Book - Comparative Embryology of the Vertebrates 3-9

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

1953 Comparative Vertebrate Embryology: 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


A. Some definitions and concepts

1. Gastrulation

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

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

One of the first to define gastrulation in a way which is more consonant with the studies mentioned in the previous paragraph was Keibel (01). He defined gastrulation in the vertebrates ('01, p. 1111) as “the process by which the entodermal, mesodermal and notochordal cells find their way into the interior of the embryo.” It is to be observed that this definition embodies the concept of migration of specific, organ-forming areas. We may restate the concept involved in this definitioq^ in a way which includes invertebrates as well as vertebrates as Gastrulation is the dynamic process during which the major, presumptive \rgan-forming areas of the blastula (Chaps. 6 and 7) become rearranged and reorganized in a way which permits their ready conversion into the body plan of the particular species. That is to say, during the process of gastrulation, the presumptive organ-forming areas of the blastula undergo axiation in terms of the body organization of the species. In some animal species, this reorganization of the blastula into the structural pattern of the gastrula results in the production of a two-layered form, for example, as in Amphioxus; in others (actually in most metazoan species) it brings about the formation of a three-layered condition^ It is apparent, therefore, as observed by Pasteels ('37b, p. 464), that “it is impossible to give a general definition of the gastrula stage.” It is obvious, also, that one cannot define gastrulation in terms of simple invagination, delamination, or the production of a two-layered condition. Many processes, involving intricate movements of cell groups, occur as outlined in the succeeding pages of this chapter.

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

  1. The blastocoel is associated with those movements in the developing blastula which produce the specific cellular configuration of the mature blastula;
  2. during gastrulation, it enables the various, presumptive organ-forming areas of the blastula to be rearranged and to migrate into the particular areas which permit their ready organization and axiation into the scheme of the body form of the particular species; and
  3. in the period of development immediately following gastrulation, it affords the initial space necessary for the tubulation of the major, organ-forming areas.

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

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

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

a. Fundamental Body Plan of the Vertebrate Animal

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

b. The Gastrula in Relation to the Primitive Body Plan

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

  1. The cargo and passengers are boarded, the engines are warmed, and the plane is taxied toward the runway.
  2. Upon reaching the starting end of the runway, the engines are accelerated, and the plane is turned around and headed in the direction of the take-off.
  3. The engines are further accelerated and the plane is moved down the runway for the take-off into the airy regions.

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

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

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

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



Primitive Body Form

1. Epidermal crescent

2. Neural crescent

3. Entodermal area

4. Two mesodermal areas

5. Notochordal crescent

1. Part of ectodermal layer

2. Elongated neural plate a part of ectoderm layer

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

4. Two mesodermal layers on either side of notochord

5. Elongated band of cells lying between mesodermal layers

1. External epidermal tube

2. Dorsally placed neural tube

3. Primitive gut tube

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

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

B. General Processes Involved in Gastrulation

Gastrulation is a nicely integrated, dynamic process; one which is controlled largely by intrinsic (i.e., autonomous) forces bound up in the specific, physicochemical conditions of the various, presumptive, organ-forming areas of the late blastula and early gastrula. These internal forces in turn are correlated with external conditions. One of the important intrinsic factors involves the so-called organization center referred to in Chapter 7. However, before consideration is given to this center, we shall define some of the major processes involved in gastrulation.

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

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

  1. morphogenetic movement of cells,
  2. the organization center and its organizing influences, and
  3. chemodifferentiation.

C. Morphogenetic Movement of Cells

1. Importance of Cell Movements During Development and in Gastrulation

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

The actual factors - physical, chemical, physiological, and mechanical - which effect cell movements are quite unknown. However, this lack of knowledge is not discouraging. In fact, it makes the problem more interesting, for cells are living entities utilizing physicochemical and mechanical forces peculiar to that condition which we call living. The living state is a problem which awaits solution.

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

2. Types of Cell Movement During Gastrulation

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

a. Epiboly

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

(2) Peripheral expansion or divergence.

b. Emboly

(1) Involution.

(2) Invagination.

(3) Concrescence (probably does not occur).

(4) Convergence.

(5) Polyinvagination.

(6) Delamination.

(7) Divergence or expansion.

(8) Extension or elongation.

(9) Blastoporal constriction.

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

3. Description of the Processes Concerned with Epiboly

Epiboly or ectodermal expansion involves the movements of the presumptive epidermal and neural areas during the gastrulative process. The general migration of these two areas is in the direction of the antero-posterior axis of the future embryonic body in ail chordate embryos. In the rounded blastula (e.g., frog, Amphioxus, etc.), the tendency to extend antero-posteriorly produces an enveloping movement in the antero-posterior direction. As a result, the presumptive epidermal and neural areas actually engulf and surround the inwardly moving presumptive notochordal, mesodermal, and entodermal areas. (Study fig. 190A-H.) In flattened blastulae the movements of epiboly are concerned largely with antero-posterior extension, associated with peripheral migration and expansion of the epidermal area. (See fig. 202.) The latter movement of the presumptive epidermal area is pronounced in teleost fishes, where the yolk is engulfed as a result of epidermal growth and expansion (figs. 210B; 21 ID).

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

4. Description of the Processes Involved in Emboly

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

a. Involution and Convergence

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

b. Invagination

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

  1. mechanical or passive infolding of cells, and
  2. active inward streaming or inpushing of cells into the blastocoelic space.

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

c. Concrescence

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

d. Cell Proliferation

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

e. Polyinvagination

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

f. Ingression

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

g. Delamination

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

h. Divergence

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

i. Extension

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

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

1. The Organization Center and the Primary Organizer

Using a transplantation technic on the beginning gastrula of the newt, it was shown by Spemann (T8) and Spemann and Mangold ('24) that the dorsallip region of the blastopore (that is, the chordamesoderm-entoderm cells in this area), when transplanted to the epidermal area of another embryo of the same stage of development, is able to produce a secondary gastrulative process and thus initiate the formation of a secondary embryo (fig. 193). Because the dorsal-lip tissue was able thus to organize the development of a second or twin embryo, Spemann and Mangold described the dorsal-lip region of the beginning gastrula as an “organizer” of the gastrulative process. In its normal position during gastrulation this area of cells has since been regarded as the organization center of amphibian development. It is to be observed in this connection that Lewis ( '07 ) performed the same type of experiment but failed to use an embryo of the same age as a host. As he used an older embryo, the notochordal and mesodermal cells developed according to their presumptive fate into notochordal and somitic tissue but failed to organize a new embryo.

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

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

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

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

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

(b) its capacity for self-organizatiotu

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

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

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

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

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

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

2. Divisions of the Primary Organizer

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

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

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

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

E. Chemodifferentiation and the Gastrulative Process

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

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

F. Gastrulation in Various Chordata

1. Arnphioxus a. Orientation

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

b. Gastrulative Movements

1) Emboly. As gastrulation begins, a marked increase in mitotic activity occurs in the cells of the dorsal crescent, composed of presumptive notochordal and neural plate cells, and also in the cells of the ventral crescent or future mesodermal tissue. The general ectodermal cells or future epidermis also are active (figs. 167, 189, 190B). The entodermal cells, however, are quiescent (Conklin, '32). Accompanying this mitotic activity, the entodermal plate gradually invaginates or folds inwardly into the blastocoel (figs. 189, 190). In doing so, the upper portion of the entodermal plate moves inward more rapidly and pushes forward toward a point approximately halfway between the polar body (i.e., the original midanimal pole of the egg) and the point which marks the anterior end of the future embryo (observe pointed end of arrow, fig. 189). Shortly after the inward movement of the entodermal plate is initiated, notochordal cells in the middorsal region of the blastopore involute, move inward along with the entoderm, and come to occupy a position in the middorsal area of the forming archenteron (fig. 190C-E). Similarly, mesodermal cells in the upper or dorsal ends of the mesodermal crescent gradually converge dorso-mediad and pass into the roof of the forming gastrocoel (archenteron) on either side of the median area occupied by the notochordal cells (fig. 190F, G). Thus the roof of the gastrocoel is composed of notochordal and mesodermal cells (fig. 195 A, B).

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

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

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

As the gastrula is extended in the antero-posterior direction, a shift occurs in the position of the mesodermal cells which form the ventral or mesodermal crescent. The ventral crescent becomes divided ventrally into two halves, and each half gradually moves dorsalward along the inner aspect of the lateral blastoporal lips as gastrulation is accomplished. Each arm of the original crescent in this manner converges dorso-mediad toward the median notochordal cells of the dorsal blastoporal lip, and a mass of mesodermal cells comes to lie along either side of the notochordal cells. As a result of this converging movement, entodermal cells of the blastoporal area converge dorsomediad and come to occupy the ventral lip of the blastopore, together with the externally placed, epidermal cells (fig. 190G, arrow). The blastopore as a whole grows smaller and moves to a dorsal position during the latter changes (fig. 247 A-C).

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

Fig. 189. Gastrulation in Amphioxus. (Modified from Conklin, 32.) (A) Beginning gastrula. (B) Observe that entodermal (hypoblast layer) is projected roughly in direction of future cephalic end of embryo. (C G) Observe continued projection of entoderm toward cephalic end of future embryo. Note also position of polar body. In (F), (G), and (H) the gastrula begins to elongate along the antero-posterior axis of the developing embryo. (H) End of gastrular condition. Blastopore is closed by epidermal overgrowth, and neurenteric canal is formed between archenteron and forming neural tube.

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

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

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

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

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

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

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

2. Gastrulation in Amphibia with Particular Reference to the Frog

a. Introduction

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

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

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

  1. The foregut material lies toward the right at the region of the forming blastoporal lip;
  2. the stomach material is slightly to the left of this area; and
  3. the future intestinal area lies to the left and toward the vegetal pole.

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

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

The experiment pictured in figure 196 involves interchanges between two presumptive areas within the same potential germ layer, i.e., ectoderm. However, Mangold ('23) demonstrated that presumptive epidermis transplanted into the dorsal-lip area, i.e., into the presumptive mesodermal area, may invaginate and form mesodermal tissue. The converse of this experiment was performed by Lopaschov ('35) who found that presumptive mesoderm from the region of the blastoporal lip transplanted to the neural plate area of a somewhat older embryo becomes, in some cases, normally incorporated in the neural tube of the host. Similar interchanges of cells of the late blastula have demonstrated that almost any part, other than the presumptive entoderm, can be interchanged without disturbing the normal sequence of events. However, as gastrulation progresses, interchange from epidermal to neural areas continues to be possible during the early phases of gastrulation (fig. 196C, D) but not at the end of gastrulation. Similar changes occur also in the mesodermal area. Pronounced physiological changes thus occur in the presumptive organforming aieas of the entire epiblastic region during gastrulation.

b. Gastrulation

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

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

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

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

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

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

2) Epiboly. The above description is concerned mainly with emboly, that is, the inward migration of the notochord-mesoderm-entoderm-yolk complex. Allied with these active events is the downward or caudal migration of the blastoporal lips. This migration is illustrated in figure 191B-E. In this figure it may be observed that, as the marginal zone cells of mesoderm and notochord together with the entoderm and yolk pass to the inside, the forces involved in epiboly effect the expansion of the purely ectodermal portion of the cpiblast which gradually comes to cover the entire external surface of the gastrula with the exception of the immediate blastoporal area (study black and white areas in fig. 191A-E). It may be observed further that the neural crescent now is elongated along the antero-posterior, embryonic axis where it forms a shieldshaped region with the broad end of the shield located anteriorly (fig. 192A).

Fig. 192. Movements of the parts of the blastula during gastrulation in amphibia. (Cf. fig. 191.) (A) Results of epiboly. (Cf. fig. 19 1 A T.) Epidermal and neural areas envelop the other areas during gastrulation. (B) Movements of the areas of the blastula during emboly, as seen from the vegetative pole. Heavy arrows, solid and broken, show the converging movements during emboly; light arrows show the extension and divergence of cells after involution at the blastoporal margin (cf. fig. 191A~F). (C) Similar to (B), as seen from the left side.

Fig. 193. Induction of a secondary embryo. (From Spemann, '38.) (A) Host embryo shown in this figure is Triton taeniatus. A median piece of the upper lip of the blastopore of a young gastrula of T. cristatus of approximately the same age as the host was implanted into the ventro-lateral ectoderm of the host. The implanted tissue developed into notochord, somites, etc.; the neural tube was induced from the host ectoderm. (B) Cross section through embryo shown in (A).

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

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

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

A short while previous to blastoporal closure, the midregion of the neural plate area begins to fold ventrad toward the notochord, while its margins are elevated and projected dorso-mediad. The exact limits of the neural plate thus become evident (fig. 197D).

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

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

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

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

Fig. 195. Placement of the presumptive, organ-forming areas in an embryo of Amphioxus of about six to seven somites. (Modified from Conklin, '32.) (A) Section through anterior region. (J) Section through caudal end of embryo. (B-1) Successive sections going posteriorly at different body levels between (A) and (J).

Fig. 196. Ectodermal potencies of the amphibian gastrula. (A and B from Spemann, '38, after Fischer; C and D from Spemann, '38, after Spemann, '18.) (A) Induction of a secondary neural plate in the axolotl gastrula by five per cent oleic acid, emulsified in agar-agar. (B) Induction of secondary neural plate by nucleic acid from calf thymus. (C) Formation of neural plate tissue from presumptive epidermal cells transplanted into neural plate region. (D) Reverse transplant, presumptive neural plate becomes epidermal tissue.

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

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

4) Position Occupied by the Pre-chordal Plate Material. Another feature of the late gastrula remains to be emphasized, namely, the pre-chordal plate composed of entodermal and mesodermal cells integrated with the anterior end of the notochord. During gastrulation the pre-chordal plate invaginates with the entoderm and comes to occupy the roof of the foregut, just anterior to the rod-like notochord (fig. 191D-F). In this position it lies below the anterior part of the neural plate area; it functions strongly in the induction and formation of the cephalic structures, including the brain as indicated above. Because of this inductive ability, it is regarded as a principal part of the head organizer (fig. 191E-G). Eventually pre-chordal plate cells contribute to the pharyngeal area of the foregut and give origin to a portion of the head mesoderm, at least in many vertebrate species (Chap. 11, p. 523).

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

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

d. Summary of Morphogenetic Movements of Cells During Gastrulation in the Frog and Other Amphibia

1) Emboly:

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

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

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

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

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

(f ) Contractile tension or constriction. A considerable constriction or contraction around the edges of the blastopore occurs as gastrulation progresses. This particularly is true when the blastopore gradually grows smaller toward the end of the gastrulative process (Lewis, '49).

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

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

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

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

3. Gastrulation in Reptiles

a. Orientation

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

b. Gastrulation

Immediately following the formation of the hypoblast, the gastrulative phenomena begin with a rather large inpushing or invagination involving the notochordal, mesdoermal areas, particularly the pre-chordal plate and notochordal areas. This invagination extends downward and forward toward the hypoblast along the antero-posterior embryonic axis, and it produces a pouchlike structure known variously as the notochordal canal, blastoporal canal, or chordamesodermal canal (figs. 199A-C; 200A-C). The invaginated notochordal canal reposes upon the entoderm, and both fuse in the region of contact (fig. 200C). The thin layer of cells in the area of fusion soon disappears, leaving the antero-ventral end of the flattened notochordal canal exposed to the archenteric space below. After some reorganization, the notochord appears as a band, extending antero-posteriorly in the median line, associated with the entoderm on either side (fig. 201B-G). However, at the extreme anterior end of the gastrula, the notochordal material, together with the entoderm and to some extent the overlying ectoderm, presents a fused condition. Within this area the pre-chordal plate or anterior portion of the head organizer is located. In this general region of the embryo, foregut, brain, and other head structures eventually arise (fig. 199C). The original, relatively large, notochordal invagination soon becomes a small canal which extends cranio-ventrally from the upper or external opening to the archenteric space which lies below the notochord and entoderm (fig. 200B, E).

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

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

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

During emboly, the presumptive neural plate and epidermal areas are

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

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

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

4. Gastrulation in the Chick

a. Orientation

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

b. Gastrulative Changes

1) Development of Primitive Streak as Viewed from the Surface of Stained Blastoderms. The formation of the primitive streak is a progressive affair. Figure 170 pictures a pre-streak blastoderm, and it is to be observed that the entodermal layer below the epiblast is present as an irregular area in the caudal region of the area pellucida. A median, sagittal section through a comparable stage is shown diagrammatically in figure 171 A. Figure 203 A illustrates an early beginning streak normally found eight hours after incubation of the egg is initiated, while figure 203B presents a medium streak, appearing after about 12 to 13 hours of incubation. In figure 203C, a definite primitive streak appears in which the primitive groove, primitive pit, primitive folds, and Hensen's node (primitive knot) are outlined. This condition occurs after about 18 to 19 hours of incubation. This may be regarded as the mature streak. A later streak after about 19 to 22 hours of incubation is indicated in figure 203 D. Observe that the head process or rudimentary notochord extends anteriorly from Hensen's node, while the mesoderm is a deeper-shaded area emanating from the antero-lateral aspect of the streak. The clear proamnion region may be observed at the anterior end of the area pellucida. In the proamnion area, mesoderm is not present at this time between the ectodermal and entodermal layers.

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

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

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

As the streak differentiates anteriorly by addition of cells to its anterior end, it also elongates posteriorly by cellular additions to its caudal end. The carbon-marking experiments of Spratt demonstrated further that, during the formation of the streak up to about the condition present at 20 to 22 hours of incubation (figs. 2021, K; 203D), almost the entire posterior half of the pellucid area, consisting of presumptive pre-chordal plate, notochord, and mesoderm, is brought into the streak and involuted to the inside between the hypoblast and epiblast (figs. 202F-H; 204). This condition of development is often referred to as the “head-process stage” (stage 5, Hamburger and Hamilton, '51). At this stage the approximate, antero-posterior limits of the future embryonic body of the chick, exclusive of the extra-embryonic tissue, are shown by the general area beginning just anterior to the head process and extending for a short distance posterior to Hensen's node (figs. 203D; 205D, E).

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

  1. the area of Hensen's node and primitive pit concerned with invaginative movements of pre-chordal plate mesoderm and notochordal cells and
  2. the body of the streak.

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

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

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

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

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

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

(b) cell proliferation in situ within the streak.

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

  1. The development of the original streak was suppressed; or
  2. a new, secondary, primitive streak was induced.

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

Certain experiments made by Spratt ('46) lend added evidence of the importance of the hypoblast in primitive-streak formation. In eight experiments in which the hypoblast was removed before streak formation, six cases failed to produce a streak, whereas in two instances a beginning streak was formed. It may be that in the latter two cases, the induction of morphogenetic movements within the epiblast cells occurred previous to hypoblast removal. These experiments are too few to permit a definite conclusion; however, they are suggestive and serve to bolster the conclusion made by Waddington. In a second set of experiments performed by Spratt, chick blastoderms in the prestreak and early-streak stages were inverted and marked with carbon particles. The results showed that the hypoblast moves forward in the median line below the epiblast layer. He also demonstrated that this forward movement of the hypoblast “precedes the anterior differentiation of the primitive streak.” Spratt further observed that: When the movement of the hypoblast deviated to the left or to the right, the primitive streak similarly deviated. This evidence “strongly suggests that the hypoblast influences the development of the primitive streak in the overlying epiblast (Spratt, '46).

Fig. 203. Surface-view drawings of photographs of developing primitive streak. (From Hamburger and Hamilton, '51, after Spratt.) (A) Initial streak, short, conical thickening at posterior end of blastoderm. (Hamburger and Hamilton, '51, stage 2.) (B) Intermediate streak. Thickened streak area approaches center of area pellucida. (Hamburger and Hamilton, '51, stage 3.) (C) Definitive streak (average length, 1.88 mm.).

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

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

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

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

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

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

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

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

(e) Once formed, the initial streak grows anteriad in the median line by: (1) cell proliferation in situ, and by the addition of

(2) converging cells from the epiblast layer.

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

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

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

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

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

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

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

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

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

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

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

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

5. Gastrulation in Mammals

a. Orientation

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

b. Gastrulation in the Pig Embryo

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

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

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

  1. formative or embryonic mesoderm, which remains within the confines of the embryonic or germinal disc and
  2. distally placed non-formative or extra-embryonic mesoderm.

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

In conclusion, therefore, we may assume that, during gastrulation in the pig embryo, emboly and epiboly are comparable and quite similar to these activities in the chick.

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

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

Fig. 209. Longitudinal and transverse sections of the early embryonic (germ) disc of the pig. (C and D after Streeter, '27.) (A) Early, pre-streak, germ disc, showing caudal thickening of epiblast layer. (B) Early streak germ disc, showing thickened caudal edge of disc and beginning migration of mesodermal cells (see fig. 208A). (C) Transverse section through late gastrula, showing three germ layers. Observe that entoderm is attached to either side of median notochordal rod. (D) Longitudinal section through pre-somite, pig blastoderm, showing the relation of notochord to Hensen's node, entoderm, and pre-chordal plate.

c. Gastrulation in Other Mammals

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

  1. In one group an enlarged notochordal canal or pouch is formed which pushes anteriad in the midline between the hypoblast and epiblast; and
  2. in others an abortive notochordal canal or primitive pit is developed, and the notochordal cells are invaginated and proliferated from the thickened anterior aspect of the pit, that is, from the primitive knot or primitive node (Hensen's node).

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

6. Gastrulation in Teleost and Elasmobranch Fishes

a. Orientation

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

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

b. Gastrulation in Teleost Fishes

1) Emboly. As the time of gastrulation approaches, the entire outer edge of the blastodisc begins to thicken and, thereby, forms a ring-like area around the edge of the disc, known as the germ ring (figs. 210C; 21 IB). At the caudal edge of the blastoderm, the germ-ring thickening is not only more pronounced, but it also ‘extends inward for some distance toward the center of the blastoderm (fig. 211 A, B). This posterior prominence of the germ ring forms the embryonic shield.

As gastrulation begins, the entodermal cells of the primary hypoblast at the caudal edge of the embryonic shield stream forward below the epiblast toward the anterior end of the blastodisc (figs. 210A, D). Coincident with this forward movement of the primary hypoblast, a small, crescent-shaped opening appears at the caudal edge of the embryonic shield; this opening forms the dorsal lip of the blastopore (figs. 210A; 211 A, B).

Fig. 210. Gastrulation in teleost fishes. (A) Sagittal section of early gastrula. (Modified slightly from Wilson, 1889.) (B) Midsagittal section through late teleost gastrula. The dorsal and ventral lips of the blastopore are shown approaching each other. (Modified slightly from Wilson, 1889.) (C) Beginning gastrula of early blastoderm of brook trout, Salvelinus. Observe inward (forward) migration of primary hypoblast cells and thickened mass of cells which arises at posterior margin. (After Sumner, '03.) (D)

Later stage in gastrulation of brook trout. (After Sumner, '03.) (E) Transverse section of late gastrula of brook trout, showing the three germ layers. (After Sumner, '03.) (F) Transverse section through late gastrula of sea bass. (After Wilson, 1889.) (G) Midsagittal section through closing blastopore of sea bass. (After Wilson, 1889.) (H) Longitudinal section through late gastrula of the brook trout. (After Sumner, '03.)

In teleost fishes with a primary hypoblast arranged as in Fundulus (fig. 180A, B), as the entodermal cells of the hypoblast move anteriad from the deeper portions of the blastodisc, the entodermal cells exposed at the caudal edge of the epiblast move over the blastoporal lip (i.e., involute) and migrate forward as a part of the entoderm already present in the deeper layer. (See arrows, fig. 180B.) The primary hypoblast thus becomes converted into the secondary hypoblast. In teleosts with a primary hypoblast or entodermal arrangement similar to Salmo (fig. 181 A), the secondary hypoblast is formed by the forward migration and expansion of the entodermal mass located in the caudal area of the embryonic shield. In both Fundulus and Salmo following the initial forward movement of the entodermal cells, the pre -chordal plate cells together with the notochordal cells move caudally and involute over the dorsal blastoporal lip, passing to the inside. (See arrows, figs. 180B; 18 IB.) The pre -chordal plate and notochordal cells migrate forward along the midline of the forming embryonic axis. The pre-chordal plate cells lie foremost, while the notochordal cells are extended and distributed more posteriorly. The presumptive mesoderm in the meantime converges toward the dorso-lateral lips of the blastopore (figs. 180B; 18 IB, see arrows), where it involutes, passing to the inside between the entoderm or secondary hypoblast and epiblast. Within the forming gastrula, the mesoderm becomes arranged along the upper aspect of the entoderm and on either side of the median, notochordal material (fig. 210E, F). The mesoderm in this way becomes inserted between the flattened entoderm (secondary hypoblast) and the outside ectodermal layer (Oppenheimer, '36; Pasteels, '36; Sumner, '03; Wilson, 1889).

During the early phases of gastrulation, the involuted entodermal, notochordal, and mesodermal tissues may superficially appear as a single, thickened, cellular layer. As gastrulation progresses, however, these three cellular areas separate or delaminate from each other. When this separation occurs, the notochordal cells make their appearance as a distinct median rod of cells, while the mesoderm is present as a sheet of tissue on either side of the notochord. The entoderm may form two sheets or lamellae, one on either side of the notochord and below the mesodermal cellular areas (fig. 21 OF) or it may be present as a continuous sheet below the notochord and mesoderm (fig. 210E, H). The entodermal lamellae, when present, soon grow mediad below the notochord and fuse to form one complete entodermal layer (Wilson, 1889).

2) Epiboly. Emboly involves for the most part the movements of cells in the caudal and caudo-lateral areas of the blastoderm, i.e., the embryonic portion of the germ ring. However, while the involution of cells concerned with the development of the dorsal, axial region of the embryo occurs, the margins of the blastodisc beyond the dorsal-lip area, that is, the extra-embryonic, germ-ring tissue, together with the presumptive epidermal area, proceeds to expand rapidly. This growth and expansion soon bring about an engulfment of the yolk mass (figs. 210B; 211C-F). The blastoporal-lip area (i.e., edge of germ ring) ultimately fuses at the caudal trunk region (figs. 210G; 21 IF). As the blastoporal region becomes narrower, a small vesicular outpocketing, known as Kupffer's vesicle, makes its appearance at the ventro-caudal end of the forming embryo at the terminal end of the solid, post-anal gut (fig. 210G) . This vesicle possibly represents a vestige of the enteric portion of the neurenteric canal found in Amphioxus, frog, etc. A certain amount of mesodermal involution occurs around the edges of the germ ring, in some species more than in others (fig. 21 OA, B, peripheral mesodermal involution).

As the cellular dispositions involved in extra-embryonic expansion of the epidermal and germ-ring areas are established, the presumptive, neural plate material (figs. 179, 180, 181) becomes greatly extended antero-posteriorly in the dorsal midline (figs. 210A, H; 21 IE), where it forms into a thickened, elongated ridge or keel. The latter gradually sinks downward toward the underlying notochordal tissue (fig. 210E, F). Also, by the time that the yolk mass is entirely enveloped, the somites appear within the mesoderm near the notochordal axis, and the developing body as a whole may be considerably delimited from the surrounding blastodermic tissue (fig. 21 IG). Therefore, if the envelopment of the yolk mass is taken as the end point of gastrulation in teleosts, the stage at which gastrulation is completed does not correspond to the developmental condition found at the termination of gastrulation in the chick, frog, and other forms. That is, the embryo of the teleost fish at the time of blastoporal closure is in an advanced stage of body formation and corresponds more truly with a chick embryo of about 35 to 40 hours of incubation, whereas the gastrulative processes are relatively complete in the chick at about 20 to 22 hours of incubation.

3) Summary of the Gastrulative Processes in Teleost Fishes:

a) Emboly:

( 1 ) Formation of the secondary hypoblast. The secondary hypoblast forms as a result of the forward migration, expansion, and proliferation of the entodermal cells lying at the caudal margin of the embryonic shield. This forward migration of the entoderm (primary hypoblast) occurs below the upper layer or epiblast and thus produces an underlying entodermal layer or secondary hypoblast.

(2) Pre-chordal plate and notochordal involution. As the formation of the secondary hypoblast is initiated, the presumptive pre-chordal plate and notochordal cells move posteriad and converge toward the dorsal lip of the blastopore, where they involute and pass anteriad in the median line between the hypoblast and epiblast. The hypoblast or entodermal layer may be separated into two flattened layers or lamellae, one on either side of the notochord in some species. However, there is considerable variation among different species as to the degree of separation of the entodermal layer; in the sea bass it appears to be definitely separated, whereas in the trout it is reduced to a single layer of entodermal cells lying below the notochord. The pre-chordal plate, entoderm, and anterior notochord merge into a uniform mass below the cranial end of the neural plate.

Fig. 211. Gastrulation in teleost fishes. (A-F after Wilson, 1889; G from Kerr, '19, after Kopsch.) (A) Sea bass, 16 hours, embryonic shield becoming evident, marks beginning of germ ring. (B) Germ ring well developed. Surface view of blastoderm of 20 hours. (C) Side view of blastoderm shown in (B). (D) Side view, 25 hours.

(E) Surface view, 25 hours. (F) Side view, 31 hours. (G) Late gastrula of trout, Salma fario.

( 3 ) Mesodermal convergence and involution. Along with the migration of notochordal cells, the presumptive mesoderm converges posteriad to the dorso-lateral lips of the blastopore, where it involutes and moves to the inside on either side of the median, notochordal mass and above the forming, secondary hypoblast.

b) Epiboly. The germ-ring tissue and the outer areas of the presumptive epidermal cells gradually grow around the yolk mass and converge toward the caudal end of the developing embryo. Associated with this migration of cells is the anterior-posterior extension of the presumptive neural plate material to form an elongated, thickened, median ridge.

4) Developmental Potencies of the Germ Ring of Teleost Fishes. The germ ring or thickened, marginal area of the teleost late blastula and early gastrula has interested embryologists for many years. It was observed in Chapter 8 that various regions of the marginal area of the blastoderm of the teleost fish have a tendency to form embryos. Luther ('36), working on the trout (Salmo), found that all sectors of the blastula were able to differentiate all types of tissue, i.e., they proved to be totipotent. However, in the early gastrula, only the sector forming the embryonic shield and the areas immediately adjacent to it were able to express totipotency. As gastrulation progresses, this. limitation becomes more marked. In other words, a generalized potency around the germ ring, present during blastulation, becomes restricted when the embryonic shield of the gastrula comes into prominence. The evidence set forth in the previous chapter indicates that the possibility for twinning in the trout becomes less and less as the gastrular condition nears. The restriction of potency thus becomes a function of a developmental sequence.

In the case of Fundulus, Oppenheimer ('38) found that various areas of the germ ring, taken from regions 90 degrees or 180 degrees away from the dorsal blastoporal lip, were able to differentiate many different embryonic structures if transplanted into the embryonic shield area. Oppenheimer concludes that: “Since under certain conditions the germ-ring can express potencies for the differentiation of many embryonic organs, it is concluded that its normal role is limited to the formation of mesoderm by the inhibiting action of the dorsal lip.” The results obtained by Luther serve to support this conclusion.

c. Gastrulation in Elasmobranch Fishes

In figure I79B the presumptive major organ-forming areas of the blastoderm of the shark, Scyllium canicula, are delineated. The arrows indicate the general directions of cell migration during gastrulation. In figure 212A-G are shown surface views of the dorsal-lip area of different stages of blastodermic development in this species, while figure 213A-G presents median, sagittal sections of these blastoderms during inward migration of the presumptive organ-forming cells. It is to be observed that the dorsal-lip region of the blastoderm is the focal area over which the cells involute and migrate to the inside.

Fig. 212. Surface views of developing blastoderms of Scyllnim canicula.

Fig. 213. Sagittal sections of blastoderms shown in figure 212A-G, with corresponding letters, showing migration of presumptive organ-forming areas. (See also fig. 179.) (B)

Dorsal lip is shown to left. (H~M) Transverse sections of embryo of Squalus acanthias, similar to stages shown in 212F and G, for Scyllium. (H) Section through anterior head fold. (M) Section through caudal end of blastoderm. H-M original drawings from prepared slides.

Fig. 214. Gastrulation in the gymnophionan Amphibia and in the bony ganoid, Amia calva. (A, B, C, after Brauer, 1897; D, E, after Dean, 1896.) Sections A~C through developing embryo of Hypogeophis alternans. (A) Middle gastrula, sagittal section. Observe that gastrocoel forms by a separation of the entodermal cells. Blastococl forms similarly through delamination of entoderm from the overlying epiblast and by spaces which appear between the cells in situ. (B) Transverse section through late gastrula. (C) Sagittal section through late gastrula. (D) Late gastrula of Amia. Mass of yolk in center is uncleaved; cellular organization is progressing peripherally around yolk mass. (E) Later gastrula of Amia. The blastopore is closing, but a large yolk mass still remains uncleaved.

In figure 21 3A, B, and C, two general areas of entoderm are shown:

(a) that exposed at the surface (cf. fig. 179), and

(b) the entoderm lying in the deeper areas of the blastoderm (cf. fig. 179, cells in black).

According to Vandebroek, '36, the deeper lying entoderm is extra-embryonic entoderm (in fig. 213, this deeper entoderm is represented as a black area with fine white stipple), whereas the entoderm exposed at the caudal portion of the blastoderm in figure 179A and B, and figure 213A is embryonic entoderm.

The later distribution of the major presumptive organ-forming areas of the shark blastoderm is shown in figure 213E-M. In figure 213, observe the periblast tissue connecting the blastoderm with the yolk substrate.

As the notochordal, cntodermal, and mesodermal cells move inward during emboly, the presumptive epidermal and neural areas become greatly expanded externally by the forces of epiboly as shown in figures 213B-E, and 213H. (Compare the positions of these two areas in fig. 179B.)

The general result of the gastrulative processes in the shark group is to produce a blastoderm with three germ layers similar to that shown in figure 21 3L and M. The notochordal and pre-chordal plate cells occupy the median area below the neural plate as shown in figure 21 3E and F; the mesoderm and entoderm lie on either side of the median notochord as shown in figure 213M. A little later the entoderm from either side of the notochord grows mediad to establish a complete floor of entoderm below the notochord as represented in figure 21 3L.

7. Intermediate Types of Gastrulative Behavior

In certain forms, such as the ganoid fish, Amia, and in the Gymnophiona among the Amphibia, the gastrulative processes present distinct peculiarities. In general, gastrulation in the bony ganoid fish, Amia calva, presents a condition of gastrulation which is intermediate between that which occurs in the teleost fishes and the gastrulative procedures in the frog or the newt. For example, a blastodisc-like cap of cells is found at the end of cleavage in the bony ganoid. This cap gradually creeps downward around the yolk masses which were superficially furrowed during the early cleavages. This process resembles the cellular movement occurring during epiboly in teleost fishes. In addition, the entodermal, notochordal, and mesodermal materials migrate inward in much the same way as occurs in the teleost fishes, although the formation of the primitive archenteron resembles to a degree the early invaginative procedure in the frog. However, a distinctive process of entodermal formation occurs in Amia, for some of the entodermal cells arise as a separation from the upper portion of the yolk substance where yolk nuclei are found. (See fig. 214D, E; consult Eycleshymer and Wilson, '06.)

The gastrulative processes in the gymnophionan Amphibia are most peculiar, particularly the behavior of the entoderm. But little study has been devoted to the group; as a result, our knowledge is most fragmentary. Elusive and burrowing in their habits and restricted to a tropical climature, they do not present readily available material for study. Brauer, 1897, described blastulation and gastrulation in Hypogeophis alternans. Our information derives mainly from this source.

In some respects gastrulation in Hypogeophis is similar to that in teleost and bony ganoid fishes, while other features resemble certain cellular activities in other Amphibia and possibly also in higher vertebrates. For example, the blastoderm behaves much like the flat blastoderm of teleost fishes, for a dorsal blastoporal lip or embryonic portion of the germ ring is formed toward which the notochordal and mesodermal materials presumably migrate, involute, and thus pass to the inside below the epiblast layer (tig. 21 4A, B). Also, the rapid epiboly of the presumptive epidermal area around the yolk material (or yolk cells) is similar to that of teleost fishes and of the bony ganoid, Amia (fig. 214C-E). However, the behavior of the entodermal cells differs markedly from that of teleosts. In the first place, there is a double delamination whereby the solid blastula is converted into a condition having a blastocoel and a gastrocoel (fig. 214A), These processes occur concurrently with the gastrulative phenomena. Blastocoelic formation resembles somewhat the delaminative behavior of the entoderm in reptiles, birds, and mammals, for the entodermal layer separates from the deeper areas of the epiblast layer. The formation of the gastrocoel (archenteron) is a complex affair and is effected by a process of hollowing or space formation within the entodermal cell mass as indicated in figure 2 1 4A. The arrangement of the entodermal cells during later gastrulative stages resembles the archenteron in the late gastrula of other Amphibia. The archenteron possesses a heavily yolked floor, with the roof of the foregut region complete, but that of the archenteron more posteriorly is incomplete, exposing the notochord to the archenteric space (fig. 214A-C).

G. The Late Gastrula as a Mosaic of Specific, Organ-forming Territories

It was observed above that the presumptive organ-forming areas of the late blastula become distributed in an organized way along the notochordal axis during gastrulation. Further, while an interchangeability of different parts of the epiblast of the late blastula is possible without upsetting normal development, such exchanges are not possible in the late gastrula. For during gastrulation, particular areas of the epiblast become individuated by activities or influences involved with induction or evocation. (The word “evocation” was introduced by Waddington and it has come to mean: “That part of the morphogenetic effect of an organizer which can be referred back to the action of a single chemical substance, the evocator.” See Needham, '42, p. 42.) As a result, the gastrula emerges from the gastrulative process as a general mosaic of self-differentiating entities or territories. (See Spemann, '38, p. 107.)

It necessarily follows, therefore, that the production of specific areas or territories of cells, each hax^ing a tendency to differentiate into a specific structure, and the axiation of these areas along the primitive axis of the embryo are two of the main functions of the gastrulative process. In figure 205A-E, diagrams are presented relative to the chick embryo showing the results of experiments made by Rawles ('36), Rudnick ('44), and others. (See Rudnick, '44.) These experiments were made to test the developmental potencies of various limited areas of the chick blastoderm. A considerable overlapping of territories is shown, which stems, probably, from the fact that transplanted pieces often show potencies which are not manifested in the intact embryo. Therefore, these maps should be regarded not with finality but merely as suggesting certain developmental tendencies.

H. Autonomous Theory of Gastrulative Movements

Our knowledge concerning the dynamics of gastrulation in the Chordata is based largely on the classical observations of cell movement made by Conklin ('05) in Styela, the same author ('32) in Arnphioxus, Vogt ('29) in various Amphibia, Oppenheimer ('36) in Fundulus, Pasteels ('36, '37b) in trout and chick, Vandebroek ('36) in the shark, and Spratt ('46) in the chick. For detailed discussions, concerning the morphodynamics of the gastrulative period, reference may be made to the works published by Roux (1895), Spemann ('38), Pasteels ('40), Waddington ('40), and Schechtman ('42).

The theory popularly held, regarding the movements of the major presumptive organ-forming areas of the late blastula, is that a strict autonomy is present among the various groups of cells concerned with the gastrulative process. Spemann ('38) p. 107, describes this theory of autonomy as follows:

Each part has already previously had impressed upon it in some way or other direction and limitation of movement. The movements are regulated, not in a coarse mechanical manner, through pressure and pull of the single parts, but they are ordered according to a definite plan. . . . After an exact patterned arrangement, they take their course according to independent formative tendencies which originate in the parts themselves.

There are some observations, on the other hand, which point to an interdependence of the various cell groups. For example, we have referred to the observations of Waddington ('33) and Spratt ('46) which suggest that the movements of the mesoderm in the bird embryo are dependent upon the inductive influence of the entoderm. Similarly, Schechtman ('42) points out that presumptive notochordal material does not have the power to invaginate (involute) to the inside when transplanted to the presumptive ectodermal area, but it does possess the autonomous power to elongate into a slender column of cells.

Fig. 215. Direction of entodermal projection in relation to egg polarity during gastrulation in various Chordata. (A) Amphioxus. (B) Frog. (C) Urodele amphibia. (D) Chick. For diagrammatic purposes, the positions to the right of the median egg axis in the diagrams arbitrarily are considered as clockwise positions, whereas those to the left are regarded as counterclockwise.

Fig. 216. Exogastrulation in the axolotl (Amphibia). (From Huxley and De Beer, *34, after Holtfreter: Biol. Zentralbl., 53: 1933.) (A, B) Mass outward or exogastrular

movements of entoderm and mesoderm, resulting in the separation of these organforming areas from the epidermal, neural areas shown as a sac-like structure in upper part of figure. (C) Section of (B). Exogastrulation of this character results when the embolic movements of gastrulation are directed outward instead of inward. Observe that neural plate does not form in the ectodermal area.

I. Exogastrulation

It was demonstrated by Holtfreter ('33) and also by others that embryos may be made to exogastrulate, i.e., the entoderm, notochord, and mesoderm evaginate to the outside instead of undergoing the normal processes involved in emboly (fig. 216). For example, in the axolotl, Ambystoma mexicanurn, if embryos are placed in a 0.35 per cent Ringer's solution, exogastrulation occurs instead of gastrulation, and the entodermal, mesodermal and notochordal areas of the blastula lie outside and are attached to the hollow ectodermal vesicle. The exogastrulated material, therefore, never underlies the ectodermal cells but comes to lie outside the neural plate and skin ectodermal areas of the gastrula (fig. 216B).

Therefore, the phenomenon of exogastrulation indicates strongly that the presumptive, neural plate and epidermal areas of the late blastula and early gastrula are dependent upon the normal gastrulative process for their future realization in the embryo. Exogastrulation also clearly separates the parts of the forming gastrula which are concerned with emboly from those which are moved by the forces of epiboly. That is, exogastrulation results when the jorces of epiboly are separated from the forces normally concerned with emboly. Normal gastrulation is concerned with a precise and exact correlation of these two sets of forces.

J. Pro-chordal Plate and Cephalic Projection in Various Chordates

It is evident from the descriptions presented in this chapter that the initial invaginative movements in gastrulation begin in the region of the dorsal lip of the blastopore in Amphioxus, fishes, and Amphibia. This initial movement of cells in the region of the dorsal lip consists in the projection forward, toward the future head region of the embryo, of foregut entoderm, pre-chordal plate mesoderm, and notochordal cells. The foregut entoderm, pre-chordal mesoderm, and the anterior extremity of the notochord come to lie beneath the anterior portion of the neural plate. The complex of anterior foregut entoderm and pre-chordal mesoderm lies in front of the anterior limits of the notochord — hence, the name pre-chordal plate. As such it represents, as previously observed, a part of the head organizer (see p. 401 ), the complete organization of the vertebrate head being dependent upon anterior chordal (notochordal), as well as pre-chordal, factors.

In higher vertebrates a different situation prevails during gastrulation. As observed in Chapter 7, the late blastula consists of a lower hypoblast and an upper epiblast in a flattened condition, the hypoblast having separated from the lower parts of the epiblast. The separation of the hypoblast occurs shortly before the gastrulative rearrangement of the major, presumptive, organ forming areas begins. The organization of the blastoderm (blastula) is such that presumptive pre -chordal plate mesoderm and notochordal areas lie far anteriorly toward the midcentral part of the epiblast. In other words, a contiguous relationship between presumptive pre-chordal entoderm (i.e., anterior foregut entoderm) and presumptive pre-chordal mesoderm and the presumptive notochord at the caudal margin of the blastula does not exist. Consequently, a different procedure is utilized in bringing the foregut entoderm, pre-chordal mesoderm, and anterior notochord together. That is, the head-organizer materials must be assembled together in one area underneath the cephalic portion of the neural plate. This is accomplished by two methods:

  1. The use of a large invaginative process, the notochordal canal, which projects pre-chordal plate mesoderm and notochord cranio-ventrad toward the foregut entoderm in the hypoblast below, as described in figure 200 relative to the reptiles or in figure 207B of the human embryo and
  2. the use of another and less dramatic method for getting the headorganizer materials together, the vestigial invaginative process which produces the primitive pit and Hensen's nodal area.

The latter mechanism succeeds in getting pre-chordal plate mesoderm and notochord down between the epiblast and hypoblast and forward to unite with the anterior part of the foregut entoderm. (See Adelmann, '22, '26; Pasteels, '37b.)

It is not clear whether the invaginative behavior which produces the primitive pit or notochordal canal is an autonomous affair or whether it may be dependent upon the inductive activities of the entoderm below. More experimentation is necessary to decide this matter. The work of Waddington ('33), however, leads one to conjecture that inductive activities may be responsible.

Regardless of the factors involved, cephalogenesis or the genesis of the head is dependent upon the assemblage of anterior foregut, pre-chordal mesoderm, and anterior notochordal cells beneath the cephalic portion of the neural plate as described on page 401.

K. Blastoporal and Primitive-streak Comparisons

From the considerations set forth above, it is clear that the area of the notochordal canal or primitive pit (i.e., Hensen's nodal area) corresponds to the general region of the dorsal lip of the blastopore of lower vertebrates, whereas the dorso-lateral and lateral lips of the blastopore of lower forms correspond to the body of the primitive streak in higher vertebrates (Adelmann, '32).


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