Book - Comparative Embryology of the Vertebrates 3-6

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Nelsen OE. Comparative embryology of the vertebrates (1953) Mcgraw-Hill Book Company, New York.

   Comparative Vertebrate Embryology 1953: 1. The Period of Preparation | 2. The Period of Fertilization | 3. The Development of Primitive Embryonic Form | 4. Histogenesis and Morphogenesis of the Organ Systems | 5. The Care of the Developing Embryo | Figures
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Part III The Development of Primitive Embryonic Form

Part III - The Development of Primitive Embryonic Form: 6. Cleavage (Segmentation) and Blastulation | 7. The Chordate Blastula and Its Significance | 8. The Late Blastula in Relation to Certain Innate Physiological Conditions: Twinning | 9. Gastrulation | 10. Tubulation and Extension of the Major Organ-forming Areas: Development of Primitive Body Form | 11. Basic Features of Vertebrate Morphogenesis

Cleavage (Segmentation) and Blastulation

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.

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 Schleiden 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 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 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).

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


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 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 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 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.

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.

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 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.

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.

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 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 (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).

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.

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.

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

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 I/2 hours after copulation. (H) Inner cell mass and blastocoelic space in embryo, approximately 90 hours after copulation. Entoderm cells have not yet appeared.

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 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


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.

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.

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

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.)

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 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).

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.

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 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.)

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.

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 of 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).

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.

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

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.

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 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.

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.

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 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).

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.

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.

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 horizontal. 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.

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

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.

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,
  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, concerning 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.

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

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.

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.

(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 central 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).

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

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.

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.

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). However, 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 blastoderm 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 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.

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.

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

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.

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 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.

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.

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.

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 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 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.

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 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.

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.

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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