within the major, organ-forming areas. In consequence, various future organs and parts of organs have their respective fates rigidly, and irrevocably determined at the end of gastrulation. The gastrula thus becomes a loose mosaic of specific, organ-forming areas (figs. 194, 205). Consequently, the areas of the beginning gastrula which possess competence (Chap. 8) become more and more restricted as gastrulation proceeds. Chemodifferentiation apparently occurs largely through inductive (evocative) action.

F. Gastrulation in Various Chordata

1. Arnphioxus a. Orientation

Consult figures 167, 189, and 190 and become familiar with the animalvegetal pole axis of the egg, the presumptive organ-forming areas, etc.

b. Gastrulative Movements

1) Emboly. As gastrulation begins, a marked increase in mitotic activity occurs in the cells of the dorsal crescent, composed of presumptive notochordal and neural plate cells, and also in the cells of the ventral crescent or future mesodermal tissue. The general ectodermal cells or future epidermis also are active (figs. 167, 189, 190B). The entodermal cells, however, are quiescent (Conklin, ’32). Accompanying this mitotic activity, the entodermal plate gradually invaginates or folds inwardly into the blastocoel (figs. 189, 190). In doing so, the upper portion of the entodermal plate moves inward more rapidly and pushes forward toward a point approximately halfway between the polar body (i.e., the original midanimal pole of the egg) and the point which marks the anterior end of the future embryo (observe pointed end of arrow, fig. 189). Shortly after the inward movement of the entodermal

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403

plate is initiated, notochordal cells in the middorsal region of the blastopore involute, move inward along with the entoderm, and come to occupy a position in the middorsal area of the forming archenteron (fig. 190C-E). Similarly, mesodermal cells in the upper or dorsal ends of the mesodermal crescent gradually converge dorso-mediad and pass into the roof of the forming gastrocoel (archenteron) on either side of the median area occupied by the notochordal cells (fig. 190F, G). Thus the roof of the gastrocoel is composed of notochordal and mesodermal cells (fig. 195 A, B).

2) Epiboly. As the above events come to pass, the potential epidermal and neural cells proliferate actively, and both areas gradually become extended in an antero-posterior direction. In this way the neural ectoderm becomes elongated into a median band which lies in the middorsal region of the gastrula (figs. 190A-H; 247B-F), while the epidermal area covers the entire gastrula externally with the exception of the neural area.

Thus, the general result of this proliferation, infolding, and involution of the presumptive entodermal, notochordal, and mesodermal cells, together with the extension and proliferation of the ectodermal cells is the production of a rudimentary double-layered embryo or gastrula (figs. 189, 190). Ectodermal cells (epidermal and neural) form the external layer (fig. 190G). The internal layer is composed of notochordal cells in the dorso-median area with two narrow bands of mesodermal cells lying along either side of the median notochordal band of cells while the remainder of the internal layer is composed of entodermal cells (figs. 190G; 195 A, B). At the blastoporal end of this primitive gastrula are to be found proliferating notochordal, mesodermal, entodermal, and ectodermal cells.

3) Antero-posterior Extension of the Gastrula and Dorsal Convergence of the Mesodermal Cells, The processes associated with epiboly bring about an antero-posterior extension of the ectodermal layer of cells. Similarly, the cells which arc moved inward by embolismic forces are projected forward toward the future cephalic end of the embryo and become extended along the median embryonic axis. Epiboly and emboly, accompanied by rapid cell proliferation at the blastoporal-lip area, thus effect an antero-posterior elongation of the developing gastrula (figs. 189H; 190H).

As the gastrula is extended in the antero-posterior direction, a shift occurs in the position of the mesodermal cells which form the ventral or mesodermal crescent. The ventral crescent becomes divided ventrally into two halves, and each half gradually moves dorsalward along the inner aspect of the lateral blastoporal lips as gastrulation is accomplished. Each arm of the original crescent in this manner converges dorso-mediad toward the median notochordal cells of the dorsal blastoporal lip, and a mass of mesodermal cells comes to lie along either side of the notochordal cells. As a result of this converging movement, entodermal cells of the blastoporal area converge dorso

GASTRULATION

mediad and come to occupy the ventral lip of the blastopore, together with the externally placed, epidermal cells (fig. 190G, arrow). The blastopore as a whole grows smaller and moves to a dorsal position during the latter changes (fig. 247 A-C).

4) Closure of the Blastopore. See Chapter 10, neuralization in Amphioxus.

DORSAL LIP OF BL

m

1 ^.

•^j

sM!

Fig. 189. Gastrulation in Amphioxus. (Modified from Conklin, ’32.) (A) Beginning

gastrula. (B) Observe that entodermal (hypoblast layer) is projected roughly in direction of future cephalic end of embryo. (C G) Observe continued projection of entoderm toward cephalic end of future embryo. Note also position of polar body. In (F), (G), and (H) the gastrula begins to elongate along the antero-posterior axis of the developing embryo. (H) End of gastrular condition. Blastopore is closed by epidermal overgrowth, and neurenteric canal is formed between archenteron and forming neural tube.

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of gastrulation, although mitoses occur in other regions as well. During later stages of gastrulation, the entire complex of cells around the blastoporal region divides actively.

(c) Involution. Notochordal cells converge to the midregion of the dorsal blastoporal lip and then turn inward (involute) over the lip area to the inside.

(d) Extension. General elongation of the embryonic rudiment as a whole occurs, including the neural plate area.

(e) Convergence. Mesodermal cells converge toward the middorsal area of the blastopore. The path of this convergence is along the lateral lips of the blastopore, particularly the inner aspects of the lips. This movement is pronounced toward the end of gastrulation when each half of the mesodermal crescent moves dorsad toward the middorsal area of the blastopore. The mesoderm thus comes to lie on either side of the notochordal material at the dorsal lip of the blastopore.

(f) Constriction of the blastopore. During later phases of gastrulation, the blastopore grows smaller (fig. 247A-D), associated with a constriction of the marginal region of the blastoporal opening, particularly of the entodermal and epidermal layers. The movement of the mesoderm described in (e) above plays a part in this blastoporal change.

2) Epiboly. The caudal growth of the entire ectodermal layer of cells, epidermal and neural, and their antero-posterior extension is a prominent feature of gastrulation in Amphioxus.

(Further changes in the late gastrula, together with the closing of the blastopore, are described in Chapter 10. See tubulation of neural plate, etc.)

2. Gastrulation in Amphibia with Particular Reference TO THE Frog

a. Introduction

1) Orientation. A line drawn from the middle region of the animal pole to the midvegetal pole constitutes the median axis of the egg. In the anuran Amphibia the embryonic axis corresponds approximately to the egg axis. That is, the midanimal pole of the egg represents the future anterior or anterodorsal end of the embryo, while the midvegetal pole area denotes the posterior region.

As indicated previously (Chap. 7), the very late blastula is composed of presumptive organ-forming areas arranged around the blastocoelic space. The yolk-laden, future entodermal cells of the gut or digestive tube form the hypoblast and are concentrated at the vegetal pole. Presumptive notochordal and mesodermal cells constitute a marginal zone of cells which surrounds the upper region of the presumptive entodermal organ-forming area (fig. 169C-F). The presumptive notochordal area is in the form of a crescent.

GASTRULATION IN VARIOUS CHORDATA

407

whose midportion is located just above the future dorsal lip of the early gastrula, while the mesoderm lies to each side of the notochordal cells, extending along the margin of the entoderm toward the corresponding mesodermal zone of the other side (fig. 169D, F). The presumptive neural crescent occupies a region just dorsal and anterior to the notochordal area. The remainder of the animal pole is composed of presumptive epidermis. The presumptive notochordal, neural plate, and epidermal areas are oriented along the general direction of the future antero-posterior embryonic axis, the notochordal tissue being the more posterior. Moreover, the midregion of the notochordal and neural crescents at this time lies at the dorsal region of the future embryo (fig. 194A). The presumptive entodermal area, on the other hand, does not have the same orientation as that of the above areas. In contrast, its axiation is at right angles to the future embryonic axis (fig. 194A). If one views a very early gastrula of the anuran amphibian in such a way that the beginning blastoporal lip is toward the right (fig. 194A), then:

( 1 ) The foregut material lies toward the right at the region of the forming blastoporal lip;

(2) the stomach material is slightly to the left of this area; and

(3) the future intestinal area lies to the left and toward the vegetal pole.

Therefore, one aspect of the gastrulative processes is to bring the entodermal area into harmony with the future embryonic axis and, in doing so, to align its specific, organ-forming subareas along the antero-posterior axis of the embryo. In other words, the entodermal material must be revolved about 90 degrees in a counterclockwise direction from the initial position occupied at the beginning of gastrulation (compare fig. 194A, B).

2) Physiological Changes Which Occur in the Presumptive Organ-forming Areas of the Late Blastula and Early Gastrula as Gastrulation Progresses. A striking physiological change is consummated in the presumptive organforming areas of the epiblastic portion of the late blastula during the process of gastrulation. This change has been demonstrated by transplantation experiments. For example, if presumptive epidermis of the very late blastula and early gastrula is transplanted by means of a micropipette to the presumptive neural area and vice versa, the material which would have formed epidermis will form neural tissue, and presumptive neural cells will form epidermis (fig. 196C, D). (See Spemann, M8, ’21; Mangold, ’28.)

\ The experiment pictured in figure 196 involves interchanges between two presumptive areas within the same potential germ layer, i.e., ectoderm. However, Mangold (’23) demonstrated that presumptive epidermis transplanted into the dorsal-lip area, i.e., into the presumptive mesodermal area, may invaginate and form mesodermal tissue. The converse of this experiment was performed by Lopaschov (’35) who found that presumptive mesoderm from the region of the blastoporal lip transplanted to the neural plate area of a

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somewhat older embryo becomes, in some cases, normally incorporated in the neural tube of the host. Similar interchanges of cells of the late blastula have demonstrated that almost any part, other than the presumptive entoderm, can be interchanged without disturbing the normal sequence of events. However, as gastrulation progresses, interchange from epidermal to neural areas continues to be possible during the early phases of gastrulation (fig. 196C, D) but not at the end of gastrulation. Similar changes occur also in the mesodermal area. Pronounced physiological changes thus occur in the presumptive organforming aieas of the entire epiblastic region during gastrulation.

b. Gastrulation

1) Emboly. As gastrulation begins, a small, cleft-like invagination appears in the entodermal material of the presumptive foregut area. This invagination is an active inpushing of entodermal cells which fold inward and forward toward the future cephalic end of the embryo (fig. 191B-E). The upper or dorsal edge of the cleft-like depression visible at the external surface forms the dorsal lip of the blastopore (fig. 19 IB). In this connection, study diagrams in figure 197. The pre-chordal plate cells are associated with the forming dorsal roof of the archenteron and, therefore, form a part of the invaginated material shortly after this process is initiated.

As the entodermal material migrates inward and the initial dorsal lip is formed, notochordal cells move posteriad to the dorsal lip and involute to the inside in close association with the pre-chordal plate cells. Also, the more laterally situated, notochordal material converges toward the dorsal lip and gradually passes to the inside, as gastrulation progresses, where it lies in the mid-dorsal region of the embryo. (See arrows, figs. 188 A; 19 1C, D). Here it begins to elongate antero-posteriorly (i.e., it becomes extended) and forms a narrow band of cells below the forming neural plate (fig. 191C-G).

With the continuance of gastrulation, the entodermal material moves more extensively inward (cf. fig. 191C-E) and the entodermal mass of yolk-laden cells below the site of invagination begins to sink or rotate inwardly. The dorsal blastoporal lip, therefore, widens considerably (fig. 197 A, B). In many Amphibia the inner surface of the entoderm, as it progresses inward, forms a cup-like structure which actually engulfs the blastocoelic fluid (fig. 191B-D). It is not clear whether this cup-like form is produced by active inward migration of entodermal cells or whether it may be due in part, at least, to constrictive forces at the blastoporal lip.

Synchronized with the events described above, the presumptive somitic mesoderm, located externally along either side of the notochordal area of the early gastrula, migrates (converges) toward the forming dorso-lateral lips of the blastopore (fig. 197 A, B, broken arrows). Upon reaching the blastoporal edge, the mesoderm moves over the lip (involutes) to the inside. However, the mesoderm does not flow over the lip to the inside as a part of the entoderm

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Coincident with the lateral extensions of the original dorsal lip of the blastopore to form the lateral lips, a more extensive convergence and involution of presumptive mesoderm located in the lateral portions of the mesodermal crescent occurs (fig. 197 A, B). The latter mesoderm eventually forms the lateral area of the hypomeric mesoderm of the future embryo (figs. 191G; 198B, C). As the lateral lips of the blastopore continue to form in the ventral direction, they eventually reach a point where they turn inward toward the median axis and thus form the ventral lip of the blastopore (fig. 197C). A rounded blastopore, circumscribing the heavily, yolk-laden, entodermal cells, thus is formed. Associated with the formation of the ventro-lateral and ventral blastoporal lips is the convergence and involution of the ventro-lateral and ventral mesoderm of the gastrula (fig. 191D-F). Accompanying the inward migration of the entoderm in the region of the dorso-lateral lip of the blastopore, there is, presumably, an inward rotation of the entodermal mass which lies toward the ventral blastoporal area. The result of this entodermal movement is the production of a counterclockwise rotation of the entodermal, organ-forming rudiments, as indicated in figure 194B, compared to their relative positions at the beginning of gastrulation, shown in figure 194A. (This counterclockwise rotation is present to a degree also in Amphioxus (fig. 190A-F). In this way, the particular, organ-forming areas of the entoderm become arranged anteroposteriorly in a linear fashion along the embryonic axis. The foregut material now is situated toward the anterior end of the developing embryo, while the stomach, liver, small intestine, and hindgut regions are placed progressively posteriad with the hindgut area near the closing blastopore (fig. 194B). The yolk material lies for the most part within the ventral wall of this primitive archenteron.

Associated with the axiation of the entodermal rudiments is the axiation of the notochord-mesoderm complex. For example, the anterior segment of the notochord and the pre-chordal plate (i.e., the head organizer) are located anteriorly in the gastrula, while the more posterior portions of the notochord and adjacent mesoderm (i.e., the trunk organizer) are located in the developing trunk region (fig. 19 IG). The mesoderm adjacent to the notochord eventually will form the somites or primitive mesodermal segments of the embryo (figs. 19 IG; 217E; 224F). Experimentation, using the Vogt method of staining with vital dyes, has demonstrated that the future, anterior, presumptive somites lie closer to the blastoporal lips in the beginning gastrula, whereas the more posterior, presumptive somites are situated at a greater distance from the blastoporal area. Because of this arrangement, the first or anterior pair of presumptive somites moves inward first, the second pair next, etc. The mesoderm of the future somites in this way is arranged along the notochord in an orderly sequence from the anterior to the posterior regions of the gastrula (fig. 169, somites 1, 2, 3, 4, etc.). Consequently, axiation and extension of the somitic mesoderm occur along with the antero-posterior arrangement of

GASTRULATION IN VARIOUS CHORDATA

411

the notochordal material. A similar distribution is effected in other regions of the mesoderm. Therefore, axiation and antero-posterior extension of the entoderrnal, notochordal, and mesodermal cells are conspicuous results of the activities which effect emboly.

2) Epiboly. The above description is concerned mainly with emboly, that is, the inward migration of the notochord-mesoderm-entoderm-yolk complex. Allied with these active events is the downward or caudal migration of the blastoporal lips. This migration is illustrated in figure 191B-E. In this figure it may be observed that, as the marginal zone cells of mesoderm and notochord

Fig. 192. Movements of the parts of the blastula during gastrulation in amphibia. (Cf. fig. 191.) (A) Results of epiboly. (Cf. fig. 19 1 A T.) Epidermal and neural areas envelop

the other areas during gastrulation. (B) Movements of the areas of the blastula during emboly, as seen from the vegetative pole. Heavy arrows, solid and broken, show the converging movements during emboly; light arrows show the extension and divergence of cells after involution at the blastoporal margin (cf. fig. 191A~F). (C) Similar to

(B), as seen from the left side.

SECONDARY EMBRYO

Fig. 193. Induction of a secondary embryo. (From Spemann, ’38.) (A) Host embryo

shown in this figure is Triton taeniatus. A median piece of the upper lip of the blastopore of a young gastrula of T. cristatus of approximately the same age as the host was implanted into the ventro-lateral ectoderm of the host. The implanted tissue developed into notochord, somites, etc.; the neural tube was induced from the host ectoderm. (B) Cross section through embryo shown in (A).

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Fig. 194. Developmental tendencies of entodermal area and their reorientation during gastnilation. (A) Developmental tendencies of entodermal area of young anuran gastrula. (B) Counterclockwise rotation of approximately 90° of the entodermal area during gastrulation.

together with the entoderm and yolk pass to the inside, the forces involved in epiboly effect the expansion of the purely ectodermal portion of the cpiblast which gradually comes to cover the entire external surface of the gastrula with the exception of the immediate blastoporal area (study black and white areas in fig. 191A-E). It may be observed further that the neural crescent now is elongated along the antero-posterior, embryonic axis where it forms a shieldshaped region with the broad end of the shield located anteriorly (fig. 192A).

A study of figure 191 E and F shows that a rotation of the entire gastrula occurs in the interim between E and F. This rotation is induced by the inward movement of the entoderm and yolk, depicted in figure 191C-E, with a subsequent shift in position of the heavy mass of yolk from the posterior pole of the embryo to the embryo’s ventral or belly region. Most of the blastocoel and its contained fluid is “engulfed” by the inward moving entoderm, as indicated in figure 191C-E, some of the blastocoelic lluid and blastocoelic space passes over into the gastrocoel. The region of the entodermal yolk mass shown to the left in figure 19 IE, therefore, is more dense and heavier than the area shown to the right. The heavier region of the gastrula seeks the lower level; hence the rotation of the entire gastrula, and the new position assumed in figure 191F.

As the blastopore progressively grows smaller, it eventually assumes a small, rounded appearance (fig. 197A-E), and the remnants of the presumptive mesoderm pass over the lips of the blastopore before it closes. In doing so, the presumptive tail mesoderm converges dorsally and becomes located inside the dorso-lateral portion of the closed blastopore near the lateral aspects of the posterior end of the folding neural plate.

A short while previous to blastoporal closure, the midregion of the neural plate area begins to fold ventrad toward the notochord, while its margins are

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413

elevated and projected dorso-mediad. The exact limits of the neural plate thus become evident (fig. 197D).

3) Embryo Produced by the Gastrulative Processes. The general result of epiboly and emboly in the Amphibia is the production of an embryo of three germ layers with a rounded or oval shape. The potential skin ectoderm and infolding, neural plate area form the external layer (fig. 192A). Underneath this external layer are the following structural regions of the middle or mesodermal layer:

(a) Below the developing nerve tube is the elongated band of notochordal cells;

(b) on either side of the notochord is the somitic (somite) mesoderm;

(c) lateral to the somitic area is the mesoderm of the future kidney system; and

Fig. 195. Placement of the presumptive, organ-forming areas in an embryo of Amphioxus of about six to seven somites. (Modified from Conklin, ’32.) (A) Section

through anterior region. (J) Section through caudal end of embryo. (B-1) Successive sections going posteriorly at different body levels between (A) and (J).

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SECONDARY NE UR A L I Z A T I 0 N SECONDARY NEUR AL I Z AT ION

Fig. 196. Ectodermal potencies of the amphibian gastrula. (A and B from Spemann, ’38, after Fischer; C and D from Spemann, ’38, after Spemann, ’18.) (A) Induction of

a secondary neural plate in the axolotl gastrula by five per cent oleic acid, emulsified in agar-agar. (B) Induction of secondary neural plate by nucleic acid from calf thymus. (C) Formation of neural plate tissue from presumptive epidermal cells transplanted into neural plate region. (D) Reverse transplant, presumptive neural plate becomes epidermal tissue.

(d) still more lateral and extending ventrally are the lateral plate and ventral mesoderm (figs. 191F-I; 198A-C; 221).

The third or inner germ layer of entoderm is encased within the mesodermal or middle germ layer. The entodermal layer is an oval-shaped structure containing a small archenteric cavity filled with fluid. Its ventral portion is heavily laden with yolk substance. Also, the future trunk portion of the archenteric roof is incomplete, the narrow notochordal band forming a part of its middorsal area (figs. 19 IF; 194B; 219D). Within each of these germ layers are to be found restricted areas destined to be particular organs. Each layer may be regarded, therefore, as a general mosaic of organ-forming tendencies.

4) Position Occupied by the Pre-chordal Plate Material. Another feature of the late gastrula remains to be emphasized, namely, the pre-chordal plate composed of entodermal and mesodermal cells integrated with the anterior end of the notochord. During gastrulation the pre-chordal plate invaginates with the entoderm and comes to occupy the roof of the foregut, just anterior to the rod-like notochord (fig. 191D-F). In this position it lies below the anterior part of the neural plate area; it functions strongly in the induction and formation of the cephalic structures, including the brain as indicated above. Because of this inductive ability, it is regarded as a principal part of the head

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organizer (fig. 191E-G). Eventually pre-chordal plate cells contribute to the pharyngeal area of the foregut and give origin to a portion of the head mesoderm, at least in many vertebrate species (Chap. 11, p. 523).

c. Closure of the Blastopore and Formation of the Neurenteric Canal

The closure of the blastopore and formation of the neurenteric canal is described in Chapter 10, p. 471.

d. Summary of Morphogenetic Movements of Cells During Gastrulation

in the Frog and Other Amphibia

1) Emboly:

(a) Invagination. Invagination in the Amphibia appears to consist of two phases: (1) an active infolding or forward migration of the future foregut, stomach, etc., areas, and (2) an insinking and inward rotation of future intestinal and heavily laden, yolk cells.

(b) Convergence. This activity is found in the presumptive, notochordal and mesodermal cells as they move toward the blastoporal lips. A dorsal convergence toward the dorsal, blastoporal-lip area is particularly true of the more laterally placed parts of the notochordal crescent and to some extent also of the somitic and lateral plate mesoderm. The tail mesoderm tends to converge toward the dorsal blastoporal area when the blastopore nears closure.

(c) Involution. An inward rolling or rotation of cells over the blastoporal lips to the inside is a conspicuous part of notochordal and mesodermal cell migration.

(d) Divergence. After the mesodermal cells have migrated to the inside, there is a particular tendency to diverge on the part of the lateral plate and ventral mesoderm. The lateral plate mesoderm diverges laterally and ventrally, while the ventral mesoderm diverges laterally in the ventral or belly area of the gastrula.

(e) Extension. The phenomenon of extension or elongation is a characteristic feature of all gastrulative processes in the chordate group. Before arriving at the blastoporal lips, the converging notochordal and mesodermal cells may undergo a stretching or extending movement. That is, convergence and stretching are two prominent movements involved in the migration of the marginal zone or chordamesodermal cells as they move toward the blastoporal lip. After these materials have involuted to the inside, the chordal cells stretch antero-posteriorly and become narrowed to a cuboidal band in the midline, and the lateral plate mesoderm stretches anteriorly as it diverges laterally. Antero-posterior extension of the somitic mesoderm also occurs.

(f ) Contractile tension or constriction. A considerable constriction or contraction around the edges of the blastopore occurs as gastrulation pro

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gresses. This particularly is true when the blastopore gradually grows smaller toward the end of the gastrulative process (Lewis, ’49).

2) Epiboly. Intimately associated with and aiding the above processes involved in emboly are the movements concerned with epiboly. These movements result from cell proliferation, associated with a marked antero-posterior extension and expansion of the presumptive epidermal and neural plate areas. These changes are integrated closely with the inward migration of cells of the marginal zone (i.e., chordamesoderm ) , and the presumptive epidermal and neural areas approach closer and closer to the blastoporal edge, until finally, when mesodermal and notochordal cells have entirely involuted, ectodermal cells occupy the rim of the blastopore as it closes (figs. 192A; 220D).

Fig. 197. History of the blastopore and adjacent posterior areas of developing embryo of the frog, Rana pipiens. (A) Dorsal lip of blastopore. Arrows show direction of initial invagination to form the dorsal lip. (B) Dorso-lateral and lateral-lip portions of the blastopore are added to original dorsal-lip area by convergence of mesodermal cells (arrows) and their involution at the edge of the lip. Entodermal material is invaginating. (C) Blastopore is complete; yolk plug is showing. (D) Toward the end of gastrulation. Blastopore is small; neural plate area becomes evident as neural folds begin their elevation. (E) Neural folds are slightly elevated; blastopore is very small; size of blastopore at this time is quite variable. (F) Blastopore has closed; neural folds are well developed; neurenteric passageway between neural folds and dorsal evagination of archenteric space into ttiil-bud area is indicated by N.C. (G) New caudal opening is forming, aided by proctodaeal invagination, PR.; tail rudiment elevation is indicated. (H) Proctodaeal opening and tail rudiment arc shown.

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Fig. 198. Anterior extension (migration) of the mesoderm from the blastoporal-lip area after involution at the lip in the urodele, Pleurodeles. (A-<^) Progressive inward migration of the mantel of mesoderm, indicated by the white area stippled with coarse dots. (A) Early gastrula. (B) Late gastrula. (C) Beginning neurula.

As a result, the presumptive epidermal and neural plate areas literally engulf the inwardly moving cells.

3. Gastrulation in Reptiles

a. Orientation

The reptilian blastoderm, as gastrulation begins, is composed of an upper epiblast and a lower hypoblast as indicated previously in Chapter 7 (fig. 174A-D). The formation of the hypoblast as a distinct layer proceeds in a rapid fashion and immediately precedes the formation of a large notochordal canal and subsequent cell migration inward. The two events of entodermal layer (hypoblast) formation and the inward migration of notochordal and mesodermal cells thus are closely and intimately correlated in reptiles. This close relationship is true particularly of the turtle group. The upper layer or epiblast of the reptilian blastoderm is a composite aggregation of presumptive epidermal, neural, notochordal, and mesodermal cells (fig. 174E, F), arranged in relation to the future, antero-posterior axis of the embryo. It is possible that some entodermal material may be located superficially in the epiblast in the turtle as gastrulation begins (Pasteels, ’37a).

b. Gastrulation

Immediately following the formation of the hypoblast, the gastrulative phenomena begin with a rather large inpushing or invagination involving the notochordal, mesdoermal areas, particularly the pre-chordal plate and notochordal areas. This invagination extends downward and forward toward the hypoblast along the antero-posterior embryonic axis, and it produces a pouchlike structure known variously as the notochordal canal, blastoporal canal, or chordamesodermal canal (figs. 199A-C; 200A-C). The invaginated noto

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Fig. 199, Surface views of blastoderm of the turtle, Chrysemys picta, during gastrulation. Darkened area in the center shows the embryonic shield, the region of the notochordal canal in the area of the primitive plate. (A) Young gastrula. External opening of notochordal canal is wide. (B) Later gastrula. External opening of notochordal canal is horseshoe-shaped; internal opening of canal is indicated by small crescentic light area in front of external opening. (C) Very late gastrula. Notochord is indicated in center; head fold is beginning at anterior extremity of blastoderm.

chordal canal reposes upon the entoderm, and both fuse in the region of contact (fig. 200C). The thin layer of cells in the area of fusion soon disappears, leaving the antero-ventral end of the flattened notochordal canal exposed to the archenteric space below. After some reorganization, the notochord appears as a band, extending antero-posteriorly in the median line, associated with the entoderm on either side (fig. 201B-G). However, at the extreme anterior end of the gastrula, the notochordal material, together with the entoderm and to some extent the overlying ectoderm, presents a fused condition. Within this area the pre-chordal plate or anterior portion of the head organizer is located. In this general region of the embryo, foregut, brain, and other head structures eventually arise (fig. 199C). The original, relatively large, notochordal invagination soon becomes a small canal which extends cranio-ventrally

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from the upper or external opening to the archenteric space which lies below the notochord and entoderm (fig. 200B, E).

Posterior to the opening of the notochordal canal is the thickened primitive plate (primitive streak), composed of converged presumptive mesodermal cells (fig. 199). This converged mass of cells involutes to the inside along the lateral borders of the notochordal canal and also posterior to this opening. However, most of the mesoderm of the future body of the embryo apparently passes inward with the notochordal material during the formation of the notochordal canal, where it comes to lie on either side of the median notochordal band between the ectoderm and the entoderm. These general relationships of notochord, ectoderm, mesoderm, and entoderm are shown in figure 201A-H.

The extent to which the original notochordal inpushing is developed varies in different reptilian species. In lizards and snakes its development is more pronounced than in turtles (cf. fig. 200A, D).

During emboly, the presumptive neural plate and epidermal areas are

Fig. 200. Sagittal section of reptilian bIa.stoderms to show notochordal inpushing (notochordal canal or pouch). (A) Section of early gastrulative procedure in Clernmys leprosa. (After Pasteels, ’36b, slightly modified.) (B) Original from slide, Chrysemys picta, showing condition after notochordal canal has broken through into archenteric space. (C) Notochordal canal of the lizard, Platydactylus. (D) Later stage of (C). (E) After notochordal canal has broken through into archenteric space. (OE, after Will, 1892.)

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A

B.

NEUR AL PLATE EPIDERMIS

NOTOCHORD

D.

INTERNAL OPENING OF

NOTOCHORDAL CANAL

NOTOCHORDAL CANAL

Fig. 201. Transverse sections of the late turtle gastrula as indicated by lines in fig. 199C.

elongated antero-posteriorly by the forces of epiboly. Meanwhile, the external opening of the notochordal canal changes in shape and together with the primitive plate moves caudally (fig. 199). As gastrulation draws to a close, the neural plate area begins to fold inward, initiating the formation of the neural tube.

4. Gastrulation in the Chick a. Orientation

As described in Chapter 7, a twodayered blastoderm (blastula) composed of an epiblast and a hypoblast is present, with the hypoblast more complete at the posterior end of the blastoderm than at its extreme anterior and anterolateral margins (figs. 171A; 202A). The epiblast over the posterior half of the blastoderm is composed of presumptive notochordal and mesodermal cells, and anteriorly in the epiblast are found the presumptive epidermal and neural areas (figs. 173 A; 202A).

b. Gastrulative Changes

1) Development of Primitive Streak as Viewed from the Surface of Stained Blastoderms. The formation of the primitive streak is a progressive affair. Figure 170 pictures a pre-streak blastoderm, and it is to be observed that the entodermal layer below the epiblast is present as an irregular area in the

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caudal region of the area pellucida. A median, sagittal section through a comparable stage is shown diagrammatically in figure 171 A. Figure 203 A illustrates an early beginning streak normally found eight hours after incubation of the egg is initiated, while figure 203B presents a medium streak, appearing after about 12 to 13 hours of incubation. In figure 203C, a definite primitive streak appears in which the primitive groove, primitive pit, primitive folds, and Hensen’s node (primitive knot) are outlined. This condition occurs after about 18 to 19 hours of incubation. This may be regarded as the mature streak. A later streak after about 19 to 22 hours of incubation is indicated in figure 203 D. Observe that the head process or rudimentary notochord extends anteriorly from Hensen’s node, while the mesoderm is a deeper-shaded area emanating from the antero-lateral aspect of the streak. The clear proamnion region may be observed at the anterior end of the area pellucida. In the proamnion area, mesoderm is not present at this time between the ectodermal and entodermal layers.

2) Cell Movements in the Epiblast Involved in Primitive-streak Formation as Indicated by Carbon-particle Marking and Vital-staining Experiments.

Recent experiments by Spratt (’46), using carbon particles as a marking device, have demonstrated that epiblast cells from the posterior half of the prestreak blastoderm gradually move posteriad and rnediad as gastrulation proceeds (figs. 202, 204, black arrows). Before the actual appearance of the streak, mesodermal cells begin to appear between the epiblast and hypoblast at the posterior margin of the area pellucida. (See fig. 202B, involuted mesoderm). As cellular convergence posteriorly toward the median line continues, the primitive streak begins to form as a median thickening posteriorly in the pellucid area (fig. 202C, observe posterior median area indicated in white). The rudimentary primitive streak formed in this manner gradually advances anteriorly toward the central region of the pellucid area of the blastoderm (fig. 202D, E). In the thickened area of the developing primitive streak, shown in white at the posterior median portion of the blastoderm in figure 202C, there are about three to four cell layers of epiblast together with about the same number of layers of mesoderm below. At its anterior end the streak is thinner.

The anterior end of this early streak gradually grows forward as a result of cell proliferation in situ and by cells added through convergence of cells from antero-lateral areas (Spratt, ’46). Some of the cells at the anterior end of the forming streak may involute or ingress from the epiblast into the space between the hypoblast and epiblast and thus come to lie at the anterior end of the forming streak, while other cells ingress laterally between these two layers (fig. 202C-E, K-O).

As the streak differentiates anteriorly by addition of cells to its anterior end, it also elongates posteriorly by cellular additions to its caudal end. The carbon-marking experiments of Spratt demonstrated further that, during the

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formation of the streak up to about the condition present at 20 to 22 hours of incubation (figs. 2021, K; 203D), almost the entire posterior half of the pellucid area, consisting of presumptive pre-chordal plate, notochord, and mesoderm, is brought into the streak and involuted to the inside between the hypoblast and epiblast (figs. 202F-H; 204). This condition of development is often referred to as the “head-process stage” (stage 5, Hamburger and Hamilton, ’51). At this stage the approximate, antero-posterior limits of the future embryonic body of the chick, exclusive of the extra-embryonic tissue, are shown by the general area beginning just anterior to the head process and extending for a short distance posterior to Hensen’s node (figs. 203D; 205D, E).

As indicated in figure 202, there are two parts to the primitive streak:

(1) the area of Hensen’s node and primitive pit concerned with invaginative movements of pre-chordal plate mesoderm and notochordal cells and

(2) the body of the streak.

The former area appears to arise independently in the center of the pellucid area^ while the body of the streak is formed at the median, caudal margin of the pellucid area, from whence it grows anteriad to unite with Hensen’s node.

Fio. 202. Migration of cells during gastrulation in the chick. Drawing to the left of the midline represents a surface view; to the right of the midline the epiblast layer has been removed. (A-F) To the left of the midline based on data provided by Spratt, *46. (J) Represents lateral, sectional view of (F)-(G), viewed from the left side. Arrows indicate direction of cell migration. (K)* Indicates a left lateral view of (I), with the epiblast cut away midsagitally throughout most of the left side of the blastoderm. (L-O) Transverse sections of (K), as indicated on (K).

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The body of the streak serves as the “door” through which migrating mesodermal cells other than the cells of the pre-chordal plate-notochordal area pass from the epiblast layer downward to the space between the epiblast and hypoblast.

Using the Vogt method of vital staining, Pasteels (’37b) was able to demonstrate morphogenetic movements of cells into the primitive streak area and thence to the inside similar to that described by Spratt (fig. 202G-I).

The evidence derived from the carbon-particle-marking technic and that of vital staining, therefore, strongly suggests that the primitive streak of the chick forms as a result of:

(a) converging movements of the epiblast cells toward the median line of the posterior half of the pellucid area and

(b) cell proliferation in situ within the streak.

3) Cell Movements in the Hypoblast and the Importance of Those Movements in Primitive-streak Formation. The hypoblast or entodermal layer of the blastula appears to play a significant role relative to the formation of the primitive streak in the bird. Various lines of evidence point to this conclusion. For example, Waddington (’33) reported the results of experiments in which he separated the epiblast from the hypoblast of early chick and duck embryos in the early, primitive-streak stage. He then replaced the two layers so that their longitudinal axes were diametrically reversed, that is, the anterior part of the entoderm (hypoblast) lay under the posterior part of the epiblast, while the posterior part of the entoderm lay below the anterior region of the epiblast. The following results were obtained:

(1) The development of the original streak was suppressed; or

(2) a new, secondary, primitive streak was induced.

During later development, in some cases, the secondary streak disappeared; in others, it persisted and a double monster was produced. In other instances the primary primitive streak disappeared and the secondary streak persisted. The general conclusion set forth by Waddington is as follows: the entoderm does not induce the differentiation of a definite tissue, but rather, it induces the form-building movements which lead to the development of the primitive streak.

Certain experiments made by Spratt (’46) lend added evidence of the importance of the hypoblast in primitive-streak formation. In eight experiments in which the hypoblast was removed before streak formation, six cases failed to produce a streak, whereas in two instances a beginning streak was formed. It may be that in the latter two cases, the induction of morphogenetic movements within the epiblast cells occurred previous to hypoblast removal. These experiments are too few to permit a definite conclusion; however, they are suggestive and serve to bolster the conclusion made by Waddington. In a

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second set of experiments performed by Spratt, chick blastoderms in the prestreak and early-streak stages were inverted and marked with carbon particles. The results showed that the hypoblast moves forward in the median line below the epiblast layer. He also demonstrated that this forward movement of the hypoblast “precedes the anterior differentiation of the primitive streak.” Spratt further observed that: When the movement of the hypoblast deviated to the left or to the right, the primitive streak similarly deviated. This evidence “strongly suggests that the hypoblast influences the development of the primitive streak in the overlying epiblast” (Spratt, ’46).

PROAMNION

PMnpyONIC AREA ir^^-PRIMITIVE

(HENSEN'S NODE) '•PRIMITIVE PIT

A - E M

TIVE GROOVE I T I V E FOLD

AREA OPA C A

AREA PELLUCIOi MATURE PRIMITIV

Fig. 203. Surface-view drawings of photographs of developing primitive streak. (From Hamburger and Hamilton, ’51, after Spratt.) (A) Initial streak, short, conical thickening at posterior end of blastoderm. (Hamburger and Hamilton, ’51, stage 2.) (B)

Intermediate streak. Thickened streak area approaches center of area pellucida. (Hamburger and Hamilton, ’51, stage 3.) (C) Definitive streak (average length, 1.88 mm.).

Primitive groove, primitive fold, primitive pit, and Hensen’s node are present. (Hamburger and Hamilton, ’51, stage 4.) (D) Head-process stage (19 to 22 hours of incu bation). Notochord or head process visible as area of condensed mesoderm extending anteriorly from Hensen’s node. Proamnion area is indicated in front portion of area pellucida; head fold is not yet present. (Hamburger and Hamilton, ’51, stage 5.)

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Fig. 204. Movements in the epiblast layer of the chick during gastrulation and primitive-streak formation. (Modified slightly from Spratt, ’46.) (A) Pre-streak con dition. Carbon particles are placed as indicated at a, b, c, d, e, f, and g. (B-G) Observe migration of carbon particles. (C) Short streak. (E) Medium broad streak. (G) Long streak. (See fig. 203C.)

4) Primitive Pit and Notochordal Canal* If one compares the notochordal canal, formed during gastrulation in the reptilian blastoderm, with that of the primitive pit in the chick, the conclusion is inevitable that the primitive pit of the chick blastoderm represents an abortive notochordal canal. The lizard, turtle, and chick thus represent three degrees of notochordal canal development (figs. 200A, D; 202J). In certain birds, such as the duck, a notochordal canal very similar to that of the turtle gastrula, is formed.

5) Resume of Morphogenetic Movements of Cells During Gastrulation in the Chick. In view of the foregoing facts relative to primitive-streak formation, steps in the gastrulative procedure in birds may be described as follows:

(a) Shortly after the incubation period is initiated, hypoblast material at the caudal end of the blastula starts to move in the median line toward the future cephalic end of the embryo. This activity may be regarded as a gastrulative streaming of the hypoblast. (This streaming movement probably represents the chick’s counterpart of the forward movement of the entodermal area in the dorsal-lip region of the frog embryo. )

(b) After this movement of the hypoblast is inaugurated, cells from the epiblast layer immediately overlying the moving hypoblast pass downward toward the hypoblast. That is, epiblast cells begin to involute and come to lie between the epiblast and hypoblast; from this new

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position the involuted cells migrate laterally and anteriorly between the hypoblast and epiblast.

(c) In conjunction with the foregoing activities, epiblast cells (presumptive mesoderm) from the posterior half of the epiblast of the pellucid area migrate posteriad, converging from either side toward the median line (fig. 204A-G).

(d) These converging cells begin to pile up in the posterior median edge of the pellucid area (fig. 204C), where they produce a raphe-like thickening which marks the beginning of the primitive streak (fig. 204C-G). The beginning streak first makes its appearance at about seven to eight hours after the start of incubation in the egg of the chick (fig. 204C).

(e) Once formed, the initial streak grows anteriad in the median line by: (1) cell proliferation in situ, and by the addition of (2) converging cells from the epiblast layer.

(f) Also, the primitive streak apparently grows posteriad by cell proliferation and the addition of converging cells.

(g-) When the migrating cells of the epiblast reach the primitive streak, they involute and pass downward to the space between the epiblast and hypoblast. From this new position they move laterad and anteriad on either side of the midline, diverging to form a broad, middle layer of mesodermal cells.

(h) As the primitive streak grows anteriad in the epiblast, it eventually approaches the presumptive pre-chordal plate and presumptive notochordal areas.

(i) The pre-chordal plate and notochordal cells then invaginate to form the primitive pit; the latter represents a shallow or vestigial notochordal canal, a structure strongly developed in reptiles and some birds, and occasionally in mammals.

(j) Notochordal cells from the notochordal crescent converge to the pit area and probably pass downward in the walls of the pit, whence they ingress and move forward in the median line (fig. 202 A-G, J, K). The definitive primitive streak is formed after about 18 to 19 hours of incubation. At about 20 to 22 hours of incubation, the prospective, notochordal material (e.g., the head process) has already invaginated. At this time it represents a mass of cells in the median line intimately associated with the neural plate ectoderm above the pre-chordal plate cells and the entoderm below (fig. 2021, K). As the primitive streak recedes posteriad (see p. 431 ), the notochordal material gradually separates from the surrounding, pre-chordal plate cells and also from the neural plate material. Eventually the notochordal cells become a distinct median mass which elongates rapidly (i.e., undergoes extension) as the nodal area and the primitive streak recede caudally (Spratt, ’47).

Fig. 205. (See facing page for legend.)

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(k) Somitic mesoderm (i.e., the mesoderm of the future somites) apparently passes inward between the epiblast and hypoblast from the anterolateral portions of the primitive streak. It migrates forward and becomes extended along either side of the notochordal cells during the period of primitive-streak recession. The nephric and lateral plate mesoderm involutes along the middle portions of the streak, and this mesoderm becomes extended antcro-posteriorly. The hypomeric or lateral plate mesoderm also diverges laterally. The extra-embryonic mesoderm moves inward along the postero-lateral portions of the streak; it migrates laterally and anteriorly (fig. 2021, extra-embryonic mesoderm).

Fig. 205. Three-germ-layered blastoderm or late gastrula of chick, showing the mosaic distribution of developmental tendencies. (A-C after Rawles, ’36; D and E after Rudnick, ’44, from various sources.) (A-C) The lines transversely placed across embryo are at levels of 0.3 mm. and 0.7 mm. from the center one, considered as 0.0 mm. (A) Ectodermal or external layer: neural plate area is indicated in black, epidermal area in white. (B) Mesodermal or middle germ layer. (C) Entodermal or inner germ layer. (D) Ectodermal layer shown on left, mesodermal and entodermal on right. (E) Superficial or ectodermal layer shown at left, deeper layer, at right. {Note: These diagrams should be considered only in a suggestive way; final knowledge relative to exact limits of potencies, especially in the mesodermal layer, should be more thoroughly explored.)

Fig. 206. Recession of the primitive streak of the chick and growth of the embryo in front of Hensen’s node. Marked cell groups represented by heavy dots; dashes opposite these are reference marks placed on the plasma clot to permit orientation. This diagram based upon 6 different types of carbon-marking experiments with the generalized results 6-15 hours following explantation. In type I, the stippled area is invaginated as indicated. Observe especially type VI, the history of the three areas marked by the three heavy dots placed on the blastoderm at the head-process stage. It is to be observed that the embryo as a whole arises from the area in front of Hensen’s node. (After Spratt, ’47.)

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(1) While the above activities take place, the area pellucida becomes elongated posteriorly. The entire pellucid area thus becomes piriform, i.e., pear-shaped (figs. 202F-I; 203C).

(m) This change in shape of the pellucid area is associated primarily with the activities involved in epiboly which accompany the embolic activities observed above. Epiboly brings about the elongation of the presumptive neural crescent, converting it into an elongated band of cells. It also effects the expansion and antero-posterior extension of the overlying presumptive, neural plate and epidermal cells. The latter behavior is intimately associated with the antero-posterior extension of the notochordal and mesodermal cellular areas mentioned in (j) and (k) on pp. 427, 428.

(n) Most of the gastrulative processes in the chick are completed at about 20 to 22 hours after incubation starts. At this time the blastoderm is in the head-process stage. The so-called head process or “notochordal process” represents the rudimentary notochord which projects forward from the primitive streak. (See (j) on p. 427.) At this time the various, specific, organ-forming areas appear to be well established (figs. 2021; 205A-E). (See Rawles, ’36; Rudnick, ’44.) From this time on the primitive streak regresses caudally, as the embryo and embryonic tissues develop in front of it. The caudal regression of the streak is shown in figure 206. Spratt (’47) concludes that as the streak regresses, it becomes shortened by transformation of its caudal end into both embryonic and extra-embryonic ectoderm and mesoderm. Finally, the anterior end of the streak, that is, the primitive knot or Hensen’s node together with possibly some condensation of adjacent streak tissue (Rudnick, ’44), forms the end bud. The latter, according to Homdahl (’26) gives origin to the posterior portion of the embryo caudal to somite 27 and to the tail. The remains of the end bud come to a final resting place at the end of the tail.

5. Gastrulation in Mammals a. Orientation

In the mammals, the formative area of the blastocyst (blastula) is located at one pole and is known as the embryonic or germ disc. It consists of a lower hypoblast and an upper epiblast. This embryonic disc is connected to the non-formative or trophoblast cells around its edges (figs. 176, 177, 178). In some species the embryonic disc is superficial and uncovered by trophoblast cells (pig, cat, rabbit, opossum), while in others, it is sequestered beneath a covering of trophoblast (human, monkey, rat). (See figs. 177, 178.)

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b. Gastrulation in the Pig Embryo

In the pig embryo, two centers of activity are concerned with the formation of the primitive streak, namely, a caudal area of mesodermal proliferation which forms the body of the primitive streak and an anterior primitive knot or Hensen’s node. The similarity of behavior of these two portions of the primitive streak in the chick and pig suggests strongly that their formation by a convergence of superficial epiblast cells occurs in the pig as it does in the chick. Hensen’s node, originally described by Hensen (1876) in the rabbit and guinea pig, is a thickened area of the epiblast in the midline near the middle of the embryonic disc. As in the chick, the body of the primitive streak takes its origin at the caudal end of the embryonic disc, where the first appearance of the streak is indicated by a thickening of the epiblast (fig. 209 A, B). From this thickened region, cells are budded off between the epiblast and hypoblast, where they migrate distad as indicated by the lightly stippled areas in figure 208. The streak ultimately elongates, continuing to give origin to cells between the hypoblast and epiblast. Eventually, the anterior neck region of the body of the streak merges with fdensen’s node (fig. 208E, F). From the anterior aspects of the primitive (Hensen’s) node, cells are proliferated off between the epiblast and hypoblast, and a depression or pit, the primitive pit, appears just caudal to the node.

The proliferation of cells from the nodal area deposits a median band of cells which merges anteriorly with the hypoblast below. More caudally, the hypoblast becomes attached to either side of the median band of cells (fig. 209C). The median band of nodal cells thus forms part of two regions, viz., an anterior, pre-chordal plate region, where the nodal cells are merged with hypoblast (entoderm), and an elongated notochordal band or rod of cells extending backward between the hypoblast cells (fig. 209C) to Hensen’s node, where it unites with the hypoblast posteriorly (fig. 209D). Unlike the condition in the chick, the notochordal rod, other than in the pre-chordal plate area, is exposed to the archenteric space below (fig. 209C). It simulates strongly that of the reptilian blastoderm as gastrulation draws to a close.

In the meantime, mesodermal cells from the primitive streak migrate forward between the hypoblast and epiblast along either side of the notochord in the form of two wing-like areas (figs. 208H, I; 209C). Other mesodermal cells migrate posteriad and laterad. Consequently, one is able to distinguish two main groups of mesodermal cells:

( 1 ) formative or embryonic mesoderm, which remains within the confines of the embryonic or germinal disc and

(2) distal ly placed non-formative or extra-embryonic mesoderm.

The former will give origin to the mesoderm of the embryonic body, while from the latter arises the mesoderm of the extra-embryonic tissues.

In conclusion, therefore, we may assume that, during gastrulation in the

Fig. 208. Development of primitive streak, notochord, and mesodermal migration in the pig. (After Streeter, ’27.) (A) Primitive .streak represented as thickened area at

caudal end of embryonic (germ) disc. Migrating mesoderm shown in heavy stipple. (B-E) Later stages of streak development. Observe mass migration of mesoderm. The mesoderm outside the germ disc is extra-embryonic mesoderm. (F) Forward growing, primitive streak makes contact with Hensen’s node. (G-I) Observe elongation of notochord accompanied by recession of primitive streak shown in (I). Observe in (I) that an embryo with three pairs of somites has formed anterior to Hensen’s node. Compare with Spratt’s observation on developing chick, fig. 206, type VI.

434

GASTRULATION IN VARIOUS CHORDATA

435

Fig. 209. Longitudinal and transverse sections of the early embryonic (germ) disc of the pig. (C and D after Streeter, ’27.) (A) Early, pre-streak, germ disc, showing caudal

thickening of epiblast layer. (B) Early streak germ disc, showing thickened caudal edge of disc and beginning migration of mesodermal cells (see fig. 208A). (C) Transverse

section through late gastrula, showing three germ layers. Observe that entoderm is attached to either side of median notochordal rod. (D) Longitudinal section through pre-somite, pig blastoderm, showing the relation of notochord to Hensen’s node, entoderm, and pre-chordal plate.

pig embryo, emboly and epiboly are comparable and quite similar to these activities in the chick.

c. Gastrulation in Other Mammals

Though the origin of notochordal and pre-chordal plate cells in the pig simulates the origin of these cells in the chick, their origin in certain mammals, such as the mole (Heape, 1883) and the human (fig. 207), resembles the condition found in reptiles, particularly in the lizards, where an enlarged notochordal pouch or canal is elaborated by an invaginative process. Consequently, in reptiles, birds, and mammals, two main types of presumptive prechordal plate-notochordal relationships occur as follows:

( 1 ) In one group an enlarged notochordal canal or pouch is formed which pushes anteriad in the midline between the hypoblast and epiblast; and

(2) in others an abortive notochordal canal or primitive pit is developed, and the notochordal cells are invaginated and proliferated from the

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thickened anterior aspect of the pit, that is, from the primitive knot or primitive node (Hensen’s node).

Another peculiarity of the gastrulative procedure is found in the human embryo. In the latter, precocious mesoderm is elaborated during blastulation presumably from the trophoblast. Later this mesoderm becomes aggregated on the inner aspect of the trophoblast layer, where it forms the internal layer of the trophoblast. This precocious mesoderm gives origin to much of the extra-embryonic mesoderm. However, in the majority of mammals, embryonic and extra-embryonic mesoderm arise from the primitive streak as in the chick.

6. Gastrulation in Teleost and Elasmobranch Fishes a. Orientation

Gastrulation in teleost and elasmobranch fishes shows certain similarities, particularly in the fact that in both groups the migrating cells use principally the dorsal-lip area of the blastopore as the gateway from the superficial layer to the deeper region inside and below the superficial layer. The lateral and ventral lips are used to some degree in teleosts, but the main point toward which the migrating cells move is the region of the dorsal lip of the blastopore.

As previously described (Chap. 7), the late blastular condition or blastodisc of elasmobranch and teleost fishes consists of an upper layer of formative tissue, or blastodisc (embryonic disc) and a lower layer of trophoblast or periblast tissue. The latter is associated closely with the yolk (figs. 179A; 180A; 181 A; 210A). In teleost fishes much of the presumptive entodermal, organ-forming area (the so-called primary hypoblast) is represented by cells which lie in the lower region of the caudal portion of the blastodisc (figs. 1 80A; 181 A; 2 IOC). The exact orientation of the hypoblast appears to vary with the species. In Fundulus, a considerable amount of the presumptive entoderm appears on the surface at the caudal margin of the blastodisc (fig. 180A, B). (See Oppenheimer, ’36.) However, in the trout, Salmo, presumptive entoderm lies in the lower areas of the thickened caudal portion of the disc, and the prechordal plate of presumptive entomesoderm alone is exposed (fig. 181A, B). (See Pasteels, ’36.) The position of the presumptive entoderm in the shark, Scyllium (Vandebroek, ’36), resembles that of Fundulus (fig. 170A), although some entoderm may arise by a process of delamination from the lower area of the blastodisc (fig. 179A).

b. Gastrulation in Teleost Fishes

1) Emboly. As the time of gastrulation approaches, the entire outer edge of the blastodisc begins to thicken and, thereby, forms a ring-like area around the edge of the disc, known as the germ ring (figs. 210C; 21 IB). At the caudal edge of the blastoderm, the germ-ring thickening is not only more pronounced, but it also ‘extends inward for some distance toward the center of

GASTRULATION IN VARIOUS CHORDATA

437

the blastoderm (fig. 211 A, B). This posterior prominence of the germ ring forms the embryonic shield.

As gastrulation begins, the entodermal cells of the primary hypoblast at the caudal edge of the embryonic shield stream forward below the epiblast toward the anterior end of the blastodisc (figs. 210A, D). Coincident with this forward movement of the primary hypoblast, a small, crescent-shaped opening

Fig. 210. Gastrulation in teleost fishes. (A) Sagittal section of early gastrula. (Modified slightly from Wilson, 1889.) (B) Midsagittal section through late teleost gastrula.

The dorsal and ventral lips of the blastopore are shown approaching each other. (Modified slightly from Wilson, 1889.) (C) Beginning gastrula of early blastoderm of brook

trout, Salvelinus. Observe inward (forward) migration of primary hypoblast cells and thickened mass of cells which arises at posterior margin. (After Sumner, ’03.) (D)

Later stage in gastrulation of brook trout. (After Sumner, ’03.) (E) Transverse section

of late gastrula of brook trout, showing the three germ layers. (After Sumner, ’03.) (F) Transverse section through late gastrula of sea bass. (After Wilson, 1889.) (G) Midsagittal section through closing blastopore of sea bass. (After Wilson, 1889.) (H)

Longitudinal section through late gastrula of the brook trout. (After Sumner, ’03.)

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appears at the caudal edge of the embryonic shield; this opening forms the dorsal lip of the blastopore (figs. 210A; 211 A, B).

In teleost fishes with a primary hypoblast arranged as in Fundulus (fig. 180A, B), as the entodermal cells of the hypoblast move anteriad from the deeper portions of the blastodisc, the entodermal cells exposed at the caudal edge of the epiblast move over the blastoporal lip (i.e., involute) and migrate forward as a part of the entoderm already present in the deeper layer. (See arrows, fig. 180B.) The primary hypoblast thus becomes converted into the secondary hypoblast. In teleosts with a primary hypoblast or entodermal arrangement similar to Salmo (fig. 181 A), the secondary hypoblast is formed by the forward migration and expansion of the entodermal mass located in the caudal area of the embryonic shield. In both Fundulus and Salmo following the initial forward movement of the entodermal cells, the pre -chordal plate cells together with the notochordal cells move caudally and involute over the dorsal blastoporal lip, passing to the inside. (See arrows, figs. 180B; 18 IB.) The pre -chordal plate and notochordal cells migrate forward along the midline of the forming embryonic axis. The pre-chordal plate cells lie foremost, while the notochordal cells are extended and distributed more posteriorly. The presumptive mesoderm in the meantime converges toward the dorso-lateral lips of the blastopore (figs. 180B; 18 IB, see arrows), where it involutes, passing to the inside between the entoderm or secondary hypoblast and epiblast. Within the forming gastrula, the mesoderm becomes arranged along the upper aspect of the entoderm and on either side of the median, notochordal material (fig. 210E, F). The mesoderm in this way becomes inserted between the flattened entoderm (secondary hypoblast) and the outside ectodermal layer (Oppenheimer, ’36; Pasteels, ’36; Sumner, ’03; Wilson, 1889).

During the early phases of gastrulation, the involuted entodermal, notochordal, and mesodermal tissues may superficially appear as a single, thickened, cellular layer. As gastrulation progresses, however, these three cellular areas separate or delaminate from each other. When this separation occurs, the notochordal cells make their appearance as a distinct median rod of cells, while the mesoderm is present as a sheet of tissue on either side of the notochord. The entoderm may form two sheets or lamellae, one on either side of the notochord and below the mesodermal cellular areas (fig. 21 OF) or it may be present as a continuous sheet below the notochord and mesoderm (fig. 210E, H). The entodermal lamellae, when present, soon grow mediad below the notochord and fuse to form one complete entodermal layer (Wilson, 1889).

2) Epiboly. Emboly involves for the most part the movements of cells in the caudal and caudo-lateral areas of the blastoderm, i.e., the embryonic portion of the germ ring. However, while the involution of cells concerned with the development of the dorsal, axial region of the embryo occurs, the margins of the blastodisc beyond the dorsal-lip area, that is, the extra-embryonic,

GASTRULATION IN VARIOUS CHORDATA

439

germ-ring tissue, together with the presumptive epidermal area, proceeds to expand rapidly. This growth and expansion soon bring about an engulfment of the yolk mass (figs. 210B; 211C-F). The blastoporal-lip area (i.e., edge of germ ring) ultimately fuses at the caudal trunk region (figs. 210G; 21 IF). As the blastoporal region becomes narrower, a small vesicular outpocketing, known as Kupffer’s vesicle, makes its appearance at the ventro-caudal end of the forming embryo at the terminal end of the solid, post-anal gut (fig. 210G) . This vesicle possibly represents a vestige of the enteric portion of the neurenteric canal found in Amphioxus, frog, etc. A certain amount of mesodermal involution occurs around the edges of the germ ring, in some species more than in others (fig. 21 OA, B, peripheral mesodermal involution).

As the cellular dispositions involved in extra-embryonic expansion of the epidermal and germ-ring areas are established, the presumptive, neural plate material (figs. 179, 180, 181) becomes greatly extended antero-posteriorly in the dorsal midline (figs. 210A, H; 21 IE), where it forms into a thickened, elongated ridge or keel. The latter gradually sinks downward toward the underlying notochordal tissue (fig. 210E, F). Also, by the time that the yolk mass is entirely enveloped, the somites appear within the mesoderm near the notochordal axis, and the developing body as a whole may be considerably delimited from the surrounding blastodermic tissue (fig. 21 IG). Therefore, if the envelopment of the yolk mass is taken as the end point of gastrulation in teleosts, the stage at which gastrulation is completed does not correspond to the developmental condition found at the termination of gastrulation in the chick, frog, and other forms. That is, the embryo of the teleost fish at the time of blastoporal closure is in an advanced stage of body formation and corresponds more truly with a chick embryo of about 35 to 40 hours of incubation, whereas the gastrulative processes are relatively complete in the chick at about 20 to 22 hours of incubation.

3) Summary of the Gastrulative Processes in Teleost Fishes:

a) Emboly:

( 1 ) Formation of the secondary hypoblast. The secondary hypoblast forms as a result of the forward migration, expansion, and proliferation of the entodermal cells lying at the caudal margin of the embryonic shield. This forward migration of the entoderm (primary hypoblast) occurs below the upper layer or epiblast and thus produces an underlying entodermal layer or secondary hypoblast.

(2) Pre-chordal plate and notochordal involution. As the formation of the secondary hypoblast is initiated, the presumptive pre-chordal plate and notochordal cells move posteriad and converge toward the dorsal lip of the blastopore, where they involute and pass anteriad in the median line between the hypoblast and epiblast. The hypoblast or entodermal layer may be separated into two flattened layers or lamellae, one on either side of the notochord in some species. However, there

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Fig. 211. Gastrulation in teleost fishes. (A-F after Wilson, 1889; G from Kerr, ’19, after Kopsch.) (A) Sea bass, 16 hours, embryonic shield becoming evident, marks beginning of germ ring. (B) Germ ring well developed. Surface view of blastoderm of 20 hours. (C) Side view of blastoderm shown in (B). (D) Side view, 25 hours.

(E) Surface view, 25 hours. (F) Side view, 31 hours. (G) Late gastrula of trout, Salma fario.

is considerable variation among different species as to the degree of separation of the entodermal layer; in the sea bass it appears to be definitely separated, whereas in the trout it is reduced to a single layer of entodermal cells lying below the notochord. The pre-chordal plate, entoderm, and anterior notochord merge into a uniform mass below the cranial end of the neural plate.

( 3 ) Mesodermal convergence and involution. Along with the migration of notochordal cells, the presumptive mesoderm converges posteriad to the dorso-lateral lips of the blastopore, where it involutes and moves

GASTRULATION IN VARIOUS CHORDATA

441

to the inside on either side of the median, notochordal mass and above the forming, secondary hypoblast.

b) Epiboly. The germ-ring tissue and the outer areas of the presumptive epidermal cells gradually grow around the yolk mass and converge toward the caudal end of the developing embryo. Associated with this migration of cells is the anterior-posterior extension of the presumptive neural plate material to form an elongated, thickened, median ridge.

4) Developmental Potencies of the Germ Ring of Teleost Fishes. The germ ring or thickened, marginal area of the teleost late blastula and early gastrula has interested embryologists for many years. It was observed in Chapter 8 that various regions of the marginal area of the blastoderm of the teleost fish have a tendency to form embryos. Luther (’36), working on the trout (Salmo), found that all sectors of the blastula were able to differentiate all types of tissue, i.e., they proved to be totipotent. However, in the early gastrula, only the sector forming the embryonic shield and the areas immediately adjacent to it were able to express totipotency. As gastrulation progresses, this. limitation becomes more marked. In other words, a generalized potency around the germ ring, present during blastulation, becomes restricted when the embryonic shield of the gastrula comes into prominence. The evidence set forth in the previous chapter indicates that the possibility for twinning in the trout becomes less and less as the gastrular condition nears. The restriction of potency thus becomes a function of a developmental sequence.

In the case of Fundulus, Oppenheimer (’38) found that various areas of the germ ring, taken from regions 90 degrees or 180 degrees away from the dorsal blastoporal lip, were able to differentiate many different embryonic structures if transplanted into the embryonic shield area. Oppenheimer concludes that: “Since under certain conditions the germ-ring can express potencies for the differentiation of many embryonic organs, it is concluded that its normal role is limited to the formation of mesoderm by the inhibiting action of the dorsal lip.” The results obtained by Luther serve to support this conclusion.

c. Gastrulation in Elasmobranch Fishes

In figure I79B the presumptive major organ-forming areas of the blastoderm of the shark, Scyllium canicula, are delineated. The arrows indicate the general directions of cell migration during gastrulation. In figure 212A-G are shown surface views of the dorsal-lip area of different stages of blastodermic development in this species, while figure 213A-G presents median, sagittal sections of these blastoderms during inward migration of the presumptive organ-forming cells. It is to be observed that the dorsal-lip region of the blastoderm is the focal area over which the cells involute and migrate to the inside.

Fig. 212. Surface views of developing blastoderms of Scyllnim canicula.

442

Fig. 213. Sagittal sections of blastoderms shown in figure 212A-G, with corresponding letters, showing migration of presumptive organ-forming areas. (See also fig. 179.) (B)

Dorsal lip is shown to left. (H~M) Transverse sections of embryo of Squalus acanthias, similar to stages shown in 212F and G, for Scyllium. (H) Section through anterior head fold. (M) Section through caudal end of blastoderm. H-M original drawings from prepared slides.

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NOTOCHORD

Fig. 214. Gastrulation in the gymnophionan Amphibia and in the bony ganoid, Amia calva. (A, B, C, after Brauer, 1897; D, E, after Dean, 1896.) Sections A~C through developing embryo of Hypogeophis alternans. (A) Middle gastrula, sagittal section. Observe that gastrocoel forms by a separation of the entodermal cells. Blastococl forms similarly through delamination of entoderm from the overlying epiblast and by spaces which appear between the cells in situ. (B) Transverse section through late gastrula. (C) Sagittal section through late gastrula. (D) Late gastrula of Amia. Mass of yolk in center is uncleaved; cellular organization is progressing peripherally around yolk mass. (E) Later gastrula of Amia. The blastopore is closing, but a large yolk mass still remains uncleaved.

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In figure 21 3A, B, and C, two general areas of entoderm are shown:

(a) that exposed at the surface (cf. fig. 179), and

(b) the entoderm lying in the deeper areas of the blastoderm (cf. fig. 179, cells in black).

According to Vandebroek, ’36, the deeper lying entoderm is extra-embryonic entoderm (in fig. 213, this deeper entoderm is represented as a black area with fine white stipple), whereas the entoderm exposed at the caudal portion of the blastoderm in figure 179A and B, and figure 213A is embryonic entoderm.

The later distribution of the major presumptive organ-forming areas of the shark blastoderm is shown in figure 213E-M. In figure 213, observe the periblast tissue connecting the blastoderm with the yolk substrate.

As the notochordal, cntodermal, and mesodermal cells move inward during emboly, the presumptive epidermal and neural areas become greatly expanded externally by the forces of epiboly as shown in figures 213B-E, and 213H. (Compare the positions of these two areas in fig. 179B.)

The general result of the gastrulative processes in the shark group is to produce a blastoderm with three germ layers similar to that shown in figure 21 3L and M. The notochordal and pre-chordal plate cells occupy the median area below the neural plate as shown in figure 21 3E and F; the mesoderm and entoderm lie on either side of the median notochord as shown in figure 213M. A little later the entoderm from either side of the notochord grows mediad to establish a complete floor of entoderm below the notochord as represented in figure 21 3L.

7. Intermediate Types of Gastrulative Behavior

In certain forms, such as the ganoid fish, Amia, and in the Gymnophiona among the Amphibia, the gastrulative processes present distinct peculiarities. In general, gastrulation in the bony ganoid fish, Amia calva, presents a condition of gastrulation which is intermediate between that which occurs in the teleost fishes and the gastrulative procedures in the frog or the newt. For example, a blastodisc-like cap of cells is found at the end of cleavage in the bony ganoid. This cap gradually creeps downward around the yolk masses which were superficially furrowed during the early cleavages. This process resembles the cellular movement occurring during epiboly in teleost fishes. In addition, the entodermal, notochordal, and mesodermal materials migrate inward in much the same way as occurs in the teleost fishes, although the formation of the primitive archenteron resembles to a degree the early invaginative procedure in the frog. However, a distinctive process of entodermal formation occurs in Amia, for some of the entodermal cells arise as a separation from the upper portion of the yolk substance where yolk nuclei are found. (See fig. 214D, E; consult Eycleshymer and Wilson, ’06.)

The gastrulative processes in the gymnophionan Amphibia are most pe

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culiar, particularly the behavior of the entoderm. But little study has been devoted to the group; as a result, our knowledge is most fragmentary. Elusive and burrowing in their habits and restricted to a tropical climature, they do not present readily available material for study. Brauer, 1897, described blastulation and gastrulation in Hypogeophis alternans. Our information derives mainly from this source.

In some respects gastrulation in Hypogeophis is similar to that in teleost and bony ganoid fishes, while other features resemble certain cellular activities in other Amphibia and possibly also in higher vertebrates. For example, the blastoderm behaves much like the flat blastoderm of teleost fishes, for a dorsal blastoporal lip or embryonic portion of the germ ring is formed toward which the notochordal and mesodermal materials presumably migrate, involute, and thus pass to the inside below the epiblast layer (tig. 21 4A, B). Also, the rapid epiboly of the presumptive epidermal area around the yolk material (or yolk cells) is similar to that of teleost fishes and of the bony ganoid, Amia (fig. 214C-E). However, the behavior of the entodermal cells differs markedly from that of teleosts. In the first place, there is a double delamination whereby the solid blastula is converted into a condition having a blastocoel and a gastrocoel (fig. 214A), These processes occur concurrently with the gastrulative phenomena. Blastocoelic formation resembles somewhat the delaminative behavior of the entoderm in reptiles, birds, and mammals, for the entodermal layer separates from the deeper areas of the epiblast layer. The formation of the gastrocoel (archenteron) is a complex affair and is effected by a process of hollowing or space formation within the entodermal cell mass as indicated in figure 2 1 4A. The arrangement of the entodermal cells during later gastrulative stages resembles the archenteron in the late gastrula of other Amphibia. The archenteron possesses a heavily yolked floor, with the roof of the foregut region complete, but that of the archenteron more posteriorly is incomplete, exposing the notochord to the archenteric space (fig. 214A-C).

G. The Late Gastrula as a Mosaic of Specific, Organ-forming Territories

It was observed above that the presumptive organ-forming areas of the late blastula become distributed in an organized way along the notochordal axis during gastrulation. Further, while an interchangeability of different parts of the epiblast of the late blastula is possible without upsetting normal development, such exchanges are not possible in the late gastrula. For during gastrulation, particular areas of the epiblast become individuated by activities or influences involved with induction or evocation. (The word “evocation” was introduced by Waddington and it has come to mean: “That part of the morphogenetic effect of an organizer which can be referred back to the action of a single chemical substance, the evocator.” See Needham, ’42, p. 42.) As a

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result, the gastrula emerges from the gastrulative process as a general mosaic of self-differentiating entities or territories. (See Spemann, ’38, p. 107.)

It necessarily follows, therefore, that the production of specific areas or territories of cells, each hax^ing a tendency to differentiate into a specific structure, and the axiation of these areas along the primitive axis of the embryo are two of the main functions of the gastrulative process. In figure 205A-E, diagrams are presented relative to the chick embryo showing the results of experiments made by Rawles (’36), Rudnick (’44), and others. (See Rudnick, ’44.) These experiments were made to test the developmental potencies of various limited areas of the chick blastoderm. A considerable overlapping of territories is shown, which stems, probably, from the fact that transplanted pieces often show potencies which are not manifested in the intact embryo. Therefore, these maps should be regarded not with finality but merely as suggesting certain developmental tendencies.

H. Autonomous Theory of Gastrulative Movements

Our knowledge concerning the dynamics of gastrulation in the Chordata is based largely on the classical observations of cell movement made by Conklin (’05) in Styela, the same author (’32) in Arnphioxus, Vogt (’29) in various Amphibia, Oppenheimer (’36) in Fundulus, Pasteels (’36, ’37b) in trout and chick, Vandebroek (’36) in the shark, and Spratt (’46) in the chick. For detailed discussions, concerning the morphodynamics of the gastrulative period, reference may be made to the works published by Roux (1895), Spemann (’38), Pasteels (’40), Waddington (’40), and Schechtman (’42).

The theory popularly held, regarding the movements of the major presumptive organ-forming areas of the late blastula, is that a strict autonomy is present among the various groups of cells concerned with the gastrulative process. Spemann (’38) p. 107, describes this theory of autonomy as follows:

Each part has already previously had impressed upon it in some way or other direction and limitation of movement. The movements are regulated, not in a coarse mechanical manner, through pressure and pull of the single parts, but they are ordered according to a definite plan. . . . After an exact patterned arrangement, they take their course according to independent formative tendencies which originate in the parts themselves.

There are some observations, on the other hand, which point to an interdependence of the various cell groups. For example, we have referred to the observations of Waddington (’33) and Spratt (’46) which suggest that the movements of the mesoderm in the bird embryo are dependent upon the inductive influence of the entoderm. Similarly, Schechtman (’42) points out that presumptive notochordal material does not have the power to invaginate (involute) to the inside when transplanted to the presumptive ectodermal

animal pole animalpole

Fig. 215. Direction of entodermal projection in relation to egg polarity during gastrulation in various Chordata. (A) Amphioxus. (B) Frog. (C) Urodele amphibia. (D) Chick. For diagrammatic purposes, the positions to the right of the median egg axis in the diagrams arbitrarily are considered as clockwise positions, whereas those to the left are regarded as counterclockwise.

Pig. 216. Exogastrulation in the axolotl (Amphibia). (From Huxley and De Beer, *34, after Holtfreter: Biol. Zentralbl., 53: 1933.) (A, B) Mass outward or exogastrular

movements of entoderm and mesoderm, resulting in the separation of these organforming areas from the epidermal, neural areas shown as a sac-like structure in upper part of figure. (C) Section of (B). Exogastrulation of this character results when the embolic movements of gastrulation are directed outward instead of inward. Observe that neural plate does not form in the ectodermal area.

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PRE-CHORDAL PLATE AND CEPHALIC PROJECTION

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area, but it does possess the autonomous power to elongate into a slender column of cells.

1. Exogastrulation

It was demonstrated by Holtfreter (’33) and also by others that embryos may be made to exogastrulate, i.e., the entoderm, notochord, and mesoderm evaginate to the outside instead of undergoing the normal processes involved in emboly (fig. 216). For example, in the axolotl, Ambystoma mexicanurn, if embryos are placed in a 0.35 per cent Ringer’s solution, exogastrulation occurs instead of gastrulation, and the entodermal, mesodermal and notochordal areas of the blastula lie outside and are attached to the hollow ectodermal vesicle. The exogastrulated material, therefore, never underlies the ectodermal cells but comes to lie outside the neural plate and skin ectodermal areas of the gastrula (fig. 216B).

Therefore, the phenomenon of exogastrulation indicates strongly that the presumptive, neural plate and epidermal areas of the late blastula and early gastrula are dependent upon the normal gastrulative process for their future realization in the embryo. Exogastrulation also clearly separates the parts of the forming gastrula which are concerned with emboly from those which are moved by the forces of epiboly. That is, exogastrulation results when the jorces of epiboly are separated from the forces normally concerned with emboly. Normal gastrulation is concerned with a precise and exact correlation of these two sets of forces.

J. Prc-chordal Plate and Cephalic Projection in Various Chordates

It is evident from the descriptions presented in this chapter that the initial invaginative movements in gastrulation begin in the region of the dorsal lip of the blastopore in Amphioxus, fishes, and Amphibia. This initial movement of cells in the region of the dorsal lip consists in the projection forward, toward the future head region of the embryo, of foregut entoderm, pre-chordal plate mesoderm, and notochordal cells. The foregut entoderm, pre-chordal mesoderm, and the anterior extremity of the notochord come to lie beneath the anterior portion of the neural plate. The complex of anterior foregut entoderm and pre-chordal mesoderm lies in front of the anterior limits of the notochord — hence, the name pre-chordal plate. As such it represents, as previously observed, a part of the head organizer (see p. 401 ), the complete organization of the vertebrate head being dependent upon anterior chordal (notochordal), as well as pre-chordal, factors.

In higher vertebrates a different situation prevails during gastrulation. As observed in Chapter 7, the late blastula consists of a lower hypoblast and an upper epiblast in a flattened condition, the hypoblast having separated from the lower parts of the epiblast. The separation of the hypoblast occurs shortly before the gastrulative rearrangement of the major, presumptive, organ

450

GASTRULATION

forming areas begins. The organization of the blastoderm (blastula) is such that presumptive pre -chordal plate mesoderm and notochordal areas lie far anteriorly toward the midcentral part of the epiblast. In other words, a contiguous relationship between presumptive pre-chordal entoderm (i.e., anterior foregut entoderm) and presumptive pre-chordal mesoderm and the presumptive notochord at the caudal margin of the blastula does not exist. Consequently, a different procedure is utilized in bringing the foregut entoderm, pre-chordal mesoderm, and anterior notochord together. That is, the head-organizer materials must be assembled together in one area underneath the cephalic portion of the neural plate. This is accomplished by two methods:

{ 1 ) The use of a large invaginative process, the notochordal canal, which projects pre-chordal plate mesoderm and notochord cranio-ventrad toward the foregut entoderm in the hypoblast below, as described in figure 200 relative to the reptiles or in figure 207B of the human embryo and

(2) the use of another and less dramatic method for getting the headorganizer materials together, the vestigial invaginative process which produces the primitive pit and Hensen’s nodal area.

The latter mechanism succeeds in getting pre-chordal plate mesoderm and notochord down between the epiblast and hypoblast and forward to unite with the anterior part of the foregut entoderm. (See Adelmann, ’22, ’26; Pasteels, ’37b.)

It is not clear whether the invaginative behavior which produces the primitive pit or notochordal canal is an autonomous affair or whether it may be dependent upon the inductive activities of the entoderm below. More experimentation is necessary to decide this matter. The work of Waddington (’33), however, leads one to conjecture that inductive activities may be responsible.

Regardless of the factors involved, cephalogenesis or the genesis of the head is dependent upon the assemblage of anterior foregut, pre-chordal mesoderm, and anterior notochordal cells beneath the cephalic portion of the neural plate as described on page 401.

K. Blastoporal and Primitive-streak Comparisons

From the considerations set forth above, it is clear that the area of the notochordal canal or primitive pit (i.e., Hensen’s nodal area) corresponds to the general region of the dorsal lip of the blastopore of lower vertebrates, whereas the dorso-lateral and lateral lips of the blastopore of lower forms correspond to the body of the primitive streak in higher vertebrates (Adelmann, ’32).

Bibliography

Adelmann, H. B. 1922. The significance of the prechordal plate: an interpretative study. Am. J. Anat. 31:55.

. 1926. The development of the

premandibular head cavities and the relations of the anterior end of the notochord in the chick and robin. J. Morphol. 42:371.

— . 1932. The development of the

prechordal plate and mesoderm of Amhly stoma punctatum. J. Morphol. 54:1.

Braiier, A. 1897. I. Beitrage zur Kenntniss der Entwicklungsgeschichte und der Anatomic der Gymnophionen. Zool. Jahrb. 10:389.

Conklin, E. G. 1905. The organization and cell-lineage of the ascidian egg. J. Acad. Nat. Sc., Philadelphia. 13:5.

. 1932. rhe embryology of Amphi oxus. J. Morphol. 54:69.

. 1933. The development of isolated and partially separated blastomeres of Amphioxiis. J. Exper. Zool. 64:303.

Dalcq, A. and Pasteels, J. 1937. Unc conception nouvclle des bases physiologiques de la morphogenese. Arch, biol., Paris. 48:669.

Dean, B. 1896. 7'he early development of Amia. Quart. J. Micr. Sc. New Series. 38:413.

Eycleshymer, A. C. and Wilson, J. M. 1906. The gastrulation and embryo formation in Amia calva. Am. J. Anat. 5:133.

Haeckel, E. 1874. Anthropogenic oder Entwickelungsgcschichte des Menschen. Vols. 1 and 2 in English translation, 1910, The evolution of man, translated by J. McCabe. G. P. Putnam’s Sons, New York.

Hamburger, V. and Hamilton, H. L. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49.

Heape, W. 1883. The development of the mole (Talpa europea). The formation of the germinal layers and early development of the medullary groove and notochord. Quart. J. Micr. Sc. 23:412.

Hensen, V. 1876. Beobachtungen iiber die Befruchtung und Entwicklung des Kaninchens und Meerschweinchens. Zeit. f. anat. u. Entwicklngesch. 1:213.

Holtfrcter, J. 1933. Die totalc Exogastrulation, eine Selbstablosung des Ektoderms vom Entomesoderm. Entwicklung und funktionelles Verhalten nervenloser Organe. Roux’ Arch. f. Entwick. d. Organ. 129:669.

. 1948. Concepts on the mechanism

of embryonic induction and its relation to parthenogenesis and malignancy. Symposia of the Soc. Exper. Biol. No. II, p. 17. Growth in Relation to Differentiation and Morphogenesis. Academic Press, Inc., New York and Cambridge University Press, England.

Holmdahl, D. E. 1926. Die erste Entwicklung des Korpers bei den Vogeln und Saugetieren, inkl. dem Menschen I-V. Morph. Jahrb. 54:333; 55:112.

Hubrecht, A. A. W. 1906. The gastrulation of the vertebrates. Quart. J. Micr. Sc. 49:403.

Huxley, J. S. and De Beer, G. R. 1934. The Elements of Experimental Embryology. Cambridge University Press, London.

Keibel, F. 1901. Gastrulation und Keimblattbildung der Wirbelthiere. See chap. 10 of Ergebnisse der Anatomic und Entwickelungsgeschichte. Wiesbaden.

Kerr, J. G. 1919. Textbook of Embryology. Vol. 11. Vertebrata with the Exception of the Mammalia. Macmillan & Co., Ltd., London.

Lankester, R. 1875. On the invaginate planula, or diploblastic phase of Paludina vivipara. Quart. J. Micr. Sc. 15:159.

Lewis, W. H. 1907. Transplantation of the lips of the blastopore in Rana palustris. Am. J. Anat. 7:137.

. 1949. Gel layers of cells and eggs

and their role in early development. Lecture Series, Rosco B. Jackson Memorial Laboratories, Bar Harbor, Maine.

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Lopaschov, G. 1935. Die Umgestaltung des prasumtiven Mesoderms in Hirnteile bei Tritonkeimen. Zool. Jahrb. (Abt. f. allg. Zool. u. Physiol.) 54:299.

Luther, W. 1936. Potenzpriifungen an isolierten Teilstiicken der Forellenkeimscheibe. Arch. f. Entwicklngsmech. d. Organ. 135:359.

Mangold, O. 1923. Transplantationsversuche zur Frage der Spezifitiit und der Bildung der Keimblatter. Arch. f. Entwicklngsmech. d. Organ. 100:198.

. 1928. Neue Experimente zur Analyse der fruhen Embryonal entwicklung des Amphibienkeims. Naturwissensch. 16:387.

. 1928. Probleme der Enlwicklungs mechanik. Naturwissensch. 16:661.

. 1932, Autonome und komplemen tare Induktionen bei Amphibien. Naturwissensch. 20:371.

Morgan, T. H. and Hazen, A. P. 1900. The gastrulation of Amphioxus. J. Morphol. 16:569.

Needham, J. 1942, Biochemistry and Morphogenesis. Cambridge University Press, London.

Nicholas. J. S. 1945. Blastulation, its role in pregastrular organization in Amhlystoma punctatum. J. Exper. Zool. 100:265.

Oppenheimer, J. M. 1936. Processes of localization in developing Funduliis. J. Exper. Zool. 73:405.

. 1938. Potencies for differentiation

in the teleostean germ ring. J. Exper. Zool. 79:185.

. 1947, Organization of the teleost

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Pasteels, J. 1936. Etudes sur la gastrulation des vertebres meroblastiques. I. Teleosteens. Arch, biol., Paris. 47:205.

. 1937a. Etudes sur la gastrulation

des vertebres meroblastiques. II. Reptiles. Arch, biol., Paris. 48:105.

. 1937b. Etudes sur la gastrulation

des vertebres meroblastiques. III. Oiseaux. IV. Conclusions generales. Arch, biol., Paris. 48:381.

. 1940. Un apergu comparatif de la

gastrulation chez les chordes. Biol. Rev. 15:59.

. 1945. On the formation of the primary entoderm of the duck (Anas domestica) and on the significance of the bilaminar embryo in birds. Anat. Rec. 93:5.

Rawles, M. E. 1936. A study in the localization of organ-forming areas in the chick blastoderm of the head-process stage. J. Exper. Zool. 72:271.

Roux, W. 1895. Gesammelte Abhandlungen iiber Entwicklungsmechanik der Organismen. II. Engelmann, Leipzig.

Rudnick, D. 1944. Early history and mechanics of the chick blastoderm. Quart. Rev. Biol. 19:187.

Rugh, R. 1951. The Frog, Its Reproduction and Development. The Blakiston Co., Philadelphia.

Schechtman, A. M. 1934. Unipolar ingression in Triturus torosus: a hitherto undescribed movement in the pregastrular stages of a urodele. University of California Publ., Zool. 39:303.

, 1935, Mechanism of ingression in

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. 1942. The mechanism of amphibian gastrulation. I. Gastrulation-promoting interactions between various regions of an anuran egg {Hyla regilla). University of California Publ., Zool. 51:1.

Spemann, H. 1918. fiber die Determination der ersten Organanlagen des Amphibienembryo. I VI. Arch. f. Entwicklngsmech. d. Organ. 43:448.

. 1921. Die Erzeugung tierischer

Chimaren durch heteroplastische embryonale Transplantation zwischcn Triton cristatus und T. taeniatus. Arch. f. Entwicklngsmech. d. Organ. 48:533.

. 1931. fiber den Anteil von Im plantat und Wirtskeim an der Orientierung und Beschaffenheit der induzierten Embryonalanlage. Arch. f. Entwicklngsmech. d. Organ. 123:389.

. 1938. Embryonic Development

and Induction. Yale University Press, New Haven.

and Mangold, H. 1924. Ober In duktion von Embryonalanlagen durch Implantation artfremder Organisatoren. Arch. f. mikr. Anat, 100:599.

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. 1946. Formation of the primitive

streak in the explanted chick blastoderm marked with carbon particles. J. Exper. Zool. 103:259.

. 1947. Regression and shortening

of the primitive streak in the explanted chick blastoderm. J. Exper. Zool. 104:69.

Streeter, G. L. 1927. Development of the mesoblast and notochord in pig embryos. Carnegie Inst., Washington, Publ. No. 380. Contrib. to Embryol. 19:73.

Sumner, F. B. 1903. A study of early fish development. Arch. f. Entwicklngsmech. d. Organ. 17:92.

Vandebroek, G. 1936. Les mouvements morphogenetiques au cours dc la gastrulation chez Scylliiim ccinicula. Arch, biol., Paris. 47:499.

Vintemberger, P. 1936. Sur le developpement compare des micromeres de I’ocuf de Rana fusca divise en huit: (a) Apres isolement. (b) Apres transplantation sur un socle de cellules vitellines. Compt. rend. Soc. de biol. 122:127.

Vogt, W. 1929. Gestaltungsanalyse am Amphibienkeim mit ortlicher Vitalfarbung. 11. Teil: Gastrulation und mesodermbildung bei Urodelen und Anuren. Roux’ Arch. f. Entwick. d. Organ. 120:385.

Waddington, C. H. 1933. Induction by the entoderm in birds. Arch. f. Entwicklngsmech. d. Organ. 128:502.

. 1940. Organizers and genes. Cambridge University Press, London.

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10

Tutulation anJ Extension of tke Major Or^an-forming Areas: Development of Primitive Body Form

A. Introduction

1. Some of the developmental problems faced by the embryo after gastrulation

a. Tabulation

b. Increase in size and antero-posteri(*)r extension of the tubulated, major organforming areas

c. Regional modifications of the tubulated areas

2. Common, vertebrate, embryonic body form

3. Starting point for tabulation

4. Developmental processes which accomplish tabulation

a. Immediate processes

b. Auxiliary processes

5. Blastocoelic space and body-form development

6. Primitive circulatory tubes or blood vessels

7. Extra-embryonic membranes

B. Tabulation of the neural, epidermal, entodermal, and mesodermal, organ-forming areas in the vertebrate group

1. Neuralization or the tabulation of the neural plate area

a. Definition

b. Neuralizative processes in the Vertebrata

1) Thickened keel method

2) Neural fold method

c. Closure of the blastopore in rounded gastrulae, such as that of the frog

d. Anterior and posterior neuropores; neurenteric canal

2. Epidermal tabulation

a. Development of the epidermal tube in Amphibia

b. Tabulation of the epidermal area in flat blastoderms

3. Formation of the primitive gut tube (enteric tabulation)

a. Regions of primitive gut tube or early metenteron

b. Formation of the primitive metenteron in the frog

c. Formation of the tubular metenteron in flat blastoderms

4. Tabulation (coelom formation) and other features involved in the early differentiation of the mesodermal areas

a. Early changes in the mesodermal areas

1) Epimere; formation of the somites

2) Mesomere

3) Hypomere

b. Tabulation of the mesodermal areas

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INTRODUCTION

455

C. Notochordal area

D. Lateral constrictive movements

E. Tubulation of the neural, epidermal, entodermal, and mesodermal, organ-forming areas in Amphioxus

1. Comparison of the problems of tubulation in the embryo of Amphioxus with that of the embryos in the subphylum Vertebrata

a. End-bud growth

b. Position occupied by the notochord and mesoderm at the end of gastrulation

2. Neuralization and the closure of the blastopore

3. Epidermal tubulation

4. Tubulation of the entodermal area

a. Segregation of the entoderm from the chordamesoderm and the formation of the primitive metenteric tube

b. Formation of the mouth, anus, and other specialized structures of the metenteron

5. Tubulation of the mesoderm

6. Later differentiation of the myotomic (dorsal) area of the somite

7. Notochord

F. Early development of the rudiments of vertebrate paired appendages

G. The limb bud as an illustration of the field concept of development in relation to the gastrula and the tubulated embryo

H. Cephalic flexion and general body bending and rotation in vertebrate embryos

I. Influences which play a part in tubulation and organization of body form

J. Basic similarity of body-form development in the vertebrate group of chordate animals

A. Introduction

1. Some of the Developmental Problems Faced by the Embryo After Gastrulation

a. Tubulation

One of the main problems, confronting the embryo immediately following gastrulation, is the tubulation of the major organ-forming areas, namely, epidermal, neural, entodermal, and the two, laterally placed, mesodermal areas. The epidermal, neural, and entodermal areas eventually form elongated, rounded tubes, whereas the mesodermal tubes are flattened. The epidermal and neural tubes extend the entire length of the developing embryo (fig. 217A-C), while the entodermal tube normally terminates at the beginning of the tail (fig. 21 7B, C), although in some instances it may extend even to the tail’s end (fig. 217A). Anteriorly, the entodermal tube ends along the ventral aspect of the developing head (fig. 217A, C). The two mesodermal tabulations are confined mainly to the trunk region of the embryo, but in the early embryo of the shark they continue forward into the head almost to the posterior limits of the developing eyes (fig. 217D). The condition of the mesodermal tubes in the Amphibia resembles to a degree that in the shark embryo (fig. 217B, E).

An important concept to grasp is that the tubulations of the respective areas occur synchronously or nearly so. It is true that the initial stages of the epidermal and entodermal tubulations slightly precede the other tubulations in

Fig. 217. Primary tubes (tubulations) of the primitive vertebrate body. (A) Schematic representation of epidermal, neural, and entodermal tubes in the early embryo of the shark. Observe that a well-developed, post-anal or tail gut continues to the end of the tail. (B) Gut, neural, and epidermal tubes in the amphibian type. (C) Gut, neural, and epidermal tubes in the chick and mammal type. (D) Mesodermal tube in the shark embryo. (E) Mesodermal tube in the amphibian embryo. (F) Mesodermal condition in the early bird and mammal embryo. (G) Transverse section of shark embryo, showing tubulations of major or^an-forming areas and primary coelomic conditions. (H) Transverse section of frog ^embryo shortly after closure of neural tube, showing the five fundamental body tubes oriented around the notochord.

456

Amphioxus, in the frog, and in forms having rounded gastrulae, while in the chick the neural area is precocious. Viewed in their totality, however, the tubulations of all of the major organ-forming areas are simultaneous processes with the exception of the notochord which does not become tubulated but continues as an elongated rod of cells.

b. Increase in Size and Antero-posterior Extension of the Tubulated, Major Organ-forming Areas

Another goal to be achieved by the embryo during the immediate, postgastrular period is an increase in size, together with an antero-posterior extension of the major organ-forming areas. These changes are associated with tubulation, and they aid in producing the elongated, cylindrical form typical of the chordate body,

c. Regional Modifications of the Tubulated Areas

As tubulation of the various major organ-forming areas progresses, specific, organ-forming areas or fields (see end of chapter), located along the respective primitive body tubes, begin to express themselves and develop in a specialized manner. Thus, regional differentiation of the major organ-forming areas; comprising each primitive body tube, is another feature of the postgastrular period. As a result, localized areas along each of the body tubes show changes in shape, and specific, individualized structures begin to make their appearance. For example, the neural tubulation develops the primitive parts of the brain at its anterior end, while the posterior portion of the neural tube, caudal to the brain area, begins to form the spinal cord. Thus, the primitive brain becomes a specific peculiarity of the head region. Also, the epidermal tubulation at its cranial end contributes definite structures peculiar to the head. In the pharyngeal region, special developmental features arise in the entodermal tube together with the epidermal tube and the mesoderm. In the trunk region, modifications of the entodermal and mesodermal tubes give origin to many of the structural conditions peculiar to this area, while in the tail, the neural and epidermal tubulations together with activities of the mesoderm account for the characterstic structures of the tail appendage. These special developmental features of the respective, tubulated, organ-forming areas, which arise in specific areas along the antero-posterior axis of the embryo, occur in much the same way throughout the vertebrate group with the result that common or generalized structural conditions of the tubulated organ-forming areas appear in all vertebrate embryos. That is, the primitive brains of all vertebrate embryos up to a certain stage of development resemble each other in a striking manner; the contributions of the epidermal tubulation to the head also resemble each other, ind the early development of the pharyngeal and trunk regions is similar. As a result, the early morphogenesis

INTRODUCTION

459

organ systems conform to generalized, basic plans. After the generalized plan of a particular system is established, it is modified in later development to fit the requirements of the habitat in which the particular species lives. In the cephalochordate, Amphioxus, a similar body form also develops, although it is considerably modified.

The common, generalized, primitive embryonic body form of all vertebrate embryos possesses the following characteristics:

( 1 ) It is an elongated structure, cylindrical in shape, and somewhat compressed laterally.

(2) It is composed of five, basic, organ-forming tubes, oriented around a primitive axis, the notochord (fig. 217).

(3) It possesses the following regions: (a) head, (b) pharyngeal area, (c) trunk, and (d) tail (figs. 217, 226, 227, 230, 238, 244, 246).

In Chapter 1 1 and the following chapters, various details of these common regions and other features will be considered. In this chapter, we are concerned mainly with tabulation and antero-posterior extension of the major organ-forming areas in relation to body-form development.

3. Starting Point for Tubulation

The starting point for tubulation of the major organ-forming areas and subsequent, primitive, body formation is the gastrula; which, as observed in Chapter 9, exists in two forms, namely, rounded and the flattened gastrulae (figs. 219, 232). Many heavily yolked embryos, such as the embryo of Necturns maculosus, although they form a rounded gastrula, are faced with some of the problems of the flattened gastrulae (fig. 227). The rounded gastrulae, found in the frog, Amphioxus, etc., differ from the flattened gastrulae present in the bird, reptile, mammal, and teleost and elasmobranch fishes, mainly by the fact that, at the beginning of tubulation and body formation, the epidermal and gut areas already are partially tubulated in the rounded gastrulae. That is, in the rounded blastoderm, the initial stages of tubulation occur in these two major organ-forming areas during gastrulation. This means that the ventral portion of the trunk area in rounded gastrulae is circumscribed by intact cellular layers of the embryonic trunk region, with yolk material contained within the cell layers, while, in flattened gastrulae, the ventro-lateral portions of the trunk region are spread out flat, the yolk not being surrounded by the future, ventro-lateral walls of the embryonic trunk region. These conditions are illustrated in figures 219B and C and 234A-F.

The developmental problems faced by these two groups of gastrulae, therefore, are somewhat different. Moreover, tubulation of the organ-forming areas and the development of body form in Amphioxus varies considerably from that of the rounded gastrulae of the vertebrate group. For this reason, tubulation in Amphioxus is considered separately.

460

DEVELOPMENT OF PRIMITIVE BODY FORM

Regardless of differences, however, all vertebrate gastnilae, rounded and flattened, possess three fundamental or basic regions, to wit, ( 1 ) a cephalic or head region, containing the rudiments of the future head and pharyngeal structures, (2) a trunk region, wherein lie the undeveloped fundaments of the trunk, and (3) an end-bud or tail rudiment, containing the possibilities of the future tail.

4. Developmental Processes Which Accomplish Tubulation

a. Immediate Processes

The term, immediate processes, signifies the events which actually produce the hollow tubular condition. In the case of the epidermal, enteric, and neural tubulations, the immediate process is mainly one of folding the particular,

BRAIN AREA

HEAD REGION

SENSORY PLATE

GILL - PLATE AREA

TRUNK REGION

NEURAL FOLD

TAIL REGION

PRE-CHOROAL PLATE

NEURAL ECTODERM EPIDERMAL

ECTODERM

S ODE R M \

NOTOCHORD \ D.

COELOMIC SPACE ENTODERM

NOTO CHORD NEURAL ECTODERM

VENTRAL MESODERM

Fig. 219. Relationships of the major presumptive organ-forming areas at the end of gastrulation in the anuran amphibia. (A) External view of gastrula, showing the ectodermal layer composed of presumptive epidermis (white) and presumptive neural plate (black), as viewed from the dorsal aspect. (B) Diagrammatic median sagittal section of condition shown in (A). (C) Same as (B), showing major organ-forming areas.

(D) Section through middorsal area of conditions (B) and (C), a short distance caudal to foregut and pre-chordal plate region. Observe that the notochord occupies the middorsal area of the gut roof.

INTRODUCTION

461

organ-forming area into a hollow tubular affair. With respect to the mesodermal areas, the immediate process is an internal splitting (de lamination), whereby the mesodermal area separates into an outer and an inner layer with a space or cavity appearing between the two layers. In the case of the teleost fishes, a process of internal separation of cells appears to play a part also in the neural tubulation.

b. Auxiliary Processes

Aiding the above activities which produce tubulation are those procedures which extend the tubulated areas into elongated structures. These auxiliary processes are as follows:

( 1 ) The cephalic or head rudiment, with its contained fundaments of the developing head region, grows forward as a distinct outgrowth. This anterior protrusion is known as the cephalic or head outgrowth (figs. 223A, B; 2321-L).

(2) The trunk rudiments enlarge and the trunk region as a whole undergoes antero-posterior extension (figs. 225 A; 233).

(3) The tail-bud area progresses caudally as the tail outgrowth and forms the various rudimentary structures associated with the tail (figs. 225; 230F; 238).

(4) A dorsal upgrowth (arching) movement occurs, most noticeable in the trunk area. It serves to lift the dorsal or axial portion of the trunk up above the yolk-laden area below, and the developing body tubes and primitive body are projected dorsalward (figs. 221, 224, 241).

(5) In embryos developing from rounded gastrulae, a ventral contraction and reshaping of the entire ventro-lateral areas of the primitive trunk region are effected as the yolk is used up in development. This results in a gradual retraction of this area which eventually brings the ventrolateral region of the trunk into line with the growing head and tail regions (cf. figs. 220, 223, 225 on the development of the frog, and 227 on the development of Necturus).

(6) In embryos developing from flattened gastrulae, a constriction of the ventral region of the developing trunk comes to pass. This constriction is produced by an ingrowth toward the median line of entodermal, mesodermal, and epidermal cellular layers in the form of folds, the lateral body folds. Upon reaching the midline, the cellular layers fuse as follows: The entodermal layer from one side fuses with the entodermal layer of the other; the mesodermal layers fuse similarly; and, finally, the epidermal layer from one side fuses with the epidermal layer of the opposite side. The result is a general fusion of the respective body layers from cither side, as shown in figure 24 1C and D, which establishes the ventral region of the trunk. A complete fusion throughout jLhe extent of the ventral body wall does not take place

462

DEVELOPMENT OF PRIMITIVE BODY FORM

NEURAL PLATE

.A.B. C. D. E.

NERVOUS LAYER OF

/\ EPIDERMIS NOTOCHORD

DORSAL DIVERTICULUM OF

HINDGUT

BRAIN AREA

NEUROCOEL

NEURENTERIC CANAL

DORSAL PROJECTION U ^ OF HINDGUT

CLOSING BLASTOPORE HINDGUT PROCTOOAEUM

ORAL'^

VENTRAL DIVERTlCULUf

DIVERTICULUM OF HINDGUT

, VENTRAL

_ _ DIVERTICULUM

MESENCHYME 7" - OF HINDGUT

LIVER DIVERTICULUM

Fig. 220. Beginning neural fold stage of frog embryo from prepared material. (A) Beginning neural fold stage as seen from dorsal view. (B) Sagittal section near median plane of embryo similar to that shown in (A). (C) Same as (B), showing organ forming areas. (D) Midsagittal section of caudal end of frog embryo slightly younger than that shown in fig. 223 B. Observe that the blastopore practically is closed, while the dorsal diverticulum of the hindgut connects with the neurocoel to form the neurenteric canal. Observe, also, ventral diverticulum of hindgut.

until later in development, and, as a result, a small opening remains, the umbilicus, where the embryonic and extra-embryonic tissues are continuous. This discontinuity of the embryonic layers permits the blood vessels to pass from the embryonic to the extra-embryonic regions. (Note: In the teleost fishes, although a typical, flattened, gastrular form is present, the formation of the ventral body wall of the trunk through a general retraction of tissues resembles that of the rounded gastrulae mentioned above.)

5. Blastocoelic Space and Body-form Development

During the terminal phases of gastrulation in such forms as Amphioxus and the frog, the blastocoel, as a spacious cavity, disappears for the most part. Its general area is occupied by cells which migrated into the blastocoel

INTRODUCTION

463

during gastrulation. However, the disappearance of the blastocoelic space is more apparent than real. For, while most of the original blastocoelic space is thus occupied and obliterated, a part of the original blastocoel does remain as an extremely thin, potential area between the outside ectoderm and the mesoderm-entoderm complex of cells. In flattened blastoderms, as in the chick, the actual space between the ectoderm, mesoderm, and entoderm is considerable (fig. 234E, F). To sum up: Though the blastocoelic space appears to disappear during the terminal phases of gastrulation, a residual or potential space remains between the three germ layers, more pronounced in some species than in others. This residual space gradually increases during the tubulation processes of the major organ-forming areas. In doing so, it permits not only the tubulation of these areas within the outside ectoderm, but it allows important cell migrations to occur between the various body tubes.

6. Primitive Circulatory Tubes or Blood Vessels

Accompanying the tubulations of the epidermal, neural, entodermal, and the two mesodermal areas on either side of the notochord, is the formation

BRAIN AREA OF NEURAL

DORSAL DIVERTICULUM OF HINOGUT

VENTRAL DIVERTICULUM OF HINDGuT

liver diverticulum

CLOSING BLASTOPORE

Fig. 221. Transverse sections through early neural fold embryo of the frog as shown in fig. 220A and B. (A-J) Sections are indicated in fig. 220B by lines A-J, respectively. Observe that the dorsal arching (dorsal upgrowth) movement of the dorsally situated tissues accompanies neural tube formation.

464

DEVELOPMENT OF PRIMITIVE BODY FORM

Fig. 222. Neural crest cells in Amhystoma punctatum. (A and B from Johnston: Nervous System of Vertebrates, Philadelphia, Blakiston, ’06; C-F from Stone: J. Exper. Zool., ’35.) (A) Transverse section of early neural tube of Am by stoma, neural crest

cells located dorsally and darkly shaded. (B) Later stage than (A), showing relation of neural crest cells, epidermis, and neural tube. (C-F) Neural crest cells stippled, placodes of special lateral line sense organs and cranial nerve ganglia shown in black. The neural crest cells arise from dorsal portion of neural tube at points of fusion of neural folds and migrate extensively. A considerable portion of neural crest cells descends upon the mesoderm of visceral arches as indicated in (D-F) and contributes mesodermal cells to these arches, where they later form cartilaginous tissue.

of a delicate system of ve.ssels which function for the transport of the circulatory fluid or blood. The formation of these blood vessels begins below the forming entodermal tube as two, subenteric (subintestinal) tubes or capillaries. These capillaries grow forward below the anterior portion of the forming digestive tube. Near the anterior end of the latter, they separate and pass upward on either side around the gut tube to the dorsal area, where they come together again below the notochord and join to form the rudiments of the dorsal aortae. The latter are two delicate supraenteric capillaries which extend from the forming head area caudally toward the trunk region. In the

TUBULATION OF ORGAN-FORMING AREAS

465

latter region, each rudiment of the dorsal aorta sends a small, vitelline blood vessel laterally into that portion of the gut tube or yolk area containing the yolk or other nutritional source. In the yolk area, each joins a plexus of small capillaries extending over the surface of the yolk substance. These capillaries in turn connect with other capillaries which join ultimately each of the original subintestinal blood capillaries. Below the anterior or foregut portion of the entodermal tube, the two subintestinal blood vessels fuse and thus form the beginnings of the future heart (figs. 234-237; 332). The further development of this system of primitive vessels is described in Chapter 17.

7. Extra-embryonic Membranes

Associated with the development of body form and tubulation of the major, organ-forming areas, is the elaboration of the very important extra-embryonic membranes. As the essential purpose at this time is to gain knowledge of the changes concerned with tubulation of the major organ-forming areas and the development of primitive body form, consideration of these membranes is deferred until Chapter 22. The latter chapter is concerned with various activities relating to the care and nutrition of developing embryos of various vertebrate species.

B. Tubulation of the Neural, Epidermal, Entodermal, and Mesodermal, Organ-forming Areas in the Vertebrate Group

1. Neuralization or the Tubulation of the Neural Plate Area

a. Definition

The separation of the neural plate material from the skin ectoderm, its migration inward, and its formation into a hollow tube, together with the segregation of the accompanying neural crest cells, is called neuralization.

b. Neuralizative Processes in the Vertebrata

Neuralization is effected by two general procedures in the vertebrate subphylum.

1) Thickened Keel Method. In tcleost, ganoid, and cyclostomatous fishes, the neural plate material becomes aggregated in the form of a thickened, elongated ridge or keel along the middorsal axis of the embryo (figs. 21 OF; 218C). This keel separates from, and sinks below, the overlying skin ectoderm (fig. 218A). Eventually the keel of neural cells develops a lumen within its central area and thus gradually becomes transformed into an elongated tube, coincident with the tubulations of the other major organ-forming areas (fig. 218B). In the cyclostomatous fish, Petromyzon planeri, although neuralization closely resembles the condition in teleost fishes, in certain respects the behavior of the neuralizative changes represents an intermediate condition

466

DEVELOPMENT OF PRIMITIVE BODY FORM

Fig. 223. Early neural tube stage of the frog, Rana pipiens, 2Vi to 3 mm. in length.

(A) Dorsal view, (B) Midsagittal section of embryo similar to (A). (C) Same as

(B) , showing organ-forming areas. Abbreviations: V. HD. = ventral hindgut divertic ulum; D. HD, = dorsal hindgut diverticulum; PHAR. ~ pharyngeal diverticulum of foregut. (D) Later view of (A). (E) Sec fig. 224.

between the keel method of the teleost and neural fold method of other vertebrates described below (Selys-Longchamp.s, ’10).

2) Neural Fold Method* In the majority of vertebrates, the neural (medullary) plate area folds inward (i.e., downward) to form a neural groove. This neural groove formation is associated with an upward and median movement of the epidermal layers, attached to the lateral margins of the neural plate, as these margins fold inward to form the neural folds. A change of position in the mesoderm also occurs at this time, for the upper part which forms the somites shijts late rad from the notochordal area to a position between the forming neural tube and the outside epidermis. This mesodermal migration permits the neural tube to invaginate downward to contact the notochordal area. Also, this change in position of the somitic mesoderm is a most important factor in neuralization and neural tube development as mentioned at the end of this chapter. {Note: In this stage of development, the embryo is often de

468

DEVELOPMENT OF PRIMITIVE BODY FORM

RHOMBENCEPHALON MESENCEPHALON PROSENCEPHALON

NEURAL TUBE

EPIPHYSIS

^ HEART RUDIMENT INFUNDIBULUM^^ ORAL EVAGINATION ORAL SUCKER NEURAL TUBE SUBN OTOCHORDAL HEAD GUT

(3VrOCT0DAEUM I'^ANAL OPENING RECTUM

VENTRAL MESODERM NOTOCHORD

FOREGUT’

DIVERTICULUM

Fig. 225. Structure of 3’/2- to 4-mm. embryo of Rana pipiens (about eight pairs of somites are present). (See fig. 226A and B for comparable external views of lateral and ventral aspects of 5-mm., .yy/v6i//ca embryo.) (A) External dorsal view. (B) Midsagittal view. (C) Same, showing major organ-forming areas.

scribed as a neurula, especially in the Amphibia. However, in the bird and the mammal, the embryo during this period is described in terms of the number of somitic pairs present, and this stage in these embryos is referred to as the somite stage.) Each lateral neural fold continues to move dorsad and mesad until it meets the corresponding fold from the other side. When the two neural folds meet, they fuse to form the hollow neural tube and also complete the middorsal area of the epidermal tube (cf. figs. 221, 224, 233, 234, 236, 237, 242, 245A). As a general rule, the two neural folds begin to fuse in the anterior trunk and caudal hindbrain area. The fusion spreads anteriad and posteriad from this point (figs. 223, 229, 233, 235, 242, 245A). It is important to observe that there are two aspects to the middorsal fusion process:

(a) The lateral edges of the neural plate fuse to form the neural tube; and

(b) the epidermal layer from either side fuses to complete the epidermal layer above the newly formed neural tube.

Associated with the fusion phenomena of the epidermis and of the neural tube, neural crest cells are given off or segregated on either side of the neural tube at the point where the neural tube ectoderm separates from the skin

TUBULATION OF ORGAN-FORMING AREAS

469

ectoderm (figs. 221C-E; 23 4B; 236B). The neural crest material forms a longitudinal strip of cells lying along either side of the dorsal portion of the neural tube. As such, it forms the neural or ganglionic crest. In some vertebrate embryos, as in the elasmobranch fish, Torpedo, and in the urodele, Ambystoma, the cells of the neural crest are derived from the middorsal part of the neural tube immediately after the tube has separated from the skin ectoderm (epidermis). (See fig. 222A, B.) In other vertebrates, such as the frog, chick, and human, the neural crest material arises from the general area of junction of neural plate and skin ectoderm as fusion of the neural folds is consummated (fig. 234B).

The neural crest gives origin to ganglionic cells of the dorsal root ganglia of the spinal nerves and the ganglia of cranial or cephalic nerves as described in Chapter 19. Pigment cells also arise from neural crest material and migrate extensively within the body, particularly to the forming derma or skin, peritoneal cavity, etc., as set forth in Chapter 12. A considerable part of the mesoderm of the head and branchial area arises from neural crest material (fig. 222C-F). (See Chapters 11 and 15.)

As the neural plate becomes transformed into the neural tube, it undergoes extension and growth. Anteriorly, it grows forward into the cephalic outgrowth, in the trunk region it elongates coincident with the developing trunk, while posteriorly it increases in length and forms a part of the tail outgrowth.

f. Closure of the Blastopore in Rounded Gastrulae, such as that of

the Frog

Neuralization and the infolding of the neural plate cells begins in the frog and other amphibia before the last vestiges of the entoderm and mesoderm have completed their migration to the inside. As mentioned above, the neural folds begin, and fusion of the neural tube is initiated in the anterior trunk region. From this point, completion of the neural tube continues anteriad and posteriad. As the neural tube proceeds in its development caudally, it reaches ultimately the dorsal lip of the now very small blastopore. As the neural tube sinks inward at the dorsal blastoporal lip, the epidermal attachments to the sides of the infolding neural tube fuse in a fashion similar to the fusion of the edges of the neural tube to complete the dorsal epidermal roof. Associated with this epidermal fusion at the dorsal lip of the blastopore is the fusion of the epidermal edges of the very small blastopore. The extreme caudal end of the archenteron or blastoporal canal in this manner is closed off from the outside (fig. 220D), and the posterior end of the archenteron (the future hindgut area), instead of opening to the outside through the blastoporal canal, now opens into the caudal end of the neural tube. In this way, a canal is formed connecting the caudal end of the future hindgut with the neural tube. This neurenteric union is known as the neurenteric canal.

It is to be observed in connection with the closure of the blastopore and

Fig. 226. External views of embryos of Rana sylvatica and Rana pi pie ns. (A to J after Pollister and Moore: Anat. Rec., 68; K and L after Shumway: Anat. Rec., 78.) (A, B) Lateral and ventral views of 5-mm. stage. Muscular movement is evident at this stage, expressed by simple unilateral flexure; tail is about one-fifth body length. (Pollister and Moore, stage 18.) (C, D) Lateral and ventral views of 6-rnm. stage. Primitive

heart has developed and begins to beat; tail equals one-third length of body. (Pollister and Moore, stage 19.) (E, F) Similar views of 7-mm. stage. Gill circulation is established; hatches; swims; tail equals one-half length of body. (Pollister and Moore, stage 20.) (G, H) Ten-mm. stage, lateral and dorsal views. Gills elongate; tail fin is well developed and circulation is established within; trunk is asymmetrical coincident with posterior bend in the gut tube; cornea of eyes is transparent; epidermis is becoming transparent. (Pollister and Moore, stage 22.) (1, J) Eleven-mm. stage, true tadpole shape. Oper cular fold is beginning to develop and gradually growing back over gills. (K, L) Eleven-mm. stage of R. pipiens embryo. Observe that opercular folds have grown back over external gills and developing limb buds; opercular chamber opens on left side of body only. Indicated in fig. 257B.

470

TUBULATION OF ORGAN-FORMING AREAS

471

the formation of the neurenteric canal that two important changes occur in the future hindgut area of the archenteron at this time, namely, the posterior dorsal end of the archenteron projects dorso-caudally to unite with the neural tube (fig. 220D), while the posterior ventral end of the archenteron moves ventrad toward the epidermis where it meets the epidermal invagination, the proctodaeum (fig. 220D).

d. Anterior and Posterior Neuropores; Neurenteric Canal

The fusion of the neural folds in the middorsal area proceeds anteriad and posteriad from the anterior somitic and hindbrain region as described above. At the anterior end of the forebrain when fusion is still incomplete, an opening from the exterior to the inside of the neural canal is present; it forms the anterior neuropore (figs. 229D; 23 IL; 235B; 242E-G; 245B). When fusion is complete, this opening is obliterated. The caudal end of the neural tube closes m a similar manner, and a posterior neuropore is formed (figs. 242E, G; 245). In the chick, as in the mammal, the posterior neuropore at first is a wide, rhomboidal-shaped trough, known as the rhomboidal sinus. The anterior end of the primitive streak is included within the floor of this sinus rhomboidalis (fig. 235A, B). The point of posterior neuroporal closure is at the base of the future tail in most vertebrates (fig. 245B), but, in the elasmobranch fishes, this closure is effected after the tail rudiments have grown caudally for some distance (fig. 229B-E).

The vertebrate tail arises from a mass of tissue, known variously as the tail bud, caudal bud, or end bud, and the posterior end of the neural tube comes to lie in the end-bud tissues (figs. 225, 238C). The end bud grows caudally and progressively gives origin to the tail. It consists of the following:

(a) the epidermal tube (i.e., the ectodermal covering of the end bud); within this epidermal layer are

(b) the caudal end of the neural tube;

(c) the caudal end of the notochord;

(d) mesoderm in the form of a mass of rather compact mesenchyme surrounding the growing caudal ends of the notochord and neural tube; and

(e) a caudal growth from the primitive intestine or gut.

This extension of the gut tube into the tail is called, variously, the tail gut, caudal gut or post-anal gut. It varies in length and extent of development in embryos of different vertebrate species. In some species it is joined to the neural tube; in others it is not so united. For example, the tail gut is as long as the trunk portion of the gut in the young shark embryo of 8 to 10 mm. in length, and at the caudal extremity it is confluent with the neural tube (figs. 21 7A; 229F). The confluent terminal portions of the neural and gut tubes form the neurenteric canal. This well-developed neurenteric canal extends

TUBULATION OF ORGAN-FORMING AREAS

473

around the caudal end or base of the notochord. In the developing frog on the other hand, the confluence between the neural and gut tubes is present only during the initial stages of tail formation, and it thus represents a transient relationship (fig. 223B, C). Consequently, as the tail bud in the frog embryo grows caudally, the neurenteric connection is obliterated and the tail gut disappears. On the other hand, in the European frog, Bombinator, the condition is intermediate between frog and shark embryos (fig. 228). True neurenteric canals within the developing tail are never formed in the reptile, chick, or mammal, although a tail or post-anal gut, much abbreviated, develops in these forms. (See paragraph below.) In teleost fishes, Kupffer’s vesicle possibly represents a small and transient attempt to form a neurenteric canal (fig. 210G). However, the tail gut here, with the exception of the terminally placed Kupffer’s

Fig. 227. Stages of normal development of Necturus maculosus. (Slightly modified from Eycleshymer and Wilson, aided by C. O. Whitman; Chap. 1 1 in Entwicklungsgeschichte cl. Wirheltiere, by F. Keibel, ’10.) (A) Stage 15, 14 days, 19 hours after

fertilization. Blastopore is circular and reduced; neural groove is indicated in center of figure. (B) Stage 18, 17 days, 2 hours old. Blastopore is an elongated, narrow aperture between caudal ends of neural folds; neural folds prominent and neural groove is deeper. (C) Stage 21, 18 days, 15 hours old, 3 or 4 pairs of somites. Neural folds are widely separated in head region, narrower in trunk, and coalesced in tail area. (D) Stage 22, 20 days, 10 hours, 6 pairs of somites, length about 6 mm. Observe head has three longitudinal ridges, the middle one represents developing brain, while lateral ones are common anlagen of optic vesicles and branchial arches. (E) Stage 23, 21 days, 2 hours, 10 to 12 pairs of somites, 7 mm. long. Head projects forward slightly above egg contour; end of tail is prominent; large optic vesicles protrude laterally from head area; branchial arch region is caudal to optic vesicle enlargement; anus is below tip of tail. (F) Stage 24, 22 days, 17 hours, 16 to 18 pairs of somites, 8 mm. long. Anterior half of head is free from egg contour; optic vesicles and mandibular visceral arch are well defined. (G) Stage 25, 23 days, 10 hours, 20 to 22 pairs of somites, 9 mm. long. Head is free from egg surface; tail outgrowth is becoming free; mandibular, hyoid, first branchial and common rudiment of second and third branchial arches are visible. Otic vesicle lies above hyoid arch and cleft between hyoid and first branchial arches. (H) Stage 26, 24 days, 22 hours, 23 to 24 pairs of somites, length-^10 mm. Head and caudal outgrowths are free from egg surface; heart rudiment is shown as darkened area below branchial arches; cephalic flexure of brain is prominent. (1) Stage 27, 26 days, 26 to 27 myotomes, length — 1 1 mm. Outline of body is straighter; nasal pits and mouth are well defined, mandibular arches are long; heart is prominent below branchial arches; anterior limb buds are indicated; faint outlines of posterior limb buds are evident. (J) Stage 28, 30 days, 8 hours, 30 to 31 myotomes, length — 13 mm. Trunk of embryo is straight, head and tail are depressed; surface of yolk is covered by dense network of capillaries; vitelline veins are prominent; pigment appears below epidermis; anterior limb bud projects dorsally; nuchal or neck flexure is prominent above heart and limb-bud area. (K) Stage 29, 36 days, 16 hours, 36 to 38 myotomes, length — 16 mm. Mandibular arches are forming lower jaw; nuchal and tail flexures are straightening; eye and lens are well defined; anlagen of gill filament are present on gill bars; pigment cells are evident on head areas; vitelline veins are prominent; yolk-laden, ventro-lateral portion of trunk is becoming elongated and contracted toward dorsal region of embryo. (L) Stage 30, 40 days, 20 hours, 44 to 46 myotomes, length — 18 mm. Fore and hind limb buds are prominent; nasal openings are small. (M) Stage 31, larva 49 days, 21 mm. (N) Stage 32, larva 61 days, 25 mm. (O) Stage 33, larva 70 days, 28 mm. (P) Stage 34, larva 97 days, 34 mm. (Q) Stage 35, young adult form, 126 days, 39 mm.

474

DEVELOPMENT OF PRIMITIVE BODY FORM

Fig. 228. Sagittal section, showing organ-forming areas of Bomhinator embryo. (After O. Hertwig: Lehrhuch der Entwicklungsgeschkhte des Menschen und der Wirbeltiere. 1890. Jena, G. Fischer.) Observe elongated tail gut.

vesicle, is a solid mass of cells. Thus, the shark and Bomhinator embryos, on the one hand, and the frog, chick, or mammal embryo, on the other, represent two extremes in the development of the tail gut in the vertebrate group.

In the reptiles, also in some birds, such as the duck, in the human embryo, and certain other mammals, a transient notochordal-neural canal is present which connects the enteron or gut tube with the caudal area of the forming neural tube (figs. 200B, E; 207B; 23 IG-K). This canal is occasionally referred to as a neurenteric canal. However, it is best to view this condition as a special type of development within the above group, for it is not strictly comparable to the neurenteric canal formed in the developing tail of the embryos of the frog, shark, etc., where the neurenteric canal is formed by a definite union between neural and taihgut tubes as they project caudalward into the tail rudiment.

2. Epidermal Tubulation

The formation of the external, epidermal, tubular layer of the vertebrate body is a complex procedure. Its development differs considerably in the rounded type of gastrula of the Amphibia from that in the flattened gastrula of the chick or mammal.

a. Development of the Epidermal Tube in Amphibia

In the frog and other Amphibia, tubulation of the epidermal area of the blastula begins during gastrulation. At the end of gastrulation, the changes involved in epiboly have transformed the ectodermal area of the blastula into an oval-shaped structure, surrounding the internally placed mesoderm and entoderm (fig. 219). The neural plate material occupies the middorsal area of this oval-shaped, ectodermal layer, while the future epidermal area forms the remainder. Following gastrulation, the anterior end of this oval-shaped structure, in harmony with the forming neural tube, begins to elongate and

TUBULATION OF ORGAN-FORMING AREAS

475

grows forward as the head outgrowth (figs. 220, 223, 225). A cylindrical, epidermal covering for the entire head, in this manner, is produced as the cranial or brain portion of the neural plate folds inward (invaginates). A similar outgrowth in the tail area proceeds posteriorly, although here the neural tube grows caudally by proliferative activity within the epidermal tube instead of folding into the epidermal tube as it does in the cephalic outgrowth (figs. 223, 225). Coincident with these two outgrowths, the trunk area, with its ventral, yolk-filled, entodermal cells, elongates antero-posteriorly as the neural plate folds inward. It also grows larger in harmony with the head and tail outgrowths. VAs these activities continue, yolk substance is used up, and

Fig. 229. Early stages of tubulation of neural and epidermal organ-forming areas with resultant body-form development in the shark, Squaliis acanthias (drawn from prepared slides). Neural area shown in black; epidermal area is stippled white; neural folds are outlined in white around edges of black area. (Consult also fig. 230.) (A) Embryonic

area is raised upward; neural plate is flattened; bilateral tail outgrowths are indicated. (B) Embryo is considerably elevated from extra-embryonic blastoderm; brain area is much expanded; trunk region of neural groove is pronounced. (C) Neuralization is considerably advanced; tail rudiments are converging. (D) Neural and epidermal areas are well tubulated; tail rudiments are fusing. (E) Young Squalus embryo, lying on left side; tail rudiments are fused into single caudal outgrowth. The body now consists of a flexed cephalic outgrowth, trunk region, and tail outgrowth. (F) Squalus embryo of about 10 mm. in length.

476

DEVELOPMENT OF PRIMITIVE BODY FORM

the ventro-lateral region of the trunk is retracted. A cylindrical shape of the trunk region thus is established, bringing the trunk area into harmony with the head and tail outgrowths. (Study particularly fig. 227.) The epidermal area of the late gastrula thus becomes converted into an elongated, epidermal tube which forms the external covering or primitive skin (see Chap. 12) for the developing body. In Amphibia, this primitive epidermal tube is two layered, consisting of an outer epidermal ectoderm and an inner neural ectoderm (figs. 221, 224). (See Chap. 12.) In the newly hatched larva, the epidermis is extensively ciliated in all anuran and urodele Amphibia.

b. Tubulation of the Epidermal Area in Flat Blastoderms

In the flat blastoderms of the elasmobranch fish, chick, reptile, and mammal, the formation of the external body tube involves processes more complicated than that of the frog type. The following steps are involved:

( 1 ) A head fold produces a cephalic epidermal extension above the general tissues of the blastoderm. This rudimentary fold of the epidermis contains within it a similar fold of the entodermal layer, together with the invaginating, neural plate material. The notochordal rod lies between the forming entodermal fold and developing neural tube (figs. 213F; 230A; 232I-L; 242B, C). Shortly, the primitive head fold becomes converted into a cylindrical head outgrowth of the epidermal and entodermal layers, associated with the forming neural tube and notochord (figs. 229C, D; 230C; 233). The general process is similar to that in the frog, but it is more complicated in that the head rudiment first must fold or project itself up above the extra-embryonic areas, before initiating the outgrowth process.

(2) A second procedure involved in epidermal tubulation in flattened blastoderms is the dorsal upgrowth movement of epidermal, mesodermal, and entodermal tissues. This activity lifts the trunk region of the embryo up above the general blastodermic tissues (figs. 213H-J; 234B; 241 ). In some forms, such as the chick, the dorsal upgrowth movement is more pronounced in the anterior trunk area at first, gradually extending caudad to the trunk region later (figs. 233, 235). However, in the pig, human, and shark embryos, the dorsal elevation extends along the entire trunk area, coincident with the head outgrowth, and thus quickly lifts the embryonic body as a whole up above the extra-embryonic tissues (figs. 229, 230, 242, 245).

(3) The tail outgrowth, in reptiles, birds, and mammals, begins in a manner similar to that of the head region, and a tail fold first is developed which later becomes a cylindrical projection, bounded externally with epidermal cells, within which are found the notochord, tail mesoderm, and tail portions of neural and gut tubes (figs. 238C; 239K, L; 245B).

B.

X / Gill pouches I ANO II

INFUNOiBULUM-OOk'y PRE-ORAL GUT

PRE-SOMITE MESODERM FIRST GILL CLEFT NOTOCHORD

TBirPMINAL CREST \ FACIAL ACOUSTIC CREST

X. V \ / GLOSSOPHARYNGEAL CREST S U B N 0 T 0 C H 0 R D A L ROD

vaguscrest j

dorsal AORT^^^ ENTERIC

AORTIC ARCH / / I \ "

ORAL PLATE / I HEART VITEL

rHYROlD GLAND VENTRAL AORTAE

P

YOLK STALK ' •

Fig. 230. Sagittal sections of early elasmobranch embryos. (Slightly modified from Scammon. See Chap. 12 in Entwicklungsgesclnchte d. Wirheltiere, by F. Keibel. ) (A)

Graphic reconstruction from sagittal sections of embryo of 2 mm., seen from left side (condition roughly comparable to stage between fig. 229A and B). Observe that neural plate is broad and flattened with slight elevation of neural folds. (B) Reconstruction of embryo of 2.7 mm., viewed from left side, showing mesoderm, forming gut, neural tubes, etc. (Consult (C) below.) (C) Same as (B) with mesoderm removed. Observe primitive gut and neural tubes. Note: (B) and (C) are comparable to stage shown in surface view in fig. 229C. (D, E) Same as (B) and (C), embryo 3.5 mm. in length.

(This embryo is comparable to fig. 229D.) (F) Same as (D) with mesoderm removed,

showing primitive vascular tubes and neural crest cells.

FOURT M CLE AVAGE

EIGHT-CELL STAGE^

'Tight-cell stage following third cleavage,

EGG INTACT Blastoderm removed from egg MARGINAL CELLS CENTRAL CELLS^^.

FIFTH CLEAVAGE

N l) T 0 CmOH D AL CANAL EPIMERIC MESODERM ep'permal Tube head outgrowth

( EC TO DERM 1 /

, FORM I NG â– 

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CLOSING NOTOCHORDAL CANAL ANlLRlOR r.AHDlN

NOTOCHORD

J

epidermal 1 u b e

N E U R A I T U B E^

HEART

VIT^L^LINE

Fici. 231. Series of diagrams, showing stages in the development of the turtle. (A~F) Cleavage stages after Agassiz. (G J ) Stages of gastrulation, drawn from slide preparations. (K T) Stages during development of body fiirm. (P, Q, T from Agassiz; the others are original.) (See L. Agassiz, 1857, Cont. Nat. Hist, of U. S. A., Vol. 11.)

479

Fig. 232. Early post-gastrular development in the chick. (A-H represent a late head-process stage-— stage 5 of Hamburger and Hamilton, ’51. Compare with figure 203D I-L show the beginnings of the head fold — intermediate condition between stages 7 and 8 of Hamburger and Hamilton, ’51.) (A) Surface view, showing primitive streak,

neural plate, and epidermal areas. (B-F) Cross sections of A at levels indicated on G. (G) Median sagittal section of (A). (H) Same, showing presumptive, organ forming areas of entoderm notochord, pre-chordal plate, neural plate, and primitive-streak mesoderm. (1) Surface view, demonstrating a marked antero-posterior extension of the neural plate area and beginnings of neural folds. Observe shortening of primitive streak. (J) Drawing of stained specimen. (K) Median sagittal section of (J). (L) Same,

showing major organ-forming areas. In (G) and (H) the entoderm, notochord, and overlying neural ectoderm are drawn as separate layers. Actually, however, at this stage, the three layers are intimately associated.

480

TUBULATION OF ORGAN-FORMING AREAS

481

Fig. 233, Early body-form development in chick of 3 to 4 pairs of somites. (Approximately comparable to Hamburger and Hamilton, ’51, stage 8, 26 to 29 hours of incubation.) (A) Surface view, unstained specimen. (B) Stained, transparent preparation. Observe blood islands in caudal part of blastoderm. (C) Median sagittal section. (D) Same as (C), showing organ-forming layers.

direction of the notochord is much more pronounced in the flattened blastoderms than in the rounded blastoderms of the frog, salamander, etc. (cf. figs. 224; 237). {Note: Associated with the dorsal invagination of the roof of the midgut in the frog, is the detachment of a median rod of entodermal cells from the middorsal area of the gut. This median rod of cells comes to lie between the notochord and the roof of the midgut. It is known as the subnotochordal rod (fig. 225C). (See Chapter 15.)

The development of the rudimentary hindgut is consummated by caudal

482

DEVELOPMENT OF PRIMITIVE BODY FORM

growth and extension of the posterior or tail region of the primitive archenteron of the late gastrula. These changes result in an extension of the archenteron in the direction of the developing tail and the area ventral to the tail (compare fig. 220B-D with figs. 223B, C; 225B, C).

Three general areas of the primitive gut are thus established:

(a) a tubular enlargement and outgrowth into the developing head, the primitive foregut,

(b) a tubular extension and growth in the caudal region toward the tail, the primitive hindgut, and

(c) a midgut area whose ventral wall is filled with yolk substance, while its roof or dorsal wall assumes a trough-like form extending below the notochord (figs. 223, 224, 225).

The foregut and hindgut areas at this time present the following special features:

( 1 ) Two terminal diverticula or evaginations evolve at the extreme anterior portion of the foregut; and

(2) at the extreme caudal end of the hindgut, similar evaginations occur.

In the foregut region, one of these evaginations projects toward the brain and anterior end of the notochord, while the second diverticulum, more pronounced than the dorsal evagination, moves ventrad toward the epidermis underlying the developing brain. The dorsal evagination represents the preoral or head gut. In the frog it is much abbreviated (figs. 220B, C; 225B, C). On the other hand, the antero-ventrally directed, oral, or pharyngeal, evagination is relatively large and projects toward the ectoderm underlying the brain where it forms the future pharyngeal area of the foregut (figs. 220; 223; 225B, C). Ultimately an invagination from the epidermis, the stomodaeum, becomes intimately associated with the anterior end of the pharyngeal evagination (see Chap. 13). In the hindgut region, the diverticulum which projects dorsally into the tail is the tail gut, whereas the ventral evagination toward the epidermis below the tail represents the future rectal and cloacal areas of the hindgut (figs. 220; 223; 225B, C). It shortly becomes associated with an invagination of the epidermis, the proctodaeum (fig. 223B, C). As previously mentioned, the tail gut may be well developed, as in the European frog, Bombinator (fig. 228), or quite reduced, as in the frog, Rana (fig. 225).

c. Formation of the Tubular Metenteron in Flat Blastoderms

The development of the cylindrical gut tube in those vertebrate embryos which possess flattened gastrulae is an involved, complicated affair. The developmental mechanics are not clearly understood. For example, it is not clear whether the embryonic layers, lying in front of the head fold in figure 23 2G and H, are folded slightly backward in figures 232K and L and still farther

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Fig. 234. Transverse sections of chick embryo with five pairs of somites. (This embryo is slightly older than that shown in fig. 233; a topographical sketch of this developmental stage is shown at the bottom of the figure with level of sections indicated.) Observe that a dorsal arching (dorsal upgrowth) movement of the dorsal tissues is associated with neural tube formation. See A and B.

483

484

DEVELOPMENT OF PRIMITIVE BODY FORM

caudad in figure 23 3C and D by autonomous activities within this tissue, or whether the actively growing head outgrowth proceeds so rapidly that it mechanically causes the area in front of the head fold to rotate backward under the developing foregut and thus contribute to the foregut floor. It is obvious, however, that the entodermal material, lying in front of the head fold of the embryo, is folded backward, at least slightly, and thus becomes a part of the floor of the foregut. The extent, however, varies considerably in different species. It appears to be greater in the mammal (fig. 242C) than in the chick. Another example suggesting the integration of different movements of cellular layers is presented in the formation of the floor of the hindgut of the developing pig embryo. In figure 242C, the rudiments of the fore gut and hindgut areas are established. However, in figure 242G, it is difficult to evaluate how much of the floor of the hindgut in this figure is formed by actual ingrowth forward from point “a” and to what extent the floor is formed by the rapid extension of tissues and backward growth of the caudal region of the embryo as a whole, including the allantoic diverticulum.

Special processes also aid the formation of foregut and hindgut in many instances. For example, in the chick, the floor of the foregut is established in part by a medial or inward growth and fusion of the entodermal folds along the sides of the anterior intestinal portal, as indicated by the arrows in figure 234C. A similar ingrowth of entoderm occurs in the shark embryo (fig. 213J). although here the entoderm grows in as a solid layer from either side and is not present in the form of a lateral fold, as in the chick. However, it should be observed that the formation of the hindgut in the shark embryo arises by a most interesting and extraordinary method. In the flattened gastrulae of reptiles, birds, and mammals, the hindgut is established by the formation of tail folds, involving entodermal and epidermal layers. In the shark embryo, on the other hand, an enteric groove with enteric folds is formed, and the folds eventually move ventrad and fuse to form a hollow tube beneath the notochord of the developing tail.

Though the rudimentary foregut and hindgut areas of the metenteron arise almost simultaneously in mammalian embryos, such as in the pig and human embryos, in the chick a different sequence of procedure is present. In the latter species the foregut begins its development immediately following gastrulation when the first pairs of somites are present (fig. 233). The hindgut, on the other hand, begins its development at a considerably later period when the embryo has attained many pairs of somites (fig. 238).

Once the rudimentary, pouch-like, foregut and hindgut areas have been established in embryos developing from flattened gastrulae, their further development assumes morphogenetic features similar to those in the frog embryo. For example, the foregut possesses an antero-dorsal prolongation toward the brain, the pre-oral or head gut, while slightly posterior to the pre-oral gut, the future pharyngeal area makes contact ventrally with the stomodaeal in

TUBULATION OF ORGAN-FORMING AREAS

485

vagination from the epidermal (ectodermal) tube (fig. 242G). Similarly, the caudal region of the hindgut rudiment contacts the proctodaeal invagination of the epidermal tube, while a tail gut extension continues into the tail (fig. 217).

The formation of definitive walls of the midgut area in embryos developing from the flattened gastrular condition (including the higher mammals which do not possess large amounts of yolk substance) occurs as follows:

(1) Where the entoderm of the midgut terminates on either side of the notochord at the end of gastrulation, it grows mesad from either side

Fig. 235. Chick embryo of 9 to 10 pairs of somites. (Approximating Hamburger and Hamilton, ’51, stage 10; 33 to 38 hours of incubation.) (A) Surface view, unstained. (B) Stained preparation. (C) Median sagittal section. Observe the following: heart is bent slightly to the right; three primary brain vesicles are indicated; foregut touches infundibular outgrowth of prosencephalon; first indication of downward bending of the head outgrowth, i.e., the cephalic (cranial) flexure is evident. (D) Same, showing major organ-forming areas.

486

DEVELOPMENT OF PRIMITIVE BODY FORM

Fig. 236. Transverse sections through chick embryo of about 12 to 13 pairs of somites, about 38 hours of incubation. (Approximately between stages 10 to 11 of Hamburger and Hamilton, ’51, slightly older than that shown in fig. 235.) Observe that the optic vesicles are constricting at their bases; heart is bent slightly to the right; anterior neuropore is evident. (A) Optic vesicles. (B) Stomodaeal area. (C) Anterior end of developing heart. (D) Caudal extremity of forming heart. (E) Anterior intestinal portal and forming caudal portion of the heart. (F) Well-developed somites. (G) Open neural groove.

below the notochord to complete the roof of the midgut (figs. 201 D; 209C; 21 OF; 213). This process is similar to that which occurs in the Amphibia (cf. fig. 219D).

(2) A dorsal arching or evagination of the entoderm toward the notochordal area, comparable to that found in the frog and other Amphibia, is present also. A study of figures 213H-J; 217G; 23 4B; 237E-G; 241B-D demonstrates the marked dorsal upgrowth of all the forming body layers in the trunk area. {Note: In the elasmobranch fishes, a subnotochordah rod of cells of entodermal origin is formed similar to that in the frog and other Amphibia.)

( 3 ) The ventro-lateral walls of the midgut area, in contrast to those found in the frog, are established largely by actual ingrowth of the entoderm, mesoderm, and ectoderm with subsequent fusion in the median line

Fig. 237. Chick embryo of 17 to 19 pairs of somites. (Approximating Hamburger and Hamilton, ’51, stage 13, 48 to 52 hours of incubation, sections indicated on outline drawing.) Head lies partly on left side; auditory pits are deep; cervical flexure is evident in region of rhombencephalon; cephalic flexure is marked; stomodaeum is a deep indentation touching foregut between the first pair of aortal arches; head fold of amnion reaches back to anterior part of rhombencephalon (hindbrain). (A) Anterior (telencephalic) portion of prosencephalon, showing closed neuropore; amnion is indicated. (B) Optic vesicles. (C) Anterior end of foregut, showing anterior extremity of stomodaeal invagination and first (mandibular) pair of aortal arches; notochord ends and pre -chordal plate area begins at about this section. (D) Anterior end of heart (ventral aorta); observe thin roof plate of neural tube, characteristic of the later myelencephalic (medulla) portion of rhombencephalon or hindbrain. (E) Otic (auditory) pits and anterior region of ventricular portion of heart. (F) Caudal limits of forming heart, dorsal mesocardium, neural crest cells. (G) Caudal end of heart, showing converging (vitelline) veins of the heart, sclerotome given off to notochordal area, lateral mesocardium forming. (H) Anterior trunk area, showing differentiation of somite and typically flattened condition of ectoderm, mesoderm, and entoderm. (I) Caudal trunk area, showing undifferentiated somite (epimeric mesoderm), intermediate mesoderm (mesomere), and lateral plate mesoderm (hypomere). (J) Similar to (I). (K) Caudal

trunk region, showing closing neural tube. (L) Area of Hensen’s node. (M) Primitive streak.

487

488

DEVELOPMENT OF PRIMITIVE BODY FORM

in elasmobranch fishes, reptiles, birds, and mammals. This process involves the formation of lateral body folds which fold mesially toward the median plane. (Study fig. 241 A-D.) In teleost fishes the process is different, for in this group the entoderm and mesoderm grow outward beneath the primitive epidermis (ectoderm) and soon envelop the yolk. Thus, the end result in teleosts is much the same as in the frog and Necturus, It is well to observe, at this point, that a complete retraction of the ventro-lateral walls of the midgut and body-wall tissues surrounding the yolk or yolk-sac area, as in the frog and Necturus (fig. 227), does not occur in the higher vertebrates, although in the elasmobranch and teleost fishes such retraction does occur.

Fig. 238. Chick embryo of about 27 to 28 pairs of somites. (Corresponding approximately to Hamburger and Hamilton, ’51, stage 16, 51 to 56 hours of incubation.) Forebrain (prosencephalon) is divided into telencephalon and diencephalon; epiphysis is appearing on roof of diencephalon; cephalic and cervical flexures are pronounced; tail bud is short; anterior part of body is rotated to the left back to about the thirteenth pair of somites; amnion now covers anterior three fifths of body; heart shows strong ventricular loop; three pairs of aortal arches can be seen. (A) External view. (B) Transparent wholemount." (C) Sagittal section, diagrammatic.

TUBULATION OF ORGAN-FORMING AREAS

489

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is shown on diagram.

(See Chap. 22.) In the elasmobranch fishes, this retraction of tissues contributes little to the formation of the wall of the enteron or to that of the body. However, in teleosts such contribution is considerable.

At this point reference should be made to figures 23 8C on the chick, 242C and G on the pig, and 245 B on the early human embryo to gain a visual image of the developing foregut, midgut, and hindgut areas of the primitive metenteron. Compare with the frog (fig. 225C).

490

DEVELOPMENT OF PRIMITIVE BODY FORM

4. Tubulation (Coelom Formation) and Other Features Involved in the Early Differentiation of the Mesodermal Areas

The differentiation of the mesodermal areas is an all-important feature of embryonic development, for the mesoderm contributes much to the substance of the developing body. (See Chaps. 11 and 15.) While the neural, enteric, and epidermal tubes are being established, radical changes occur within the two mesodermal layers on either side of the notochord as follows:

a. Early Changes in the Mesodermal Areas

1) Epimere; Formation of the Somites. The longitudinal mass of paraxial mesoderm which lies along the side of the notochord forms the epimere (figs. 221F, G; 234E, F). The two epimeres, one on either side of the notochord, represent the future somitic mesoderm of the trunk area. In the early post-gastrula, the epimeric mesoderm, together with the notochord, lies immediately below the neural plate. However, as neuralization is effected^ the

Fig. 240. Chick embryo of about 72 to 75 hours of incubation, about stage 20 of Hamburger and Hamilton, ’51.

492

DEVELOPMENT OF PRIMITIVE BODY FORM

area of the anterior trunk and posterior hindbrain region of the embryo. In the chick embryo (see Patterson, ’07), the most anterior segment forms first, and later segmentation progresses in a caudal direction. This probably holds true for most other vertebrates. However, in elasmobranch fishes, segmentation of the epimeric mesoderm also extends forward from the hindbrain area into the head region presenting a continuous series of somites from the eye region caudally into the tail (fig. 217D). (Study figs. 217D, 230D.) Segmentation of epimeric mesoderm appears in the head region of Amphibia. In many higher vertebrates, three pairs of somitic condensations appear in the area just caudal to the eye but at a slightly later period of development than that of the elasmobranch fishes (fig. 217D-F).

2 ) Mesomere. The narrow longitudinal band of mesoderm, adjoining the lateral border of the epimere, is the mesomere (figs. 22 IF, G; 230D; 234E, F) . This mesoderm ultimately gives origin to much of the excretory (kidney) tissue and ducts and to certain of the reproductive ducts of many vertebrates. (See Chap. 18.) Because of the origin of nephric tissue from its substance, this longitudinal band of mesoderm generally is referred to as the urogenital or nephrotomic mesoderm. Synonymous terms often used are intermediate mesoderm or intermediate cell mass. The mesomere undergoes a segmentation similar to the epimeric area in its more anterior portion where the pronephric kidney develops in higher vertebrates, while in lower vertebrates, such as the shark embryo, it may be more extensively segmented.

3) Hypomere. The remainder of the mesoderm which extends lateroventrally from the mesomere forms the hypomere or hypomeric mesoderm. It also is called the lateral plate mesoderm or lateral plate mesoblast. This portion of the mesoderm does not become segmented in present-day vertebrates. (Compare with the condition in Amphioxus described on p. 505.)

b. Tabulation of the Mesodermal Areas

Coincident with the formation of the somites, a cavity begins to appear within the mesoderm. This cavity or primitive coelomic space separates the mesoderm into two layers, an outer layer near the ectoderm and an inner layer close to the neural, notochordal, and entodermal cells. This hollowing process within the mesodermal layer is known as coelom formation or tubulation of the mesoderm. In many embryos of the lower vertebrates, there is a strong tendency for the coelomic space to form throughout the entire lateral mass of mesoderm from the epimeric area ventrad into the lateral plate mesoderm. For example, in elasmobranch (shark) embryos of about 3 to 4 mm. in length and also in many early post-gastrular amphibia, the following features of the primitive coelom are found in the trunk region of each mesodermal mass:

(1) The mesoderm possesses a cavity, continuous dorso^ventrally from the epimere into the lateral plate (figs. 217G, H; 221 E). When the epimere (and to some extent the nephrotomic region as well) under

LATERAL CONSTRICTIVE MOVEMENTS

493

goes segmentation, the coelomic space within these areas becomes segregated within the segments and, thus, is present in a discontinuous condition.

(2) The early coelomic cavity in the shark and amphibian embryo, therefore, may be divided into three parts: (a) the myocoelic portion within the epimeric mesoderm, (b) the nephrocoel within the nephrotomic mesoderm, and (c) the splanchnocoel contained within the hypomeric or lateral plate mesoderm. While the myocoelic and nephrocoelic regions of the primitive coelom may become segmented and discontinuous, that within the splanchnocoel is continuous antero-posteriorly in the trunk region.

The coelomic cavities contained within the somites of the shark and amphibian embryo are soon lost. The coelomic cavity or nephrocoel within the nephrotome is concerned with the development of the lumen within the tubules and ducts of the excretory (urinary) system, while the splanchnocoels give origin to the coelomic cavity proper of the adult. The lateral wall of the splanchnocoel near the primitive epidermis is known as the somatopleural mesoderm, and the inner or medial wall associated with the gut tube and developing heart tissues constitutes the splanchnopleural layer. The epidermis and somatopleural mesoderm together form the somatopleure, while the entoderm and splanchnopleural mesoderm form the splanchnopleure.

In the embryos of higher vertebrates, the coelomic space of the somitic portion of the primitive coelom (i.e., the myocoels) is less pronounced and appears somewhat later in development than in the shark and amphibian embryo, but it does tend to appear. This is true also of the nephrocoel or coelomic cavity within the nephrotome. (See Chap. 18.) The coelomic condition or splanchnocoel within the hypomere forms similarly in all vertebrates. These matters will be described more in detail in Chapter 20.

C. Notochordal Area

The notochord is the elongated, median band of cells of the gastrula which lies between the two mesodermal areas. The notochord thus may be regarded as a specialized, median portion of the middle germ layer of mesodermal tissue. During gastrulation and shortly after, there may be a tendency for the notochordal material in certain forms to canalize or tubulate. Later, the notochordal material becomes converted into a definite rod of notochordal cells which represents the primitive skeletal axis of the embryo. The notochord and its relation to the early skeletal system are discussed in Chapter 15.

D. Lateral Constrictive Movements

While the neural, epidermal, and entodermal tabulations are in progress, a lateral constriction or invagination of the body wall occurs on either side in all vertebrate embryos from the fishes to the mammals. These constrictions

494

DEVELOPMENT OF PRIMITIVE BODY FORM

are effected at the level of the notochord and lower margin of the somitic area from the anterior trunk region caudally into the tail. As a result, a transverse section of the early vertebrate body appears pyriform or pear shaped, with the neck of the pear directed dorsally (fig. 241 C). The constriction line is shown typically in the developing embryo of Necturus (fig. 227 ) where it extends from the lower aspect of the head outgrowth along the lower boundary of the somitic area to the base of the tail. A line, drawn across the body from the general area of the two lateral constrictions and passing through the notochord, divides the embryonic body into an upper or epaxial (epiaxial) region above the level of the notochord and a lower or hypaxial (hypoaxial) region below the level of the notochord.

£. Tubulation of the Neural, Epidermal, Entodermal, and Mesodermal, Organ-forming Areas in Amphioxus

1. Comparison of the Problems of Tubulation in the Embryo OF Amphioxus with that of the Embryos in the Sub PHYLUM Vertebrata

a. End-bud Growth

In Amphioxus, the procedures involved in tubulation of the major organforming areas and development of primitive body form differ from those in the vertebrate group. For example, in the latter group, the basic rudiments of the head, pharyngeal, trunk, and tail regions appear to be well established at the end of gastrulation. During tubulation of the major organ-forming areas, these subregions become extended in an antero-posterior direction and the rudiments of specific structures begin to express themselves. This is especially true of the head, pharyngeal, and trunk regions. The vertebrate tail, however, arises from an end-bud tissue which progressively lays down the various parts of the tail by means of a proliferative growth in the caudal direction. On the other hand, in Amphioxus, only a small portion of the anterior end of the future body is laid down during gastrulation. Further development of the epidermal, neural, enteric, and mesodermal cellular areas together with the notochord are dependent upon cell proliferation at the caudal end of the late gastrula and later embryo. Much of the body of Amphioxus, therefore, is formed by a caudal proliferative growth of end-bud cells, somewhat comparable to the end-bud growth of the tail in the vertebrate group.

b. Position Occupied by the Notochord and Mesoderm at the End of Gastrulation

A second feature of difference in the developing embryo of Amphioxus from that of the vertebrate embryo lies in the arrangement of the notochordmesoderm complex of cells in the late gastrula. In the late gastrula of

TUBULATION OF ORGAN-FORMING AREAS IN AMPHIOXUS

495

Amphioxus, this potential, third germ layer forms a part of the entodermal roof, although the studies of Conklin (’32) have demonstrated that notochord and mesoderm are distinct cellular entities even in the blastula. In contrast to this condition, the notochord and the mesoderm already are segregated as a middle germ layer between the ectoderm and the entoderm in the late vertebrate gastrula. The gastrula of Amphioxus, therefore, has the added problem of segregating the notochordal and mesodermal cells from the entoderm during tubulation of the major organ-forming areas.

2. Neuralization and the Closure of the Blastopore

In the late gastrula of Amphioxus, a longitudinal middorsal plate of cells, the neural plate, elaborated by cell division and extension during gastrulation, represents the future central nervous system (fig. 247E). As the period of gastrulation comes to its end, the blastopore decreases greatly in size (fig. 247A-D). The archenteric opening also moves dorsally, coincident with a shifting of the caudal end of the archenteron in such a way that it projects in a dorso-caudal direction (figs. 189G, H; 247H). This movement of the archenteron is associated with the migration of the mass of mesodermal cells from the two lateral areas of the blastoporal lips (fig. 247A, B) to the dorsomedial portion of the blastopore (fig. 247C), where the mesoderm comes to lie on either side of the notochord below the neural plate (fig. 247C). As these changes occur, the dorsal area of the gastrula near the blastopore becomes flattened with a subsequent depression of the neural plate (fig. 247C, D). In sagittal section, the gastrula now appears oval in shape and considerably elongated in the antero-posterior direction (fig. 189G, H); in transverse view, it is triangular, especially at the caudal end (fig. 247D).

As the above changes are brought about, the ectoderm of the ventral lip of the blastopore grows dorsad, while that of the lateral lips grows mediad. In this way, the opening of the blastopore is closed by the coming together and fusion of these ectodermal (epidermal) growths (fig. 247D-F). However, the archenteron does not lose its connection, at this time, with the outside environment of the embryo for two reasons:

( 1 ) As observed above, the caudal end of the archenteron previously had shifted in such a manner that it now projects dorso-caudally; and

(2) synchronized with the epidermal growth which closes the blastoporal opening (fig. 248A), the neural plate sinks downward, becoming detached along its margin from the epidermal area (fig. 248B-D).

The downward sinking of the neural plate and its detachment from the epidermal layer begins at the dorsal lip of the blastopore and spreads anteriad. (Compare fig. 248D with 248B and C.) Consequently, as the epidermal growth along the lateral lips of the blastopore reaches the area of the sinking neural

Fig. 242. Early development of the pig embryo (B, C, and G from Patten: Embryology of the Pig, Philadelphia, Blakiston; A is from Streeter: Carnegie Inst. Publ. No. 380; Contrib. to Embryol. 100; D, E, and F from Heuser and Streeter: Carnegie Inst. Publ. No. 394, Contrib. to Embryol. 109. All figures have been modified), (A) Early, neural groove stage. Neural area is shown in black; amnion is cut away as indicated. (B) Four-somite stage. (C) Median sagittal section, approximating the stage of development shown in (B). Observe foregut, midgut, and hindgut areas. (D) Embryo of about six pairs of somites. (E) Embryo of about 7 to 8 pairs of somites. (F) Eighteen pairs of somites. (G) Sagittal .sectional diagram of embryo slightly younger than (F), showing neural and gut tubes, amnion, allantois, and forming heart.

496

TUBULATION OF ORGAN-FORMING AREAS IN AMPHIOXUS

497

plate in the region of the dorsal blastoporal lip, it continues forward along the epidermal margins of the insinking neural plate, growing mesad and fusing in the midline over the neural plate (fig. 247E-G). In this way, the epidermal growth forms a covering for the neural plate. It follows, therefore, that the posterior end of the archenteron will now open into the space between the neural plate and its epidermal covering. This new passageway between the epidermalneural plate cavity and the archenteron is the beginning of the neurenteric canal (figs. 247H; 248A).

The flattened neural plate, canopied by the epidermal overgrowth, then begins to fold itself into the form of a tube. In doing so, its lateral edges swing gradually toward the middorsal line, as shown in figure 195. The actual grooving and tubulation of the neural plate starts at a point about midway along the embryo at the stage of development shown in figure 247F. It proceeds anteriorly and posteriorly from this point. At its extreme anterior end, the neural tube remains open to the surface as the anterior neuropore (figs. 247H; 249A-D). Eventually the caudal end of the neural plate becomes tubulated, and a definite canal is formed, connecting neural and enteric tubes. This canal is the neurenteric canal. The neurenteric canal disappears between the stage of development shown in figure 249C and that shown in figure 249D. The continued caudal growth of the neural tube is accomplished by cell proliferation from the posterior end of the tube and neurenteric canal area.

Fig. 243. Sections of pig embryo of about stage shown in fig. 242 (B) and (C). (Modified from Patten: Embryology of the Pig, 3d Ed., Philadelphia, Blakiston, ’48.) (A) Line 1, fig. 242C. (B) Line 2, fig. 242C. (C) Line 3, fig. 242C. (D) Line 4,

fig. 242C.

498

DEVELOPMENT OF PRIMITIVE BODY FORM

MAXILLARY PROCESS

ANDI BULAR ARCH

HYOID ARCH

Fig. 244. Development of body form in the pig embryo. (A and B from Keibel: Normentafel zur Entwicklungsgeschichte des Schweines (Sus scrofa domesticus). 1897. Jena, G. Fischer. C, D, and E slightly modified from Keibel, previous reference, and from Minot: A Laboratory Text-book of Embryology. 1903. Philadelphia, P. Blakiston’s Son & Co.) (A) About 4 to 5 mm. (B) About 6 mm. (C) Ten mm. (crown-rump measurement). (D) Fifteen mm. (E) Twenty mm.

3. Epidermal Tubulation

After the neural plate sinks downward and becomes separated from the outside epidermis, the medial growth of the epidermis over the neural plate completes the middorsal area of the primitive epidermal tube (fig. 247 E-H). It then comes to enclose the entire complex of growing and elongating neural,

Fig. 245. Human embryo of ten somites. (After G. W. Corner: Contrib. to Embryol Carnegie Inst., Washington, Publ. No. 394, 112.) (A) Dorsal view. (B) Median

sagittal section of model.

500

DEVELOPMENT OF PRIMITIVE BODY FORM

Fig. 246. Development of body form in human embryo. (C from Keibel and Mall: Manual of Human Embryology, Vol. I, 1910. Philadelphia and London, Lippincott. A, B, D, and E from Keibel and Elze: Normentafel zur Entwicklungsgeschichte des Menschen. Jena, 1908. G. Fischer.) (A) Early neural fold stage. Somites are beginning to form; notochordal canal is evident. (B) About nine pairs of somites. (C) His’s embryo M. (D) About 23 pairs of somites, 4-5 mm. long. (E) About 35 pairs of trunk somites, 12 mm. long.

mesodermal, and entodermal tubes and with them it continues to grow in length principally by rapid cell proliferation at the caudal end of the embryo.

4, Tubulation of the Entodermal Area

The primitive metenteron of Amphioxus is derived from the archenteron of the late gastrula as follows.

a. Segregation of the Entoderm from the Chordamesoderm and the Formation of the Primitive Metenteric Tube

The mesoderm and notochord which occupy the roof of the archenteron of the gastrula evaginate dorsally at the anterior end of the embryo and, thus, become separated from the entoderm. (Compare fig. 195 with fig. 250A.) This separation of notochord and mesoderm by dorsal evagination from the

TUBULATION OF ORGAN-FORMING AREAS IN AMPHIOXUS

501

entoderm continues slowly in a caudal direction from the anterior end until an embryonic condition is reached approximating about 13 to 14 pairs of mesodermal segments. At this level, the notochord and mesoderm become completely separated from the entoderm. As a result, the enteric or gut tube from this point in its growth posteriad is a separate entity. (See tabulation of mesoderm on p. 505. Anterior to the fourteenth somite, after the notochord and mesoderm separate from the entoderm, the latter grows medially from either side to complete the entodermal roof below the evaginated notochord and mesoderm (fig. 250A). A primitive metenteric tube thus is formed, as shown in figure 249C, whose only opening is that which leads by way of the neurenteric canal (fig. 249 A, C) into the neurocoel of the neural tube and from thence to the outside through the anterior neuropore.

NEURAL PLATE CELLS

Fig. 247. Closure of the blastopore and epidermal overgrowth of neural plate in Amphioxus (original diagrams, based on data supplied by Conklin, ’32). (A) Vegetal

pole view of early stage of gastrulation, showing general areas occupied by notochordal, entodermal, and mesodermal cells. (B) Same view of gastrula, one hour later, showing triangular form of blastopore, (C) Posterior view of late gastrula. Blastopore is now ovoid in shape and dorsally placed. Gastrula is triangular in transverse section with dorsal surface flattened. (D) Same view, later. Slight epidermal upgrowth, indicated by arrows (a and a') merges with ingrowing epidermal edges along lateral lips of blastopore (b and b') which spreads along epidermal edges of neural plate. (E) Dorsal view a brief period later than (D). Epidermal ingrowth from lateral blastoporal lips is now closing the blastoporal opening, shown in broken lines, and also is proceeding craniad along edges of sinking neural plate. (See fig. 248.) (F, G) Later stages of

epidermal overgrowth of neural plate. (H) Sagittal section of (G).

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Fig. 248. Sinking of neural plate and epidermal overgrowth of neural plate in Amphioxus. (Slightly modified from Conklin, ’32.) (A) Sagittal section of embryo comparable

to that shown in fig. 247F. (B, C, D) Sections through embryo as shown by lines B,

C, D, respectively, on (A). Observe that the neural plate begins to sink downward from region of closed blastopore and proceeds forward from this point.

b. Formation of the Mouth, Anus, and Other Specialized Structures of the Metenteron

At the anterior end of the metenteron, a broad, dorsal outgrowth occurs which continues up on either side of the notochord and becomes divided into right and left dorsal diverticula (fig. 249B, H). The left diverticulum remains small and thick-walled and later fuses with an ectodermal invagination to form the pre-oral pit, described as a sense organ. The right diverticulum, however, increases greatly in size, becomes thin-walled, and gives origin to the so-called head cavity.

The mouth develops at a time when the larva acquires about 16 to 18 pairs of mesodermal segments or somites (fig. 249D). It appears when the overlying epidermis about halfway up on the left side of the body fuses with the entoderm, a fusion which occurs just posterior to the forming pre-oral pit (left diverticulum). (See black oval fig. 249D, and fig. 249F.)

At the time that the mouth forms, the entoderm opposite the first pair of somites pushes ventrally and fuses with the ectoderm. This area of fusion finally perforates and forms the first gill slit. The gill slit, once formed, moves up on the right side of the body (fig. 249E). The entodermal area from which the first and later gill slits make their appearance is known as the branchial rudiment (fig. 249D).

At the caudal end of the larva, following the degeneration of the neurenteric canal, a small area of entoderm fuses with the ectoderm and forms the anal opening. The anus is first ventral in position, but later moves up to the left side as the caudal fin develops (fig. 249E, G).

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5. Tubulation of the Mesoderm

Tubulation of the mesoderm and the formation of a continuous anteroposterior coelom in Amphioxus differs considerably from that found in the subphylum Vertebrata. This fact becomes evident in tracing the history of the mesoderm from the time of its segregation from the entoderm of the late

Fig. 249. Various stages of development of Amphioxus. (A from Kellicott, ’13, and Conklin, ’32; B from Kellicott, ’13, slightly modified; C-I, slightly modified from Conklin, ’32.) (A) Six -somite stage, comparable to fig. 247G and H. The animal hatches about

the time that two pairs of somites are present. (B) Nine-somite stage. The larva at this stage swims by means of cilia which clothe the entire ectodermal surface. (C) About fourteen pairs of somites are present at this stage. Neurenteric canal is still patent. (D) About 16 to 18 pairs of somites. Neurenteric canal is degenerating; mouth is formed. (E) About 20 to 22 pairs of somites. Anal opening is established between this stage and that shown in (D). (F) Transverse section, showing oral opening, looking from anterior

end of animal. (G) Same through anal area. (H) Frontal section of a 24-hour larva near dorsal side showing notochord, somites (S-1, S-8, etc.) and undifferentiated tissue at caudal end. Neural tube shown at anterior end. Nine pairs of somites are present. (I) Frontal section of a 38-hour larva at the level of the notochord showing section through the neural tube at the anterior and posterior ends, i.e., in region where larva bends ventralwards. Thirteen pairs of somites are present with muscle fibrillae along the mesial borders of the somites.

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gastrula and later embryo to the stage where a continuous antero-posterior coelomic space is formed, comparable to that found in the vertebrates.

The mesoderm of the late gastrula of Amphioxus is present as a dorsomedian band of cells on either side of the notochord, and together with the notochord, occupies the dorsal area or roof of the archenteron as mentioned previously. In the region of the blastopore, the two mesodermal bands diverge ventrally and occupy the inner aspect of the lateral walls of the blastopore (fig. 190F, G; 247B). At about the time of blastoporal closure, the two mesodermal masses of cells, located along the lateral lips of the blastopore, are retracted dorsally, where they come to lie on either side of the notochord (fig. 247C). In this position the two bands of mesoderm and the notochord continue to form the dorsal region or roof of the archenteron until approximately the time when the embryo is composed of 13 to 14 pairs of mesodermal segments or somites (fig. 249C). (See Hatschek, 1893, pp. 131, 132; Willey, 1894, p. 115; Conklin, ’32, p. 106.) When the embryo reaches a stage of development wherein 15 to 16 pairs of somites are present, the notochord and mesoderm have separated entirely from the entoderm (fig. 249D). At about this period the neurenteric canal between the metenteron and the neural tube disappears (fig. 249C, D).

ENTEROCOEL

DORSAL FIN-RAY CAVITY

SPLANCMNOCOEL SPLANCHNOCOELS ^FUSE

I'’ HORIZONTAL SEPTUM

Fig. 250. Differentiation of somites in Amphioxus. (A and B from Conklin, ’32; C, E, and F after Hatschek, 1888 and 1893; D from MacBride, 1898; all figures are modified.) (A) Somites shortly after separation from entoderm. (B) Later stage, the somites grow ventrally. (C) Semitic wall begins to differentiate into a thickened, dorsal, myotomic area, located near notochord and neural tube, and thinner somatic and visceral areas. (D) Horizontal septum formed which separates dorso-myotomic portion of somite from splanchnocoelic area below. (E, F) Later stages in differentiation of myotome and myocoelic diverticulum. (See text.)

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The formation of a continuous, antero-posterior, coelomic cavity in Amphioxus may be described as follows. The mesodermal bands on either side of the notochord of the post-gastrular embryo become converted into mesodermal grooves as each mesodermal band folds inwards or evaginates into the residual blastocoelic space between the archenteron and the outside ectoderm (fig. 195). Beginning at the anterior end, these longitudinal grooves of mesoderm soon become divided into distinct segments or somites by the appearance of transverse divisions (fig. 249 A, B, H). The first and second pairs of somites are formed at the anterior ends of the mesodermal grooves at about the time that the embryo hatches and swims about by means of ciliary action.

Eventually each somite becomes entirely constricted from the notochord and entoderm. In this segregated condition the somite forms a rounded structure retaining within itself a portion of the original archenteric cavity (fig. 250A). Hence, the cavity within the somite is called an enterocoel and represents the beginnings of the coelomic cavity of later development, at least in the anterior 13 or 14 pairs of somites. (Note: It is to be observed in this connection that the primitive somite in Amphioxus is not comparable to the primitive somite of the vertebrate embryo. In the latter, the somite represents merely a segment of the epimeric mesoderm, whereas in Amphioxus it is the entire mesoderm in each half of a particular segment of the embryo.)

After hatching, the mesodermal bands continue to form into grooves as the embryo elongates, and, synchronously, successive pairs of somites are formed. At about the time 8 to 10 pairs of somites are present (fig. 249B, H), the enterocoels of the first two pairs of somites have become entirely separated from the archenteron. The enterocoels of the following six pairs of somites are small and are not as evident at first as those of the first two pairs. Ultimately a definite enterocoel is found, however, in each somite.

Posterior to the eighth or ninth pairs of somites, the forming mesodermal grooves do not show the enterocoelic pouches as plainly as the more anterior somites. Slit-like mesodermal grooves tend to be present, however, and, when the somite is entirely free from the archenteron, this slit-like cavity expands into the enterocoelic space of the somite. As the region of the fourteenth pair of somites is approached, the slit-like mesodermal groove becomes more and more indefinite. Posterior to the fourteenth or fifteenth pair of somites, the somites originate from a solid mesodermal band on either side of the notochord. An enterocoelic origin of the cavity within each somite, therefore, is not possible caudal to this area, and the coelomic space arises by a hollowing-out process similar to coelomic cavity formation in the vertebrate group.

At about the time when eight pairs of somites are established, a shift of

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the mesoderm on either side of the embryo produces a condition wherein the somites of either side may be slightly intersegmental in relation to the somites on the other side (fig. 249H).

During its later development, each somite grows ventrally (fig. 250B). That portion of the somite contiguous to the notochord and neural tube thickens and forms the myotome. The region of the somite near the epidermal ectoderm is called the somatic or parietal mesoderm, while that associated with the entoderm forms the visceral or splanchnic mesoderm (fig. 25 OC).

As the myotome enlarges, the coelomic space becomes more and more displaced ventrally, and most of it comes to lie on either side of the enteron (metenteron). (See fig. 250D.) This ventral coelomic space forms the splanchnocoel, while the dorsal space, lateral to the myotome, is known as the myocoel. Eventually, the splanchnocoels of each pair of somites push ventrally to the lower portion of the enteron, where they ultimately fuse (fig. 250D-F). Gradually the splanchnocoels of each segment fuse anteroposteriorly and in this way a continuous, antero-posterior, splanchnocoelic space below and around the gut tube is formed. Tubulation or the formation of a continuous, antero-posterior, coelomic cavity thus is effected by fusion of the splanchnocoels of the respective somites on either side (fig. 25 OF). A horizontal septum, the intercoelomic membrane also appears, separating the myocoels above from the splanchnocoelic cavity below (fig. 250D).

6. Later Differentiation of the Myotomic (Dorsal) Area OF the Somite

While the above events are taking place in the ventral portion of the somite, the upper, myotomic region undergoes profound modification.

As shown in figure 250D, the myotomic portion of the somite has two unequally developed areas;

( 1 ) a medial muscular portion, the myotome and

(2) the laterally placed, thin-walled, parietal part which surrounds the coelomic space, or myocoel.

The muscular portion enlarges rapidly and, as seen in figure 25 OE and F, forms the muscle plate or myotome of the adult. These muscle plates very early assume the typical > shape characteristic of the adult. On the other hand, the myocoelic portion contributes important connective or skeletal tissue to the framework of the body. In each segment, the wall of the myocoel gives origin to three diverticula as follows:

(a) a lower sclerotomic diverticulum,

(b) a ventral diverticulum, and

(c) a dorsal sclerotomic diverticulum.

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The lower sclerotomic diverticulum (fig. 250D, E) extends up between the myotome and the medially placed notochord and nerve cord, as diagrammed in figure 250F. Its walls differentiate into two parts:

( 1 ) an inner layer which, together with a similar contribution from the somite on the opposite side, wraps around the notochord and nerve cord and, subsequently, gives origin to a skeletogenous sheath of connective tissue which enswathes these structures; and

(2) an outer layer which covers the mesial (inner) aspect of the myotome with a fascia or connective tissue covering.

The outer surface of the myotome does not have a covering of fascia.

The ventral diverticulum extends between the lateral wall of the splanchnococl and the epidermal layer of the body wall (fig. 250E, F) and separates the parietal wall of the splanchnocoel from the epidermal wall (fig. 250F). This ventral diverticulum or dermatomic fold, together with the external or parietal wall of the myocoel above, forms the dermatome. The inner and outer layers of the ventral diverticulum gradually fuse to form the cutis or dermal layer of the integument or skin in the ventro-lateral portion of the body, whereas the parietal wall of the myocoel above gives origin to the same dermal layer in the body region lateral to the myotome. The dorsal sclerotomic diverticula form the fin-ray cavities in the dorsal fin. These cavities become entirely isolated from the rest of the myocoelic spaces. Several fin-ray cavities occupy the breadth of a single myotome. The dorsal myotomic portion of the somite thus differentiates into three main structural parts:

(a) the muscular myotome,

(b) the mesial sclerotome or skeletogenous tissue, and

(c) the latero- ventral dermatome or dermal tissue of the skin.

7. Notochord

The notochord arises as a middorsal evagination of the primitive archenteron up to about the stage of about 13 to 14 pairs of somites (fig. 195). Posterior to this region it takes its origin by proliferative growth from a separate mass of notochordal tissue, lying above the gut and between the two mesodermal masses of cells. Its origin posterior to the general area of the thirteenth to fourteenth body segments, therefore, has no relation to the entoderm. It rapidly develops into a conspicuous skeletal rod, lying below the neural tube and between the mesodermal somites and resting in a slight depression along the dorsal aspect of the metenteron or entodermal tubulation (fig. 249E, H). It continues forward in the head region, anterior to the brain portion of the neural tube (fig. 249E).

(The student is referred to the following references for further details relative to the early development of Amphioxus: Cerfontaine, ’06; Conklin, ’32;

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Hatschek, 1893; Kellicott, ’13; MacBride, 1898, ’00, ’10; Morgan and Hazen, ’00; and Willey, 1894.)

F. Early Development of the Rudiments of Vertebrate Paired Appendages

Two pairs of appendages, placed at the anterior (pectoral) and posterior (pelvic) extremities of the trunk, are common to all vertebrate groups. However, all vertebrates do not possess two pairs of paired appendages. Certain lizards of the genera Pygopus and Pseudopus have only a posterior pair of appendages, while in certain other vertebrates the opposite condition is found, the anterior pair being present without posterior appendages. The latter condition is found in certain teleost and ganoid fishes; the amphibian. Siren lacertina; the lizard, Chirotes; and among the mammals, the Sirenia and Cetacea. Again, some vertebrates are entirely apodal, e.g., cyclostomatous fishes and most snakes, although the boa constrictors and pythons possess a pair of rudimentary posterior appendages embedded in the skin and body wall. Some have rudimentary appendages only in the embryo, as the legless amphibians of the order Gymnophiona, and certain lizards. Consequently, the presence of embryonic rudiments of the paired appendages is a variable feature when the entire group of vertebrates is considered.

The rudiments of the paired appendages also are variable, relative to the time of their appearance in the vertebrate group as a whole. They are more constant in the Amniota, i.e., reptiles, birds, and mammals, in time of appearance than in the Anarnniota, i.e., fishes and amphibia. In the reptiles, birds, and mammals, the limb buds arise when primitive body form is being evolved. In the anuran amphibia, the anterior rudiments may appear and go on to a high degree of differentiation before the appearance of the posterior pair of appendages. For example, in the frog, Rana pipiens, the posterior limb buds first make their appearance a brief period before the beginning of metamorphosis of the tadpole into the adult form. However, the anterior limb buds differentiate earlier but remain concealed beneath the operculum until they become visible during the later stages of metamorphosis. In urodele amphibia, the fore limb bud is not covered by an operculum, and it is visible at the time of its initial appearance which occurs before the hind limb rudiment arises (fig. 227J-L).

In the majority of vertebrates, the limb rudiment first makes its appearance as an elongated, dorso-ventrally flattened fold of the epidermis, containing a mass of mesodermal cells within, as shown, for example, in the chick and mammalian embryos (figs. 240, 244, 246). The contained mesodermal cells may be in the form of epithelial muscle buds derived directly from the myotomes (e.g., sharks) or as a mass of mesenchyme (chick, pig, human). (See Chap. 16.) The early limb-bud fold may be greatly exaggerated in certain elasmobranch fishes, as in the rays, where the anterior and posterior fin folds

LIMB BUD AN ILLUSTRATION OF FIELD CONCEPT OF DEVELOPMENT

509

fuse together for a time, forming one continuous lateral body fold. On the other hand, in the lungfishes (the Dipnoi) and in amphibia (the Anwa and Urodela), the appendage makes its first appearance, not as an elongated fold of the lateral body wall, flattened dorso-ventrally, but as a rounded, knob-like projection of the lateral body surface (fig. 227K-M).

G. The Limb Bud as an Illustration of the Field Concept of Development in Relation to the Gastrula and the Tubulated Embryo

In Chapter 9 it was observed that the major presumptive organ-forming areas are subdivided into many local, organ-forming areas at the end of gastrulation. In the neural and epidermal areas, this subdivision occurs during gastrulation through influences associated with local inductive action. At the end of the gastrular period, therefore, each local area within the major organforming area possesses the tendency to give origin to a specific organ or a part of an organ. The restricted, localized areas within each major organforming area represent the individual, or specific, organ-forming fields. During tubulation, the major organ-forming areas with their individuated, organ-forming fields are molded into tubes, and, thus, the individual fields become arranged along each tube. Consequently, each tube possesses a series of individual, organ-forming areas or fields, distributed antero-posteriorly along the tube.

As a result of the close association of cells and substances during gastrulation and tubulation, many specific organ-forming fields are related to more than one of the body tubes. Specific organ-forming fields, therefore, may have intertubular relationships. For example, the lens field is located in the epidermal tube, but, in many species, its origin as a lens field is dependent upon influences emanating from the optic vesicle of the neural tube (see Chap. 19). Another example of an association between the parts of two contiguous tubes is the limb-bud field in the urodele, Amby stoma punctatum. As the limb-bud field in this species illustrates various aspects and properties of an organ-forming field, it will be described below in some detail.

The presumptive anterior limb disc or limb field of Amby stoma is determined as a specific limb-forming area in the middle gastrular stage (Detwiler, ’29, ’33). Later on in the embryo, it occupies a circular-shaped area within trunk segments three to six. According to Harrison (’18) and Swett (’23), its properties as a field mainly are resident in the cells of the somatic layer of the mesoderm in this area. If, for example, the somatic layer of mesoderm in this area is transplanted to another area, a well-developed limb will result. Also, the mesoderm of the dorsal half of the field forms a greater part of the limb than the other parts, with the anterior half of the limb disc next in importance. It appears, therefore, that the limb-forming potencies are greatest in the dorso-anterior half of the limb field and become less postero-ventrally. Moreover, not “all of the cells which are potentially limb forming go into

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the limb” (Swett, ’23). As demonstrated by Harrison (’18) half discs (half fields), left intact in the developing embryo or removed and transplanted to other areas, develop into normal limbs.

The above experiments of Harrison, together with those of Detwiler (’29, ’33) suggest that while the limb field is irreversibly determined at an early stage to form limb tissue, the exact determination of the various parts within the field is absent at the earlier phases of development. One kind of precise determination is present, however, for the first digit-radial aspect (i.e., the pre-axial aspect) of the limb appears to arise only from the anterior end of the field, whether the field is allowed to develop intact or is split into two parts. That is, if it is split into two portions, the anterior extremity of the posterior portion, as well as the original anterior part of the limb field, develops the pre-axial aspect of the limb. This antero-posterior polarization is present from the first period of field determination. On the other hand, the dorso-ventral polarity is not so determined; for if the transplanted limb disc is rotated 180 degrees (i.e., if it is removed and reimplanted in its normal place dorsal side down) it will develop a limb with the dorsal side up but with the antero-posterior axis reversed (Harrison, ’21). In these cases the first digit-radial aspect will appear ventral in position. This result indicates that the pre-axial aspect of the limb becomes oriented always toward the ventral aspect of the limb. However, the experiments of Swett (’37, ’39, ’41) tend to show that the reversal of the dorso-ventral axis occurs only when implanted below the myotome s; for when the rotated limb field is implanted in the somitic (myotomic) area, it will remain inverted. Factors other than those resident within the limb field itself, probably factors in the flank area, appear thus to induce the normal dorso-ventral axis when the limb disc is implanted in its normal site.

In the descriptions given above, the importance of the somatic layer of mesoderm as the seat of the limb-forming factors is emphasized. It is obvious, however, that the epidermal covering of the limbs derived from the epidermal tubulation also is important in limb formation. For example, epidermal importance is suggested by the experiments of Saunders (’49) on the developing limb bud of the chick wherein it was found that the apical ridge of ectoderm, located at the apex of the early limb bud, is essential for normal limb development.

Individual, or specific, organ-forming fields which appear in the gastrula and early tubulated embryo thus are generalized areas determined to form individual organs. As development proceeds, two main limitations are imposed upon the field:

( 1 ) The cellular contribution of the field actually entering into the organ becomes restricted; and

(2) specific parts of the field become progressively determined to form specific parts of the organ. •

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It is obvious, therefore, that the fields of influence which govern the development of specific organs may be much more extensive in cellular area than the actual cellular contributions which take part in the formation of the specific organ structures. Experiments on the forming limb of Amby stoma also have demonstrated that a particular area of the field is stronger in its limb-forming potencies than other regions of the field. This property probably is true of other fields as well.

(For a detailed discussion of the field concept in embryonic development, reference should be made to Huxley and DeBeer, ’34, Chaps. 8 and 9; Weiss, ’39, p. 289 ff.)

H. Cephalic Flexion and General Body Bending and Rotation in Vertebrate Embryos

The anterior end of the neural tubulation is prone to assume a bent or flexed contour whereby the anterior end of the neural tube is directed downward toward the ventral aspect of the embryo. This general behavior pattern is strong in vertebrate embryos with the exception of the teleost fishes. In teleost fishes this bending habit is slight. As the later development of the head progresses in other vertebrate embryos, the neural tube shows a pronounced cephalic (cranial) flexure in the region of the midbrain, in some species more than in others. (See Chap. 19.) An additional bending occurs in the posterior hindbrain area. The latter flexure is the cervical or nuchal flexure (figs. 231, 238, 240, 244, 246).

Aside from the acute bending which takes place in the formation of the cephalic and the nuchal flexures, there is a definite tendency for many vertebrate embryos to undergo a general body bending, with the result that the anterior part of the body and the caudal portion of the trunk and tail may be depressed in a ventral direction (figs. 222C-E; 227; 229F; 238; 240; 244; 246). In the frog embryo, at hatching, the opposite tendency may prevail for a brief period, and the dorsal trunk region may appear sagging or hollowed (fig. 226A, C).

In addition to these bending movements, in the embryos of higher vertebrates, a rotation or twisting (torsion) of the developing body along the antero-posterior axis is evident. In the chick embryo, for example, the head region begins to rotate toward the right at about 38 hours of incubation. Gradually this torsion continues caudally (figs. 237, 238, 239, 260). At about 70 to 75 hours, the rotational movement reaches the tail region, and the embryo then comes to lie on its left side throughout its length (fig. 240). In exceptional embryos, the rotational movement is toward the left, and the embryo comes to lie on its right side. Similar movements occur in the pig and other mammals.

This rotational movement is advantageous, particularly in long-bodied Amniota, such as the snakes, where it permits the developing embryo to coil

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in spiral form within the extra-embryonic membranes. The coiling tendency, however, is not alone confined to the snake group, for the habits of general body bending, referred to above, essentially is a coiling tendency. Viewed thus, the rotation or torsion of the developing body along its median axis is a generalized behavior pattern which permits and aids the coiling habit so prevalent among the embryos of higher vertebrates. It may be observed further that the coiling behavior is a common attitude during rest not only among snakes but also among the adults of many higher vertebrates.

I. Influences Which Play a Part in Tubulation and Organization of

Body Form

In Chapter 9, it was pointed out that the pre-chordal plate material, that is, organizer material which invaginates first during gastrulation and which comes to lie in the future head region, induces the organization of certain head structures and itself may form a part of the pharyngeal wall and give origin to head mesoderm, etc. On the other hand, the trunk-organizer material (notochord and somitic mesoderm) which moves to the inside, following the pre-chordal plate material, organizes the trunk region. The following series of experiments based upon work by Spemann, ’31, sets forth the inductive properties of these two cellular areas:

Experiment

1. Head-organizer material, taken from one embryo and placed at head level of a host embryo, will induce a secondary head, having eyes and ear vesicles

2. Head-organizer material, transplanted to trunk and tail levels in host embryos, induces a complete secondary embryo, including head

3. Trunk-organizer material (i.e., notochord and somitic mesoderm), placed at head level in host embryo, induces a complete secondary embryo, including the head structures

4. Trunk-organizer material, placed at future trunk or tail levels in host embryos, induces trunk and tail structures only

The many influences which play a part in the organization of the vertebrate head and body constitute an involved and an unsolved problem. The extreme difficulty of this general problem has long been recognized. (See Kingsbury and Adelmann, ’24.) The above-mentioned work of Spemann represents a beginning attempt to analyze this aspect of development and to understand the factors involved. It demonstrates that the organization of the neural tube and other axial areas is dependent upon specific cellular areas which migrate inward during gastrulation. However, this is but one aspect of the problem. As observed in the series of experiments above, trunk-organizer material is able to organize a complete secondary embryo, including the head, when

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513

Fig. 251. Dependency of neural tube formation upon surrounding tissues. (A) Effect of notochord without myotomes. (B) Effect of myotomes without notochord. (C) Absence of notochord and myotomes.

placed at head level in the host but can only organize trunk and tail structures when placed in trunk and tail areas of the host. In other words, there exists a mutual relationship between the level of the host tissues and the transplanted organizer material of the trunk organizer in effecting tlie formation of a head at the head level.

Another forceful example of the interrelationship of developing parts and formative expression of body structures is shown by the work of Holtfreter (’33) on the development of the neural tube. This work demonstrates that the form of the neural tube is dependent upon influences in its environment, as shown in figure 251. The presence of the later developing notochord determines a thin ventral floor of the neural canal, whereas the contiguous myotome determines a thick wall of the neural tube. Normally, in development, the notochord lies below the neural tube, while the somites with their myotomie parts come to lie lateral to the tube. That is to say, the normal bilateral symmetry of the neural tube is dependent upon the relationship, in their normal positions, of the notochord and the myotomes.

The behavior of the developing neural tube, relative to the notochord and the myotomes, demonstrates the importance of the’ migration of the somitic mesoderm from a position contiguous and lateral to the notochord at the beginning of neuralization to one which is lateral to the forming neural tube as neuralization and differentiation of the neural tube progresses.

A further illustration of the probable influence of the notochordal area in morphogenesis and organization of body form is the behavior of the developing metenteron or enteric tube. As observed previously, the gut tubulation tends to invaginate or arch upward toward the notochord not only in embryos developing from flattened gastrulae but also in amphibia. The movement of the entoderm toward the notochord strikingly resembles the behavior of the neural plate ectoderm during the formation of the neural tube. This comparison becomes more striking when one considers the manner of enteron for

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mation in the tail and hindgut regions in the shark embryo, Squalus acanthias, already mentioned, p. 484. In this species the entoderm of the developing tail actually invaginates dorsad and closes in a manner similar to the forming neural tube. That is to say, in the developing tail of the shark, two invaginations toward the notochord are evident, one from the dorsal side, which involves the formation of the neural tube, and the other from the ventral side, effecting the developing enteric tube.

The above facts suggest, therefore, that one of the main organizing influences at work during tubulation and primitive body formation emanates from the pre-chordal plate area, the notochord, and the epimeric portion of the mesoderm. From this general area or center, a chain of acting and interacting influences extends outward, one part acting upon another, to effect the formative expression of the various parts of the developing body.

J. Basic Similarity of Body-form Development in the Vertebrate Group of Chordate Animals

In the earlier portion of this chapter, differences in the general procedures concerned with tubulation and primitive body formation in round and flattened gastrulae were emphasized. However, basically all vertebrate embryos show the same tendency of the developing body to project itself upward and forward in the head region, dorsally in the trunk area and dorso-posteriad in the tail region. Literally, the embryonic body tends to lift itself up out of, and above, the area which contains the yolk and extra-embryonic tissues. This proneness to move upward and to protrude its developing head end forward and its caudal end backward is shown beautifully in the development of the embryos of the shark (figs. 229, 230), the mud puppy (fig. 227), the chick (fig. 235C), and the pig (fig. 242). The embryo struggles to be free from its bed of yolk and extra-embryonic tissue, as it were, and it reminds one of the superb imagery employed by the poet, John Milton, in his immortal poem, Paradise Lost, where he describes the development of the lion thus:

The grassy clods now calv’d; now half appear’d

The tawny lion, pawing to get free

His hinder parts, then springs as broke from bonds,

And rampant shakes his brinded mane.

In summary, therefore, although it appears that rounded and flattened gastrulae in the vertebrate group may have slightly different substrative conditions from which to start, they ail employ essentially similar processes in effecting tubulation of the respective, major organ-forming areas and in the development of primitive body form.

Bibliography

Cerfontaine, P. 1906. Recherches sur le developpement de I’ Amphioxus. Arch, biol., Paris. 22:229.

Conklin, E. G. 1932. The embryology of Amphioxus. J. Morphol. 54:69.

Dean, B. 1896. The early development of Amia. Quart. J. Micr. Sc. (New Series) 38:413.

Detwiler, S. R. 1929. Transplantation of anterior limb mesoderm from Amhlystonia embryos in the slit blastopore stage. J. Exper. Zool. 52:315.

. 1933. On the time of determination of the antero-posterior axis of the forelimb in Amhly stoma. J. Exper. Zool. 64:405.

Hamburger, V. and Hamilton, H. L. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49.

Harrison, R. G. 1918. Experiments on the development of the forelimb of Ainblystorna, a self-differentiating equipotential system. J. Exper. Zool. 25:413.

. 1921. On relations of symmetry

in transplanted limbs. J. Exper. Zool. 32:1.

Hatschek, B. 1888. Uber den Schechtenbau von Amphioxus. Anat. Anz. 3:662.

Hatschek, B. 1893. The Amphioxus and its development. Translated by J. Tuckey. The Macmillan Co., New York.

Holtfreter, J. 1933. Der Einfluss von Wirtsalter und verschiedenen Organbezirken auf die Differenzierung von angelagertem Gastrulaektoderm. Arch. f. EntwickIngsmech. d. Organ. 127:619.

Huxley, J. S. and De Beer, G. R. 1934. The Elements of Experimental Embryology. Cambridge University Press, London.

Kcllicott, W. E. 1913. Outlines of Chordate Development. Henry Holt & Co., New York.

Kingsbury, B. F. and Adelmann, H. B. 1924. The morphological plan of the head. Quart. J. Micr. Sc. 68:239.

MacBride, E. W. 1898. The early development of Amphioxus. Quart. J. Micr. Sc. 40:589.

. 1900. Further remarks on the development of Amphioxus. Quart. J. Micr. Sc. 43:351.

. 1910. The formation of the layers

in Amphioxus and its bearing on the interpretation of the early ontogenetic processes in other vertebrates. Quart. J. Micr. Sc. 54:279.

Morgan, T. H. and Hazen, A. P. 1900. The gastrulation of Amphioxus. J. Morphol. 16:569.

Needham, J. 1942. Biochemistry and Morphogenesis. Cambridge University Press, London.

Patterson, J. T. 1907. The order of appearance of the anterior somites in the chick. Biol. Bull. 13:121.

Saunders, J. W. 1949. An analysis of the role of the apical ridge of ectoderm in the development of the limb bud in the chick. Anat. Rec. 105:567.

Selys-Longchamps, M. de. 1910. Gastrulation et L)rmation des feuillets chez Petromyzon planeri. Arch, biol., Paris. 25:1.

Spemann, H. 1931. Uber den Anteil von Implantat und Wirtskeim an der Orienticrung und Beschaffenheit der induzierten Embryonalanlage. Arch. f. EntwickIngsmech. d. Organ. 123:389.

Swett, F. H. 1923. The prospective significance of the cells contained in the four quadrants of the primitive limb disc of Amhly stoma. J. Exper. Zool. 37:207.

. 1937. Experiments upon delayed

determination of the dorsoventral limb axis in Amhly stoma punctatum (Linn.). J. Exper. Zool. 75:143.

. 1939. Further experiments upon

the establishment and the reversal of prospective dorsoventral limb-axis polarity. J. Exper. Zool. 82:305.

. 1941. Establishment of definitive

polarity in the dorsoventral axis of the forelimb girdle in Amhly stoma punctatum (Linn.). J. Exper. Zool. 86:69.

Weiss, P. 1939. Principles of Development. Henry Holt & Co., New York.

Willey, A. 1894. Amphioxus and the Ancestry of the Vertebrates. The Macmillan Co., New York.

515

11

Basic Features of VerteLrate Morplio^enesis

A. Introduction

1. Purpose of this chapter

2. Definitions

a. Morphogenesis and related terms

b. Primitive, larval, and definitive body forms

1 ) Primitive body form

2) Larval body form

3) Definitive body form

3. Basic or fundamental tissues

B. Transformation of the primitive body tubes into the fundamental or basic condition of the various organ systems present in the primitive embryonic body

1. Processes involved in basic system formation

2. Fundamental similarity of early organ systems

C. Laws of von Baer

D. Contributions of the mesoderm to primitive body formation and later development

1. Types of mesodermal cells

2. Origin of the mesoderm of the head region

a. Head mesoderm derived from the anterior region of the trunk

b. Head mesoderm derived from the pre-chordal plate

c. Head mesoderm contributed by neural crest material

d. Head mesoderm originating from post-otic somites

3. Origin of the mesoderm of the tail

4. Contributions of the trunk mesoderm to the developing body

a. Early differentiation of the somites or epimere

b. Early differentiation of the mesomere (nephrotome)

c. Early differentiation and derivatives of the hypomere

1) Contributions of the hypomere (lateral plate mesoderm) to the developing pharyngeal area of the gut tube

2) Contributions of the hypomere (lateral plate mesoderm) to the formation of the gut tube and heart structures

3) Contributions of the hypomere (lateral plate mesoderm) to the external (ectodermal or epidermal) body tube

4) Contributions of the hypomere or lateral plate mesoderm to the dorsal body areas

5) Contributions of the lateral plate mesoderm to the walls of the coelomic cavity

5. Embryonic mesenchyme and its derivatives

516

INTRODUCTION

517

E. Summary of later derivatives of presumptive, major, organ-forming areas of the late blastula and gastrula

1. Neural plate area (ectoderm)

2. Epidermal area (ectoderm)

3. Entodermal area

4. Notochordal area

5. Mesodermal areas

6. Germ-cell area

F. Metamerism

1. Fundamental metameric character of the trunk and tail regions of the vertebrate body

2. Metamerism and the basic morphology of the vertebrate head

G. Basic homology of the vertebrate organ systems

1. Definition

2. Basic homology of vertebrate blastulae, gastrulae, and tubulated embryos

A. Introduction

1. Purpose of This Chapter

In this chapter, the basic morphogenetic features which give origin to the later organ systems are emphasized. These features arise from the stream of morphogenetic phenomena which come down from the fertilized egg through the periods of cleavage, blastulation, gastrulation, and tabulation. This chapter thus serves to connect the developmental processes, outlined in Chapters 6 to 10, with those which follow in Chapters 12 to 21. As such, it emphasizes certain definitions and basic structural features involved in the later morphogenetic activities which mold the adult body form.

2. Definitions

a. Morphogenesis and Related Terms

The word morphogenesis means the development of form or shape. It involves the elaboration of structural relationships. The morphogenesis of a particular shape and structure of a cell is called cytomorphosis or cytogenesis and is synonymous with the term cellular differentiation, considered from the structural aspect. In the Metazoa, the body is composed of groups of cells, each cellular group possessing cells of similar form and function. That is, each cell group is similarly differentiated and specialized. A cellular group, composed of cells similar in form (structure) and function, is called a tissue. Histology is the study of tissues, and the word histogenesis relates to that phase of developmental morphology which deals with the genesis or development of tissues. An organ is an anatomical structure, produced by an association of different tissues which fulfills one or several specialized functions. For example, the esophagus, stomach, liver, etc., are organs of the body. During development, each of the major organ-forming areas, delineated in

518

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

Chapters 6, 7, 9 and 10, produce several specific organs. Organogenesis is concerned with the formation of these specific organs. A group of organs which are associated together to execute one general function form an organ system. The digestive system, for example, has for its general function that of obtaining nourishment for the body. It is composed of a series of o’-gans integrated toward this end. The nervous system, similarly, is an assemblage of specific organs devoted to the discharge of nervous functions. So it is with the other systems of the organism. System development is concerned with the genesis of such systems. The association of various systems, integrated together for the maintenance of the body within a particular habitat, constitutes the organism. Finally, the organism acquires a particular body form because of the form, structure, and activities assumed by its organ systems as a result of their adaptation to the functional necessities of the particular habitat in which the organism lives. It should be urged further that this nice relationship between form and structure, on the one hand, and functional requirements, on the other, is a fundamental principle of development from the egg to the adult. It is a principle intimately associated with the morphogenesis of the organ systems described in Chapters 12 to 21.

During development from the egg to the adult form, three major types of body form are evolved in the majority of vertebrate species.

b. Primitive, Larval, and Definitive Body Forms (see fig. 255)

1) Primitive Body Form. The condition of primitive or generalized, embryonic body form is attained when the embryo reaches a state in which its developing organ systems resemble the respective developing organ systems in other vertebrate embryos at the same general period of development. (See p. 520.) Superficially, therefore, the general structure of the primitive embryonic body of one species resembles that of the primitive embryonic bodies of other vertebrate species. Such comparable conditions of primitive, body-form development are reached in the 10 to 15-mm. embryo of the shark, Squalus acanthias, of the frog embryo at about 5 to 7 mm., the chick at about 55 to 96 hrs. of incubation, the pig at 6 to 10 mm., and the human at 6 to 10 mm.

2) Larval Body Form. Following primitive body form, the embryo gradually transforms into a larval form. The larval form is present in the period between primitive body form and definitive body form. The larval period is that period during which the basic conditions of the various organ systems, present in primitive body form, undergo a metamorphosis in assuming the form and structure of the adult or definitive body form. In other words, during the larval period, the basic or generalized conditions of the various organ systems are changed into the adult form of the systems, and the larval period thus represents a period of transition. Embryos which develop in the water (most fishes, amphibia) tend to accentuate the larval condition, whereas those which develop within the body of the mother (viviparous teleosts,

INTRODUCTION

519

sharks, mammals) or within well-protected egg membranes (turtle, chick) slur over the larval condition.

The larval stage in non-viviparous fishes (see Kyle, ’26, pp. 74-82) and in the majority of amphibia is a highly differentiated condition in which the organs of the body are adapted to a free-living, watery existence. The tadpole of the frog, Rana pipiens, from the 6-mm. stage to the 11 -mm. stage, presents a period during which the primitive embryonic condition, present at the time of hatching (i.e., about 5 mm.), is transformed into a well-developed larval stage capable of coping with the external environment. From this time on to metamorphosis, the little tadpole possesses free-living larval features. Another example of a well-developed, free-living, larval stage among vertebrates is that of the eel, Anguilla rostrata. Spawning occurs in the ocean depths around the West Indies and Bermuda. Following the early embryonic stage in which primitive body form is attained, the young transforms into a form very unlike the adult. This form is called the Leptocephalus. The Leptocephalus was formerly classified as a distinct species of pelagic fishes. After many months in the larval stage, it transforms into the adult form of the eel. The latter migrates into fresh-water streams, the American eel into streams east of the Rockies and the European eel into the European streams (Kyle, ’26, pp. 54-58). The larval stages in most fishes conform more nearly to the adult form of the fish.

The embryo of Squalus acanthias at 20 to 35 mm. in length, the chick embryo at 5 to 8 days of incubation, the pig embryo of 12- to 18-mm. length, and the human embryo of 12 to 20-mm. length may be regarded as being in the stage of larval transition. The young opossum, when it is born, is in a late larval state. It gradually metamorphoses into the adult body form within the marsupium of the mother (Chap. 22).

3) Definitive Body Form. The general form and appearance of the adult constitute definitive body form. The young embryo of Squalus acanthias, at about 40 mm. in length, assumes the general appearance of the adult shark; the frog young, after metamorphosis, resembles the adult frog (Chap. 21), the chick of 8 to 13 days of incubation begins to simulate the form of the adult bird; the pig embryo of 20 to 35 mm. gradually takes on the body features of a pig, and the human fetus, during the third month of pregnancy, assumes the appearance of a human being. The transformation of the larval form into the body form of the adult is discussed further in Chapter 21 in relation to the endocrine system.

3. Basic or Fundamental Tissues

Through the stages of development to the period when the primitive or generalized, embryonic body form is attained, most of the cells which take part in development are closely associated. In the primitive embryonic body, this condition is found in all the five primitive body tubes and in the notochord. These closely arranged cells form the primitive epithelium. In the de

520

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

veloping head and tail regions, however, mesoderm is present in the form of loosely aggregated cells, known as mesenchyme. While the cells of the epithelial variety are rounded or cuboidal in shape with little intercellular substance or space between the cells, mesenchymal cells tend to assume stellate forms and to have a large amount of intercellular substance between them. The primitive vascular or blood tubes are composed of epithelium in the sense that the cells are closely arranged. However, as these cells are flattened and show specific peculiarities of structure, this tissue is referred to as endothelium. Also, while the cells of the early neural tube show the typical epithelial features, they soon undergo marked changes characteristic of developing neural tissue. The primitive or generalized, embryonic body thus is composed of four fundamental tissues, viz., epithelial, mesenchymal, endothelial, and neural tissues.

B. Transformation of the Primitive Body Tubes into the Fundamental or Basic Condition of the Various Organ Systems Present in the Primitive Embryonic Body

1. Processes Involved in Basic System Formation

As the primitive body tubes (epidermal, neural, enteric, and mesodermal) are established, they are modified gradually to form the basis for the various organ systems. While the notochordal axis is not in the form of a tube, it also undergoes changes during this period. The morphological alterations, which transform the primitive body tubes into the basic or fundamental structural conditions of the systems, consist of the following:

(a) extension and growth of the body tubes,

(b) saccular outgrowths (evaginations) and ingrowths (invaginations) from restricted areas of the tubes,

(c) cellular migrations away from the primitive tubes fo other tubes and to the spaces between the tubes, and

(d) unequal growth of different areas along the tubes.

As a result of these changes, the primitive neural, epidermal, enteric, and mesodermal tubes, together with the capillaries or blood tubes and the notochord, experience a state of gradual differentiation which is directed toward the production of the particular adult system to be derived from these respective basic structures. The primitive body tubes, the primitive blood capillaries, and the notochord thus come to form the basic morphological conditions of the future or gut ^ systems. The basic structural conditions of the various systems are described in Chapters 12 to 21.

2. Fundamental Similarity of Early Organ Systems

The general form and structure of each primitive embryonic system, as it begins to develop in one vertebrate species, exhibits a striking resemblance

LAWS OF VON BAER

521

to the same system in other vertebrate species. This statement is particularly true of the gnathostomous vertebrates (i.e., vertebrates with jaws). Consequently, we may regard the initial generalized stages of the embryonic or rudimentary systems as fundamental or basic plans of the systems, morphologically if not physiologically. The problem which confronts the embryo of each species, once the basic conditions of the various systems have been established, is to convert the generalized basic condition of each system into an adult form which will enable that system to function to the advantage of the particular animal in the particular habitat in which it lives. The conversion of the basic or primitive condition of the various systems into the adult form of the systems constitutes the subject matter of Chapters 12 to 21.

The basic conditions of the various organ systems are shown in the structure of shark embryos from 10 to 20 mm. in length, frog embryos of 5 to 10 mm., chick embryos from 55 to 96 hrs., pig embryos from 6 to 10 mm., crownrump length, and human embryos of lengths corresponding to 6 to 10 mm. That is to say, the basic or generalized conditions of the organ systems are present when primitive or generalized embryonic body form is developed. It is impossible to segregate any particular length of embryo in the abovementioned series as the ideal or exact condition showing the basic condition of the systems, as certain systems in one species progress faster than those same systems in other species. However, a study of embryos of these designations serves to provide an understanding of the basic or fundamental conditions of the various systems (figs. 257-262; also fig. 347A).

C. Laws of von Baer

As indicated above, the species of the vertebrate group as a whole tend to follow strikingly similar (although not identical) plans of development during blastulation, gastrulation, tubulation, the development of the basic plan of the various systems and primitive body form. As observed in the chapters which follow, the fundamental or basic plan of any particular, organ-forming system, in the early embryo of one species, is comparable to the basic plan of that system in other species throughout the vertebrate group. However, after these basic parallelisms in early development are completed, divergences from the basic plan begin to appear during the formation of the various organ systems of a particular species.

The classical statements or laws of Karl Ernst von Baer (1792-1876) describe a tendency which appears to be inherent in the developmental procedure of any large group of animals. This developmental tendency is for generalized structural features to arise first, to be remodeled later and supplanted by features specific for each individual species. To interpret these laws in terms of the procedure principle mentioned in Chapter 7, it may be assumed that general, or common, developmental procedures first are utilized, followed by

522

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

specific developmental procedures which change the generalized conditions into specific conditions.

The laws of von Baer ( 1 828-1837, Part I, p. 224) may be stated as follows:

(a) The general features of a large group of animals appear earlier in development than do the special features;

(b) after the more general structures are established, less general structures arise, and so on until the most special feature appears;

(c) each embryo of a given adult form of animal, instead of passing through or resembling the adult forms of lower members of the group, diverges from the adult forms, because

(d) the embryo of a higher animal species resembles only the embryo of the lower animal species, not the adult form of the lower species.

D. Contributions of the Mesoderm to Primitive Body Formation and Later Development

The mesoderm is most important to the developing architecture of the body. Because the mesoderm enters so extensively into the structure of the many organs of the developing embryo, it is well to point out further the sources of mesoderm and to delineate the structures and parts arising from this tissue.

1. Types of Mesodermal Cells

Most of the mesoderm of the early embryo exists in the form of epithelium (see p. 519). As development proceeds, much of the mesoderm loses the close arrangement characteristic of epithelium. In doing so, the cells separate and assume a loose connection. They also may change their shapes, appearing stellate, oval, or irregular, and may wander to distant parts of the body. This loosely aggregated condition of mesoderm forms the primitive mesenchyme. Though most of the mesoderm becomes transformed into mesenchyme, the inner layer of cells of the original hypomeric portion of the mesodermal tubes retains a flattened, cohesive pattern, described as mesothelium. Mesothelium comes to line the various body cavities, for these cavities are derived directly from the hypomeric areas of the mesodermal tubes (Chap. 20).

2. Origin of the Mesoderm of the Head Region

The primary cephalic outgrowth (Chap. 10), which later forms the head structures, contains two basic regions, namely, the head proper and the pharyngeal or branchial region. During its early development, the heart lies at the ventro-caudal extremity of the general head region; it recedes gradually backward as the head and branchial structures develop. The exact origin of the mesoderm which comes to occupy the head proper and pharyngeal areas varies in different gnathostomous vertebrates. The general sources of the head mesoderm may be described in the following manner.

CONTRIBUTIONS OF THE MESODERM TO PRIMITIVE BODY FORMATION

523

a. Head Mesoderm Derived from the Anterior Region of the Trunk

The mesoderm of the branchial area in lower vertebrates, such as the snarks and, to some degree, the amphibia, represents a direct anterior extension of the mesoderm of the trunk (figs. 217D, E; 230D; 252E) . It is divisible into two parts: (1) a ventro-lateral region, the hypomeric or lateral plate mesoderm, and (2) a dorsal or somitic portion. The latter represents a continuation into the head region of the epimeric (somitic) mesoderm of the trunk. That portion of the mesoderm of the branchial area which may be regarded specifically as part of the mesoderm of the head proper is the mesoderm associated with the mandibular and hyoid visceral arches, together with the hyoid and mandibular somites located at the upper or dorsal ends of the hyoid and mandibular visceral arches (fig. 217D, E).

In the higher vertebrates (reptiles, birds, and mammals), the mesoderm of the branchial region appears early, not as a continuous epithelium, as in the shark and amphibian embryo, but as a mass of mesenchyme which wanders into the branchial area from the anterior portion of the developing trunk region (figs. 217F; 23 3B; 234B). This mesenchyme assumes branchial region characteristics, for it later condenses to form the mandibular, hyoid, and more posteriorly located, visceral arches. Also, mesenchymal condensations appear which correspond to the pre-otic head somites formed in the early shark embryo. For example, in the chick, there is an abducent condensation, which corresponds to the hyoid somite of the shark embryo, and a superior oblique condensation corresponding probably to the mandibular somite of the shark embryo (cf. fig. 217D, F). (See also Adelmann, ’27, p. 42.) Both of these condensations give origin to eye muscles (Chap. 16). Somewhat similar condensations of mesenchyme which form the rudiments of eye muscles occur in other members of the higher vertebrate group.

b. Head Mesoderm Derived from the Pre-chordal Plate

The term pre-chordal plate mesoderm signifies that portion of the head mesoderm which derives from the pre-chordal plate area located at the anterior end of the foregut. The pre-chordal plate mesoderm is associated closely with the foregut entoderm and anterior extremity of the notochord in the late blastula and gastrula in the fishes and amphibia. However, in reptiles, birds, and mammals, this association is established secondarily with the foregut entoderm by means of the notochordal canal and primitive-pit invaginations during gastrulation. (See Chap. 9 and also Hill and Tribe, ’24.)

(Note: It is advisable to state that Adelmann, ’32, relative to the 19-somite embryo of the urodele Ambystoma pimctatum, distinguishes between a prechordal mesoderm, which forms the core of the mandibular visceral arch, and the pre-chordal plate mesoderm, which earlier in development is associated with the dorsal anterior portion of the foregut entoderm. See figure 252E.)

During the period when the major organ-forming areas are being tubulated.

NEURAL ECTODERM

.RANCHIAL POUCHBRANCHIAL GROOVE OR gill- SL 1 T AREA

HYPOMERICl MESOOERMAI^ CONTRIBUTION ^ TO LATERAL BODY WALL

MESENCHYMAL CONTRIBUTION FROM SPLANCHNIC LAYER OF HYPOMERE

Fig. 252. Mesodermal contributions to developing body. (A-D) Sections through developing chick of 48-52 hrs. of incubation. (A) Section through somites of caudal trunk area showing primitive area of mesoderm and coelomic spaces. (B) Section through anterior trunk area depicting early differentiation of somite. (C) Section through trunk area posterior to heart revealing later stage of somite differentiation than that shown in B. (D) Section through developing heart area. Observe dermomyotome, sclerotomic mesenchyme, and mesenchymal contributions of hypomere to forming body substance. (E) Mesodermal contributions to anterior end of developing embryo of Ambystoma of about 19 somites. (Redrawn and modified from Adelmann: 1932, J. Morphol. 54.) (F) Frontal section of early post-hatching larva of Rana pipiens show ing mass of mesoderm lying between gut, epidermal and neural tubes, together with the contributions of the mesoderm to the visceral arches.

524

CONTRIBUTIONS OF THE MESODERM TO PRIMITIVE BODY FORMATION

525

the pre-chordal plate mesoderm separates as a mass of mesenchyme from the antero-dorsal aspect of the foregut, anterior to the cephalic terminus of the notochord (fig. 232G, H). It migrates forward as two groups of mesenchyme connected at first by an interconnecting bridge of mesenchyme. Eventually these two mesenchymal masses become separated and each forms a dense aggregation of mesodermal cells over the mandibular visceral arch and just caudal to the eye (fig. 252E). In the shark embryo and in the chick it gives origin to the pre-mandibular somites (condensations) which probably give origin to the eye muscles innervated by the oculomotor or third cranial nerves. In Ambystoma, Adelmann (’32, p. 52) describes the pre-chordal plate mesoderm as giving origin to “the eye muscles” and “probably much of the head mesenchyme ahead of the level of the first (gill) pouch, but its caudal limit cannot be exactly determined.” Thus it appears that a portion of the head mesoderm in the region anterior to the notochordal termination is derived from the pre-chordal plate mesoderm in all vertebrates.

c. Head Mesoderm Contributed by Neural Crest Material

A conspicuous phase of the development of the head region in vertebrate embryos is the extensive migration of neural crest cells which arise in the middorsal area as the neural tube is formed (Chap. 10; fig. 222C-F). Aside from contributing to the nervous system (Chap. 19), a portion of the neural crest material migrates extensively lateroventrally and comes to lie within the forming visceral (branchial) arches, contributing to the mesoderm in these areas (figs. 222C-F; 230D, F). Also, consult Landacre (’21); Stone (’22, ’26, and ’29); and Raven (’33a and b). On the other hand, Adelmann (’25) in the rat and Newth (’51 ) in the lamprey, Lampetra planeri, were not able to find evidence substantiating this view. However, pigment cells (melanophores) of the skin probably arise from neural crest cells in the head region of all vertebrate groups.

d. Head Mesoderm Originating from Post-otic Somites

There is good evidence that the musculature of the tongue takes its origin in the shark embryo and lower vertebrates from cells which arise from the somites of the trunk area, immediately posterior to the otic (ear) vesicle, from whence they migrate ventrad to the hypobranchial region and forward to the area of the developing tongue (fig. 253). In the human embryo, Kingsbury (’15) suggests this origin for the tongue and other hypobranchial musculature. However, Lewis (’10) maintains that, in the human, the tongue musculature arises from mesenchyme in situ.

3. Origin of the Mesoderm of the Tail

In the Amphibia, the tail mesoderm has been traced by means of the Vogt staining method to tail mesoderm in the late blastular and early gastrular

526

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

stages. At the time of tail-rudiment formation, this mesoderm forms two bilateral masses of cells located within the “tail bud” or “end bud.” These cellular masses proliferate extensively as the tail bud grows caudally and give origin to the mesoderm of the tail. Similarly, in other vertebrates, the mesoderm of the future tail is present as mesenchyme in the terminal portion of the tail bud. These mesenchymal cells proliferate, as the tail grows caudalward, and leave behind the mesoderm, which gradually condenses into the epithelial masses or segments (myotomes) along either side of the notochord and neural tube.

4. Contributions of the Trunk Mesoderm to the Developing Body

The mesoderm of the trunk area contributes greatly to the development of the many body organs and systems in the trunk region. Details of this contribution will be described in the chapters which follow, but, at this point, it is well to survey the initial activities of the mesodermal tubes of the trunk area in producing the vertebrate body.

a. Early Differentiation of the Somites or Epimere

The somites (figs. 217, 237, 252) contribute much to the developing structure of the vertebrate body. This fact is indicated by their early growth and differentiation. For example, the ventro-mesial wall of the fully developed somite gradually separates from the rest of the somite and forms a mass of mesenchymal cells which migrates mesad around the notochord and also dorsad around the neural tube (fig. 252A-C). The mesenchyme which thus arises from the somite is known as the sclerotome. In the somite of the higher vertebrates just previous to the origin of the sclerotome, a small epithelial core of cells becomes evident in the myocoel; this core contributes to the sclerotomic material (fig. 252B). As a result of the segregation of the sclerotomic tissue and its migration mesad to occupy the areas around the notochord and nerve cord, the latter structures become enmeshed by a primitive skeletogenous mesenchyme. The notochord and sclerotomic mesenchyme are the foundation for the future axial skeleton of the adult, including the vertebral elements and the caudal part of the cranium as described in Chapter 15.

After the departure of sclerotomic material, myotomic and dermatomic portions of the somite soon rearrange themselves into a hollow structure (fig. 252C, D), in which the myotome forms the inner wall and the dermatome the outer aspect. This composite structure is the dermomyotome, and the cavity within, the secondary myocoei. In many vertebrates (fishes, amphibia, reptiles, and birds), the dermatome gives origin to cells which migrate into the region of the developing dermis (Chap. 12) and contributes to the formation of this layer of the skin.

CONTRIBUTIONS OF THE MESODERM TO PRIMITIVE BODY FORMATION

527

b. Early Differentiation of the Mesomere (Nephrotome)

The differentiation of the nephrotome or intermediate mesoderm will be considered later (Chap. 18) in connection with the urogenital system.

c. Early Differentiation and Derivatives of the Hypomere

The lateral-plate mesoderm (hypomere), figure 252A, performs an extremely important function in embryological development. The cavity of the hypomere (splanchnocoel) and the cellular offspring from the hypomeric mesoderm, which forms the wall of this cavity, give origin to much of the structural material and arrangement of the adult body.

1) Contributions of the Hypomere (I^ateral Plate Mesoderm) to the Developing Pharyngeal Area of the Gut Tube. The developing foregut (Chap. 13) may be divided into four main areas, namely, (1) head gut, (2) pharyngeal, (3) esophageal, and (4) stomach areas. The head gut is small and represents a pre-oral extension of the gut; the pharyngeal area is large and expansive and forms about half of the forming foregut in the early embryo; the esophageal segment is small and constricted; and the forming stomach region is enlarged. At this point, however, concern is given specifically to the developing foregut in relation to the early development of the pharyngeal region.

In the pharyngeal area the foregut expands laterally. Beginning at its anterior end, it sends outward a series of paired, pouch-like diverticula, known as the branchial (pharyngeal or visceral) pouches. These pouches push outward toward the ectodermal (epidermal) layer. In doing so, they separate the lateral plate mesoderm which synchronously has divided into columnar masses or cells (fig. 252E, F). Normally, about four to six pairs of branchial (pharyngeal) pouches are formed in gnathostomous vertebrates, although in the cyclostomatous fish, Petromyzon, eight pairs appear. In the embryo of the shark, Squalus acanthias, six pairs are formed, while in the amphibia, four to six pairs of pouches may appear (fig. 252F). In the chick, pig, and human, four pairs of pouches normally occur (figs. 259, 261). Also, invaginations or inpushings of the epidermal layer occur, the branchial grooves (visceral furrows); the latter meet the entodermal outpocketings (figs. 252F; 262B).

The end result of all these developmental movements in the branchial area is to produce elongated, dorso-ventral, paired columns of mesodermal cells (figs. 252E; 253), the visceral or branchial arches, which alternate with the branchial-groove-pouch or gill-slit areas (figs. 252F; 253). The most anterior pair of visceral arches forms the mandibular visceral arches; the second pair forms the hyoid visceral arches; and the succeeding pairs form the branchial (gill) arches (figs. 239C, D; 240; 244; 246; 252E; 253). The branchial arches with their mesodermal columns of cells will, together with the contributions from the neural crest cells referred to above, give origin to the connective, muscle, and blood-vessel-forming tissues in this area.

528

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

2) Contributions of the Hypomere (Lateral Plate Mesoderm) to the Formation of the Gut Tube and Heart Structures. Throughout the length of the forming gut tube, from the oral area to the anal region, the lateral plate mesoderm (mesoblast) contributes much to the forming gut tube. This is occasioned to a great extent posterior to the pharyngeal area by the fact that the inner or mesial walls of the two hypomeres enswathe the forming gut tube as they fuse in the median plane (fig. 241), forming the dorsal and ventral mesenteries of the gut. However, in the heart area, due to the dorsal displacement of the foregut, the dorsal mesentery is vestigial or absent while the ventral mesentery is increased in extent. Each mesial wall of the hypomeric mesoderm, forming the ventral mesentery in the region of the developing heart, becomes cupped around the primitive blood capillaries, coursing anteriad in this area to form the rudiments of the developing heart. The ventral mesentery in the heart area thus gives origin to the dorsal mesocardium, the ventral mesocardium, and the rudimentary, cup-shaped, cpimyocardial structures around the fusing blood capillaries (figs. 236C-D; 254A). The primitive blood capillaries soon unite to form the rudiment of the future endocardium of the heart, while the enveloping epimyocardium establishes the rudiment of the future muscle and connective tissues of the heart (Chap. 17).

On the other hand, in the region of the stomach and continuing posteriorly to the anal area of the gut, the movement mediad of the mesial walls of the two lateral plate (hypomeric) mesodermal areas occurs in such a way as to

Fig. 253. Diagram illustrating the basic plan of the vertebrate head based upon the shark, Scy Ilium canicula. (Modified from Goodrich: 1918, Quart. Jour. Micros. Science, 63.)

CONTRIBUTIONS OF THE MESODERM TO PRIMITIVE BODY FORMATION

529

the hypomeres to the developing heart and gut structures in reptiles, birds, and mammals. Sections are drawn through the following regions: (A) Through primitive tubular heart anterior to sinus venosus. (B) Through caudal end of sinus venosus and lateral meso* cardia. (C) Through liver region. (D) Through region posterior to liver. (E) Through posterior trunk in region of urinary bladder.

envelop or enclose the gut tube. This enclosure readily occurs because in this region of the trunk, the gut tube lies closer to the ventral aspect of the embryo than in the heart area. Consequently, a dorsal mesentery above and a ventral mesentery below the primitive gut tube are formed (fig. 25 4C). The dorsal and ventral mesenteries may not persist everywhere along the gut (fig. 254D). The degree of persistence varies in different vertebrates; these variations will be mentioned later (Chap. 20) when the coelomic cavities are discussed. However, there is a persistence of the ventral mesentery below the stomach and anterior intestinal area of all vertebrates, for here the ventral mesentery (i.e., the two medial walls of the lateral plate mesoderm below the gut) contributes to the development of the liver and the pancreas. These matters are discussed in Chapter 13.

Aside from the formation of the dorsal and ventral mesenteries by the inward movement and fusion of the medial walls of the lateral plate mesoderm above and below the primitive enteron or gut tube, that part of the medial walls of the lateral plate mesoderm which envelops the primitive gut itself is of great importance. This importance arises from the fact that the entoderm of the gut only forms the lining tissue of the future digestive tract and its various glands, such as the liver, pancreas, etc., whereas mesenchymal contributions from the medial wall of the lateral plate mesoderm around the

530

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

entodermal lining give origin to smooth muscle tissue, connective tissue, etc. (figs. 254C, D; 258; 260; 262; 278C). It is apparent, therefore, that the gut throughout its length is formed from two embryonic contributions, namely, one from the entoderm and the other from the mesenchyme given off by the medial walls of the lateral plate or hypomeric mesoderm.

{Note: The word splanchnic is an adjective and is derived from a Greek word meaning entrails or bowels. That is, it pertains to the soft structures within the body wall. The plural noun viscera (singular, viscus) is derived from the Latin and signifies the same structures, namely, the heart, liver, stomach, intestine, etc., which lie within the cavities of the body. It is fitting, therefore, to apply the adjective splanchnic to the medial portion of the hypomere because it has an intimate relationship with, and is contributory to, the development of the viscera. The somatic mesoderm, on the other hand, is the mesoderm of the lateral or body-wall portion of the hypomere. The word splanchnopleure is a noun and it designates the composite tissue of primitive entoderm and splanchnic mesoderm, while the word somatopleure is applied to the compound tissue formed by the primitive lateral wall of the hypomere (somatic mesoderm) plus the primitive ectoderm overlying it. The coelom proper or spianchnocoel is the space or cavity which lies between the splanchnic and somatic layers of the lateral plate or hypomeric mesoderm. During later development, it is the cavity in which the entrails lie.

3) Contributions of the Hypomere (Lateral Plate Mesoderm) to the External (Ectodermal or Epidermal) Body Tube. The somatopleural mesoderm gives origin to a mass of cellular material which migrates outward to lie along the inner aspect of the epidermal tube in the lateral and ventral portions of the developing body (fig. 252A, D). In the dorsal and dorso-lateral regions of the body, contributions from the sclerotome and dermatome apparently aid in forming this tissue layer. The layer immediately below the epidermis constitutes the embryonic rudiment of the dermis. (See Chap. 12.)

4) Contributions of the Hypomere or Lateral Plate Mesoderm to the Dorsal Body Areas. Many cells are given off both from splanchnic and somatic layers of the hypomeric mesoderm to the dorsal body areas above and along either side of the dorsal aorta (fig. 254), contributing to the mesenchymal “packing tissue” in the area between the notochord and differentiating somite, extending outward to the dermis.

5) Contributions of the Lateral Plate Mesoderm to the Walls of the Coelomic Cavities. The pericardial, pleural, and peritoneal cavities are lined, as stated above, by an epithelial type of tissue called mesothelium (fig. 254A-E). These coelomic spaces (see Chap. 20) are derived from the fusion of the two primitive splanchnocoels or cavities of the two hypomeres. External to the mesothelial lining of the coelomic spaces, there ultimately is developed a fibrous, connective tissue layer. Thus, mesothelium and connective tissue form.

Fig. 255. This figure illustrates different types of body form in various vertebrates during embryonic development. A, D, H, M, and Q show primitive embryonic body form in the developing shark, rock fish, frog, chick, and human. B, larval form of shark; E and F, larval forms of rock fish; I and J, larval forms of frog; N and O, larval forms of chick; R, larval form of human. C, G, K, L, P, and S represent definitive body form in the above species. (Figures on rockfish development (Roccus saxatilis) redrawn from Pearson: 1938, Bull. Bureau of Fisheries, L). S. Dept, of Commerce, vol. 49; figures on chick redrawn from Hamburger and Hamilton: 1951, J. Morphol., vol. 88; figure Q, of developing human embryo, redrawn and modified from model based upon Normentafeln of Keibel and Elze: 1908, vol. 8, G. Fischer, Jena; Dimensions of human embryos in R and S, from Mall: Chap. 8, vol. 1, Human Embryology, by F. Keibel and F. P. Mall, 1910, Lippincott, Philadelphia.)

531

532

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

in general, the walls of the coelomic spaces. These two tissues arise directly from the hypomeric mesoderm.

5. Embryonic Mesenchyme and Its Derivatives

The mesenchymal cells given off from the mesodermal tubes of the trunk area, namely, (1) sclerotomic mesenchyme, (2) dermatomic mesenchyme, (3) mesenchymal contributions from the lateral plate mesoblast (hypomere) to the gut, skin, heart, and (4) the mesenchyme contributed to the general regions of the body lying between the epidermal tube, coelom, notochord, and neural tube, form, together with the head and tail mesoderm, the general packing tissue which lies between and surrounding the internal tubular structures of the embryo (fig. 254). Its cells may at times assume polymorphous or stellate shapes. This loose packing tissue of the embryo constitutes the embryonic mesenchyme. (See Chap. 15.)

This mesenchyme ultimately will contribute to the following structures of the body:

(a) Myocardium (cardiac musculature, etc.) and the epicardium or covering coelomic layer of the heart (Chap. 17),

(b) endothelium of blood vessels, blood cells (Chap. 17),

(c) smooth musculature and connective tissues of blood vessels (Chaps. 16 and 17),

(d) spleen, lymph glands, and lymph vessels (Chap. 17),

(e) connective tissues of voluntary and involuntary muscles (Chap. 16),

(f) connective tissues of soft organs, exclusive of the nerve system (Chap. 15),

(g) connective tissues in general, including bones and cartilage (Chap. 15),

(h) smooth musculature of the gut tissues and gut derivatives (Chap. 16),

(i) voluntary or striated muscles of the tail from tail-bud mesenchyme (Chap. 16),

(j) striated (voluntary) musculature of face, jaws, and throat, derived from the lateral plate mesoderm in the anterior pharyngeal region (Chap. 16),

(k) striated (voluntary) extrinsic musculature of the eye (Chap. 16),

(l) intrinsic, smooth musculature of the eye (Chap. 16),

(m) tongue and musculature of bilateral appendages, derived from somitic muscle buds (sharks) or from mesenchyme possibly of somitic origin (higher vertebrates) (Chap. 16), and

(n) chromatophores or pigment cells of the body from neural crest mesenchyme (Chap. 12).

SUMMARY OF DERIVATIVES OF ORGAN-FORMING AREAS

533

£. Summary of Later Derivatives of the Major Presumptive Organforming Areas of the Late Blastula and Gastrula

1. Neural Plate Area (Ectoderm)

This area gives origin to the following:

(a) Neural tube,

(b) optic nerves and retinae of eyes,

(c) peripheral nerves and ganglia,

(d) chromatophores and chromaffin tissue (i.e., various pigment cells of the skin, peritoneal cavity, etc., chromaffin cells of supra-renal gland),

(e) mesenchyme of the head, neuroglia, and

(f) smooth muscles of iris.

2. Epidermal Area (Ectoderm)

This area gives origin to:

(a) Epidermal tube and derived structures, such as scales, hair, nails, feathers, claws, etc.,

(b) lens of the eye, inner ear vesicles, olfactory sense area, general, cutaneous, sense organs of the peripheral area of the body,

(c) stomodaeum and its derivatives, oral cavity, anterior lobe of pituitary, enamel organs, and oral glands, and

(d) proctodaeum from which arises the lining tissue of the anal canal.

3. Entoderm AL Area

From this area the following arise:

(a) Epithelial lining of the primitive gut tube or metenteron, including: (1) epithelium of pharynx; epithelium pharyngeal pouches and their derivatives, such as auditory tube, middle-ear cavity, parathyroids, and thymus; (2) epithelium of thyroid gland; (3) epithelial lining tissue of larynx, trachea, and lungs, and (4) epithelium of gut tube and gut glands, including liver and pancreas,

(b) most of the lining tissue of the urinary bladder, vagina, urethra, and associated glands,

(c) Seessel’s pocket or head gut, and

(d) tail gut.

4. Notochordal Area

This area:

(a) Forms primitive antero-posterior skeletal axis of all chordate forms,

(b) aids in induction of central nerve tube.

534

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

(c) gives origin to adult notochord of Amphioxus and cyclostomatous fishes and to notochordal portions of adult vertebral column of gnathostomous fishes and water-living amphibia, and

(d) also, comprises the remains of the notochord in land vertebrates, such as “nucleus pulposus” in man.

5. Mesodermal Areas

These areas give origin to:

(a) Epimeric, mesomeric, and hypomeric areas of primitive mesodermal tube,

(b) epimeric portion also aids in induction of central nerve tube,

(c) muscle tissue, involuntary and voluntary,

(d) mesenchyme, connective tissues, including bone, cartilage,

(e) blood and lymphoid tissue,

(f) gonads with exception of germ cells, genital ducts, and glandular tissues of male and female reproductive ducts, and

(g) kidney, ureter, musculature and connective tissues of the bladder, uterus, vagina, and urethra.

6. Germ-cell Area

This area gives origin to:

(a) Primordial germ cells and probably to definitive germ cells of all vertebrates below mammals and

(b) primordial germ cells of mammals and possibly to definitive germ cells.

F. Metamerism

1. Fundamental Metameric Character of the Trunk and Tail Regions of the Vertebrate Body

Many animals, invertebrate as well as vertebrate, are characterized by the fact that their bodies are constructed of a longitudinal series of similar parts or metameres. As each metamere arises during development in a similar manner and from similar rudiments along the longitudinal or antero-posterior axis of the embryo, each metamere is homologous with each of the other metameres. This type of homology in which the homologous parts are arranged serially is known as serial homology. Metamerism is a characteristic feature of the primitive and later bodies of arthropods, annelids, cephalochordates, and vertebrates.

In the vertebrate group, the mesoderm of the trunk and tail exhibits a type of segmentation, particularly in the epimeric or somitic area. Each pair of somites, for example, denotes a primitive body segment. The nervous system

^OPTIC VESICLE LENS PLACODE .

^ nasal placode — —maxillary process

mandibular arch

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.

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^ NaSOMEDIAL -*

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EXTERNAL EAR

ear tubercles around hyomandibular cleft

-hyoid bone REGlONr

ih j 1

F.O. 256. Developmental features of the human fac. Modified slightly from models by B. Ziegler, Freiburg, after Karl Peter.

535

536

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

also manifests various degrees of segmentation (Chap. 19), although the origin and arrangement of the peripheral nerves in the form of pairs, each pair innervating a pair of myotomic derivatives of the somites, is the most constant feature.

In the cephalochordate, Amphioxus, the segmentation of the early mesoderm is more pronounced than that of the vertebrate group. As observed in Chapter 10, each pair of somites is distinct and entirely separate from other somitic pairs, and each pair represents all the mesoderm in the segment or metamere. That is, all the mesoderm is segmented in Amphioxus. However, in the vertebrate group, only the more dorsally situated mesoderm undergoes segmentation, the hypomeric portion remaining unsegmented.

2. Metamerism and the Basic Morphology of the Vertebrate Head

While the primitive, metameric (segmental) nature of the vertebrate trunk and tail areas cannot be gainsaid, the fundamental metamerism of the vertebrate head has been questioned. Probably the oldest theory supporting a concept of cephalic segmentation was the vertebral theory of the skull, propounded by Goethe, Oken, and Owen. This theory maintained that the basic structure of the skull demonstrated that it was composed of a number of modified vertebrae, the occipital area denoting one vertebra, the basisphenoidtemporo-parietal area signifying another, the presphenoid-orbitosphenoidfrontal area denoting a third vertebra, and the nasal region representing a fourth cranial vertebra. (Consult Owen, 1848.) This theory, as a serious consideration of vertebrate head morphology was demolished by the classic Croonian lecture given in 1858 by Huxley (1858) before the Royal Society of London. His most pointed argument against the theory rested upon the fact that embryological development failed to support the hypothesis that the bones of the cranium were formed from vertebral elements.

A factor which aroused a renewal of interest in a segmental interpretation of the vertebrate head was the observation by Balfour (1878) that the head of the elasmobranch fish, Scy Ilium, contained several pairs of pre-otic (prootic) somites (that is, somites in front of the otic or ear region). Since Balfour’s publication, a large number of studies and dissertations have appeared in an endeavor to substantiate the theory of head segmentation. The anterior portion of the central nervous system, cranial nerves, somites, branchial (visceral) arches and pouches, have all served either singly or in combination as proffered evidence in favor of an interpretation of the primitive segmental nature of the head region. However, it is upon the head somites that evidence for a cephalic segmentation mainly depends.

A second factor which stimulated discussion relative to head segmentation was the work of Locy (1895) who emphasized the importance of so-called neural segments or neuromcres (Chap. 19) as a means of determining the

ARROWS SHOW water CURRENTS

Fig. 257. Drawings of early frog tadpoles showing development of early systems. (A) Frog tadpole (R. pipiens) of about 6 7 mm. It is difficult to determine the exact number of vitelline arteries at this stage of development and the number given in the figure is a diagrammatic representation. {A') Shows right and left ventral aortal divisions of bulbus cordis. (B) Anatomy of frog tadpole of about 10-18 mm. See also figures 280 and 335.

537

540

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

primitive segmental structure of the vertebrate brain. It is to be observed that the more conservative figure 253, taken from Goodrich, does not emphasize neuromeres, for, as observed by Kingsbury (’26, p. 85), the evidence is overwhelmingly against such an interpretation. The association of the cranial nerves with the gill (branchial) region and the head somites, shown in figure 253, will be discussed further in Chapter 19.

A third factor which awakened curiosity, concerning the segmental theory of head development, is branchiomerism. The latter term is applied to the development of a series of homologous structures, segmentally arranged, in the branchial region; these structures are the visceral arches and branchial pouches referred to above. As mentioned there, the branchial pouches or outpocketings of the entoderm interrupt a non-segmented mass of lateral plate (hypomeric) mesoderm, and this mesoderm secondarily becomes segmented and located within the visceral arches. These arches when formed, other than possibly the mandibular and the hyoid arches (fig. 253), do not correspond with the dorsal somitic series. Consequently, “branchiomerism does not, therefore, coincide with somitic metamerism.” (See Kingsbury, ’26, p. 106.)

Undoubtedly, much so-called “evidence” has been accumulated to support a theory of head segmentation. A considerable portion of this evidence apparently is concerned more with segmentation as an end in itself than with a frank appraisal of actual developmental conditions present in the head (Kingsbury and Adelmann, ’24 and Kingsbury, ’26). However, the evidence which does resist critical scrutiny is the presence of the head somites which includes the pre-otic somites and the first three or four post-otic somites. While the pre-otic somites are somewhat blurred and slurred over in their development in many higher vertebrates, the fact of their presence in elasmobranch fishes is indisputable and consistent with a conception of primitive head segmentation.

Furthermore, aside from a possible relationship with head-segmentation phenomena, the appearance of the pre-otic and post-otic head somites coincides with basic developmental tendencies. As observed above, for example, there is a tendency for nature to use generalized developmental procedures in the early development of large groups of animals (see von Baer’s laws, p. 522, and also discussion relative to Haeckel’s biogenetic law in Chap. 7). Nature, in other words, is utilitarian, and one can be quite certain that if general developmental procedures are used, they will prove most efficient when all factors are considered. At the same time, while generalized procedures may be used, nature does not hesitate to mar or elide parts of procedures when needed to serve a particular end. The obliteration of developmental steps during development is shown in the early development of the mesoderm in the vertebrate group compared to that which occurs in Amphioxus. In the vertebrate embryo, as observed previously, the hypomeric mesoderm is unsegmented except in a secondary way and in a restricted area as occurs in branchiomerism. However, in Amphioxus, early segmentation of the meso

METAMERISM

541

derm is complete dorso-ventrally, including the hypomeric region of the mesoderm. It becomes evident, therefore, that the suppression of segmentation in the hypomeric area in the vertebrate embryo achieves a precocious result which the embryo of Amphioxus reaches only at a later period of development. Presumably in the vertebrate embryo, segmentation of the epimeric mesoderm is retained because it serves a definite end, whereas segmentation of the hypomeric mesoderm is deleted because it also leads to a necessary end result in a direct manner.

When applied to the developing head region, this procedure principle means this: A primitive type of segmentation does tend to appear in the pre-otic area as well as in the post-otic portion of the head, as indicated by the pre-otic and post-otic somites, and secondarily there is developed a branchial metam

GASSERIAN GANGLION I ME TENCEPHAUON

geniculate GANGLION OF NERVE Stt ACOUSTIC GANGLION OF NERVE :

MYf lencephalon OTIC VESICLE

SUPERIOR GANGLION OF NERVE H JUGULAR GANGLION OF NERVE X PETROSAL GANGLION OF NERVE IX ^

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NERVE

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pharyngeal pouch in-<: pharyngeal POUCHBC; thyroid BODY BUL0US COROIS

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DORSAL aorta NOTOCHORD stomach LIVER

ventral pancreasdorsal pancreas gall blaode MESONEPHROS- —

MIDGUT AN DUCT

glomeruli

COLLECTING DUCT HINDGUT

SPINAL GANGLION

Fig. 259. Chick embryo reconstruction of about 100 hrs. of incubation with special reference to the nervous and urinary systems. See also fig. 336D.

bation. Reference should 5

BASIC HOMOLOGY OF ORGAN SYSTEMS

545

erism (branchiomerism) . However, all these segmental structures serve a definite end. In other areas, head development proceeds in a manner which obscures segmentation, for the probable reason that segmentation does not fit into the developmental pattern which must proceed directly and precociously to gain a specific end dictated by problems peculiar to head development.

{Note: For a critical analysis of the supposed facts in favor of segmentation, together with a marshaling of evidence against such an interpretation, consult Kingsbury and Adelmann (’24) and for a favorable interpretation of the segmental nature of the head region, see Goodrich (’18) and Delsman (’22). Figure 253 is taken from Goodrich (’18), and the various structures which favor a segmental interpretation of the head region are shown.)

G. Basic Homology of the Vertebrate Organ Systems

1. Definition

Homology is the relationship of agreement between the structural parts of one organism and the structural parts of another organism. An agreeable relationship between two structures is established if:

( 1 ) the two parts occupy the same relative position in the body,

(2) they arise in the same way embryonically and from the same rudiments, and

(3) they have the same basic potencies.

By basic potency is meant the potency which governs the initial and fundamental development of the part; it should not be construed to mean the ability to produce the entire structure. To the basic potency, other less basic potencies and modifying factors may be added to produce the adult form of the structure.

2. Basic Homology of Vertebrate Blastulae, Gastrulae, and Tubulated Embryos

In Chapters 6 and 7, the basic conditions of the vertebrate blastula were surveyed, and it was observed that the formative portion of all vertebrate blastulae presents a basic pattern, composed of major presumptive organforming areas oriented around the notochordal area and a blastocoelic space. During gastrulation (Chap. 9), these areas are reoriented to form the basic pattern of the gastrula, and although round and flattened gastrulae exist, these form one, generalized, basic pattern, composed of three germ layers arranged around the central axis or primitive notochordal rod. Similarly, in Chapter 10, the major organ-forming areas are tubulated to form an elongated embryo, composed of head, pharyngeal, trunk, and tail regions. As tubulation is effected in much the same manner throughout the vertebrate series and as the pre-chordal plate mesoderm, foregut entoderm, notochord, and somitic meso

546

BASIC FEATURES OF VERTEBRATE MORPHOGENESIS

geniculate ganglion of seventh nerve

ACOUSTIC GANGLION OF EIGHTH NERVE AUDITORY VESICLE

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PORTAL VEIN DORSAL AORTA

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GLOMERULUS MESONEPHRIC TUBULE

DORSAL AORTA MESONEPHRIC DUCT

Fig. 261. Drawings of pig embryos of about 9.5 to 12 mm. (A) Reconstruction of about 9.5 to 10 mm. pig embryo with special emphasis on the arterial system.

derm appear to be the main organizing influence throughout the series (Chap. 10), the conclusion is inescapable that the tubulated embryos of all vertebrates are homologous basically, having the same relative parts, arising in the same manner, and possessing the same basic potencies within the parts. To this conclusion must be added a caution, namely, that, although the main segments or specific organ regions along each body tube of one species are homologous with similar segments along corresponding tubes of other species, variations may exist and non-homologous areas may be insinuated or homologous areas

BASIC HOMOLOGY OF ORGAN SYSTEMS

547

may be deleted along the respective tubes. Regardless of this possibility, a basic homology, however, appears to exist.

During later development through larval and definitive body-form stages, a considerable amount of molding or plasis by environmental and intrinsic factors may occur. An example of plasis is given in the development of the forelimb rudiment of the fish, frog, bird, and pig. In the definitive form, these structures assume different appearances and are adapted for different func

METENCEPHALON

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ESOPH AGU S VALVES OF

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GALL BLADDER

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MESONEPHRIC KIDNE

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ju / POSTERIUS ~ — ^INFUNDIBULUM OIEUCEPHALON

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CLOACA

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B.

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TISSUE SPINAL GANGLION

Fig. 261 — (Continued} (B) Median sagittal section of 10 mm. embryo.

VEIN OF maxillary REGION (BRANCH OF INTERNAL JUGULAR)

OTIC VESICLE

VEIN OF

MANDIBULAR REGION BRANCH OF EXTERNAL JUGULAR)

INTERNAL JUGULAR VEIN DORSAL

jSEGMENTAL VEINS EXTERNAL JUGULAR VEIN

LEFT DUCT OF CUVIER RIGHT VITELLINE VEIN LIVER DUCTUS VENOSUS HEPATIC VEINS PORTAL VEIN

UMBILICAL

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'TRANSVERSE ANASTOMOSIS OF SITBCAROINALS

POSTERIOR CARDINAL VEIN

PIG EMBRYO SHOWING RIGHT HALF OF VENOUS SYSTEM

Fig. 261 — (Continued) (C) Lateral view of 12 mm. embryo showing venous system. (C is redrawn and modified from Minot; 1903, A Laboratory Text-book of Embryology, Blakiston, Philadelphia.)

548

Fig. 262. Sections and stereograms of 10 mm. pig embryo.

MCSCNCHYME^

Ibl— (Continued) Sections and stereograms of 10 mm. pig embryo

BIBLIOGRAPHY

551

tional purposes. Basically, however, these structures are homologous, although plasis produces adult forms which appear to be different.

A further statement should be added, concerning that type of molding or plasis of a developing structure which produces similar structures from conditions which have had a different genetic history. For example, the bat’s fore limb rudiment is molded to produce a structure resembling superficially that of the bird, although modern bats and birds have arisen through different lines of descent. Similarly, the teeth of certain teleost fishes superficially resemble the teeth of certain mammals, an effect produced from widely diverging lines of genetic descent. These molding effects or homoplasy, which produce superficially similar structures as a result of adaptations to certain environmental conditions, are called convergence, parallelism, and analogy. An example of experimental homoplasy is the induction of eye lenses in the embryo by the transplantation of optic-cup material to a place in the epidermis which normally does not produce a lens.

{Note: For a discussion of homology, homogeny, plasis, convergence, etc., see Tait, ’28.)

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