Book - Comparative Embryology of the Vertebrates 3-10

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

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

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Part III The Development of Primitive Embryonic Form

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

Development of Primitive Body Form

A. Introduction

1. Some of the Developmental Problems Faced by the Embryo After Gastrulation

a. Tubulation

One of the main problems, confronting the embryo immediately following gastrulation, is the tubulation of the major organ-forming areas, namely, epidermal, neural, entodermal, and the two, laterally placed, mesodermal areas. The epidermal, neural, and entodermal areas eventually form elongated, rounded tubes, whereas the mesodermal tubes are flattened. The epidermal and neural tubes extend the entire length of the developing embryo (fig. 217A-C), while the entodermal tube normally terminates at the beginning of the tail (fig. 21 7B, C), although in some instances it may extend even to the tail's end (fig. 217A). Anteriorly, the entodermal tube ends along the ventral aspect of the developing head (fig. 217A, C). The two mesodermal tabulations are confined mainly to the trunk region of the embryo, but in the early embryo of the shark they continue forward into the head almost to the posterior limits of the developing eyes (fig. 217D). The condition of the mesodermal tubes in the Amphibia resembles to a degree that in the shark embryo (fig. 217B, E).

An important concept to grasp is that the tubulations of the respective areas occur synchronously or nearly so. It is true that the initial stages of the epidermal and entodermal tubulations slightly precede the other tubulations in Amphioxus, in the frog, and in forms having rounded gastrulae, while in the chick the neural area is precocious. Viewed in their totality, however, the tubulations of all of the major organ-forming areas are simultaneous processes with the exception of the notochord which does not become tubulated but continues as an elongated rod of cells.

Fig. 217. Primary tubes (tubulations) of the primitive vertebrate body. (A) Schematic representation of epidermal, neural, and entodermal tubes in the early embryo of the shark. Observe that a well-developed, post-anal or tail gut continues to the end of the tail. (B) Gut, neural, and epidermal tubes in the amphibian type. (C) Gut, neural, and epidermal tubes in the chick and mammal type. (D) Mesodermal tube in the shark embryo. (E) Mesodermal tube in the amphibian embryo. (F) Mesodermal condition in the early bird and mammal embryo. (G) Transverse section of shark embryo, showing tubulations of major or^an-forming areas and primary coelomic conditions. (H) Transverse section of frog ^embryo shortly after closure of neural tube, showing the five fundamental body tubes oriented around the notochord.

b. Increase in Size and Antero-posterior Extension of the Tubulated, Major Organ-forming Areas

Another goal to be achieved by the embryo during the immediate, postgastrular period is an increase in size, together with an antero-posterior extension of the major organ-forming areas. These changes are associated with tubulation, and they aid in producing the elongated, cylindrical form typical of the chordate body,

c. Regional Modifications of the Tubulated Areas

As tubulation of the various major organ-forming areas progresses, specific, organ-forming areas or fields (see end of chapter), located along the respective primitive body tubes, begin to express themselves and develop in a specialized manner. Thus, regional differentiation of the major organ-forming areas; comprising each primitive body tube, is another feature of the postgastrular period. As a result, localized areas along each of the body tubes show changes in shape, and specific, individualized structures begin to make their appearance. For example, the neural tubulation develops the primitive parts of the brain at its anterior end, while the posterior portion of the neural tube, caudal to the brain area, begins to form the spinal cord. Thus, the primitive brain becomes a specific peculiarity of the head region. Also, the epidermal tubulation at its cranial end contributes definite structures peculiar to the head. In the pharyngeal region, special developmental features arise in the entodermal tube together with the epidermal tube and the mesoderm. In the trunk region, modifications of the entodermal and mesodermal tubes give origin to many of the structural conditions peculiar to this area, while in the tail, the neural and epidermal tubulations together with activities of the mesoderm account for the characterstic structures of the tail appendage. These special developmental features of the respective, tubulated, organ-forming areas, which arise in specific areas along the antero-posterior axis of the embryo, occur in much the same way throughout the vertebrate group with the result that common or generalized structural conditions of the tubulated organ-forming areas appear in all vertebrate embryos. That is, the primitive brains of all vertebrate embryos up to a certain stage of development resemble each other in a striking manner; the contributions of the epidermal tubulation to the head also resemble each other, ind the early development of the pharyngeal and trunk regions is similar. As a result, the early morphogenesis organ systems conform to generalized, basic plans. After the generalized plan of a particular system is established, it is modified in later development to fit the requirements of the habitat in which the particular species lives. In the cephalochordate, Amphioxus, a similar body form also develops, although it is considerably modified.

The common, generalized, primitive embryonic body form of all vertebrate embryos possesses the following characteristics:

1. It is an elongated structure, cylindrical in shape, and somewhat compressed laterally.
2. It is composed of five, basic, organ-forming tubes, oriented around a primitive axis, the notochord (fig. 217).
3. It possesses the following regions: (a) head, (b) pharyngeal area, (c) trunk, and (d) tail (figs. 217, 226, 227, 230, 238, 244, 246).

In Chapter 11 and the following chapters, various details of these common regions and other features will be considered. In this chapter, we are concerned mainly with tabulation and antero-posterior extension of the major organ-forming areas in relation to body-form development.

3. Starting Point for Tubulation

The starting point for tubulation of the major organ-forming areas and subsequent, primitive, body formation is the gastrula; which, as observed in Chapter 9, exists in two forms, namely, rounded and the flattened gastrulae (figs. 219, 232). Many heavily yolked embryos, such as the embryo of Necturns maculosus, although they form a rounded gastrula, are faced with some of the problems of the flattened gastrulae (fig. 227). The rounded gastrulae, found in the frog, Amphioxus, etc., differ from the flattened gastrulae present in the bird, reptile, mammal, and teleost and elasmobranch fishes, mainly by the fact that, at the beginning of tubulation and body formation, the epidermal and gut areas already are partially tubulated in the rounded gastrulae. That is, in the rounded blastoderm, the initial stages of tubulation occur in these two major organ-forming areas during gastrulation. This means that the ventral portion of the trunk area in rounded gastrulae is circumscribed by intact cellular layers of the embryonic trunk region, with yolk material contained within the cell layers, while, in flattened gastrulae, the ventro-lateral portions of the trunk region are spread out flat, the yolk not being surrounded by the future, ventro-lateral walls of the embryonic trunk region. These conditions are illustrated in figures 219B and C and 234A-F.

The developmental problems faced by these two groups of gastrulae, therefore, are somewhat different. Moreover, tubulation of the organ-forming areas and the development of body form in Amphioxus varies considerably from that of the rounded gastrulae of the vertebrate group. For this reason, tubulation in Amphioxus is considered separately.

Regardless of differences, however, all vertebrate gastnilae, rounded and flattened, possess three fundamental or basic regions, to wit, ( 1 ) a cephalic or head region, containing the rudiments of the future head and pharyngeal structures, (2) a trunk region, wherein lie the undeveloped fundaments of the trunk, and (3) an end-bud or tail rudiment, containing the possibilities of the future tail.

4. Developmental Processes Which Accomplish Tubulation

a. Immediate Processes

The term, immediate processes, signifies the events which actually produce the hollow tubular condition. In the case of the epidermal, enteric, and neural tubulations, the immediate process is mainly one of folding the particular, organ-forming area into a hollow tubular affair. With respect to the mesodermal areas, the immediate process is an internal splitting (delamination), whereby the mesodermal area separates into an outer and an inner layer with a space or cavity appearing between the two layers. In the case of the teleost fishes, a process of internal separation of cells appears to play a part also in the neural tubulation.

Fig. 219. Relationships of the major presumptive organ-forming areas at the end of gastrulation in the anuran amphibia. (A) External view of gastrula, showing the ectodermal layer composed of presumptive epidermis (white) and presumptive neural plate (black), as viewed from the dorsal aspect. (B) Diagrammatic median sagittal section of condition shown in (A). (C) Same as (B), showing major organ-forming areas. (D) Section through middorsal area of conditions (B) and (C), a short distance caudal to foregut and pre-chordal plate region. Observe that the notochord occupies the middorsal area of the gut roof.

b. Auxiliary Processes

Aiding the above activities which produce tubulation are those procedures which extend the tubulated areas into elongated structures. These auxiliary processes are as follows:

1. The cephalic or head rudiment, with its contained fundaments of the developing head region, grows forward as a distinct outgrowth. This anterior protrusion is known as the cephalic or head outgrowth (figs. 223A, B; 2321-L).
2. The trunk rudiments enlarge and the trunk region as a whole undergoes antero-posterior extension (figs. 225 A; 233).
3. The tail-bud area progresses caudally as the tail outgrowth and forms the various rudimentary structures associated with the tail (figs. 225; 230F; 238).
4. A dorsal upgrowth (arching) movement occurs, most noticeable in the trunk area. It serves to lift the dorsal or axial portion of the trunk up above the yolk-laden area below, and the developing body tubes and primitive body are projected dorsalward (figs. 221, 224, 241).
5. In embryos developing from rounded gastrulae, a ventral contraction and reshaping of the entire ventro-lateral areas of the primitive trunk region are effected as the yolk is used up in development. This results in a gradual retraction of this area which eventually brings the ventrolateral region of the trunk into line with the growing head and tail regions (cf. figs. 220, 223, 225 on the development of the frog, and 227 on the development of Necturus).
6. In embryos developing from flattened gastrulae, a constriction of the ventral region of the developing trunk comes to pass. This constriction is produced by an ingrowth toward the median line of entodermal, mesodermal, and epidermal cellular layers in the form of folds, the lateral body folds. Upon reaching the midline, the cellular layers fuse as follows: The entodermal layer from one side fuses with the entodermal layer of the other; the mesodermal layers fuse similarly; and, finally, the epidermal layer from one side fuses with the epidermal layer of the opposite side. The result is a general fusion of the respective body layers from cither side, as shown in figure 24 1C and D, which establishes the ventral region of the trunk. A complete fusion throughout jLhe extent of the ventral body wall does not take place until later in development, and, as a result, a small opening remains, the umbilicus, where the embryonic and extra-embryonic tissues are continuous. This discontinuity of the embryonic layers permits the blood vessels to pass from the embryonic to the extra-embryonic regions. (Note: In the teleost fishes, although a typical, flattened, gastrular form is present, the formation of the ventral body wall of the trunk through a general retraction of tissues resembles that of the rounded gastrulae mentioned above.)

Fig. 220. Beginning neural fold stage of frog embryo from prepared material. (A) Beginning neural fold stage as seen from dorsal view. (B) Sagittal section near median plane of embryo similar to that shown in (A). (C) Same as (B), showing organ forming areas. (D) Midsagittal section of caudal end of frog embryo slightly younger than that shown in fig. 223 B. Observe that the blastopore practically is closed, while the dorsal diverticulum of the hindgut connects with the neurocoel to form the neurenteric canal. Observe, also, ventral diverticulum of hindgut.

5. Blastocoelic Space and Body-form Development

During the terminal phases of gastrulation in such forms as Amphioxus and the frog, the blastocoel, as a spacious cavity, disappears for the most part. Its general area is occupied by cells which migrated into the blastocoel during gastrulation. However, the disappearance of the blastocoelic space is more apparent than real. For, while most of the original blastocoelic space is thus occupied and obliterated, a part of the original blastocoel does remain as an extremely thin, potential area between the outside ectoderm and the mesoderm-entoderm complex of cells. In flattened blastoderms, as in the chick, the actual space between the ectoderm, mesoderm, and entoderm is considerable (fig. 234E, F). To sum up: Though the blastocoelic space appears to disappear during the terminal phases of gastrulation, a residual or potential space remains between the three germ layers, more pronounced in some species than in others. This residual space gradually increases during the tubulation processes of the major organ-forming areas. In doing so, it permits not only the tubulation of these areas within the outside ectoderm, but it allows important cell migrations to occur between the various body tubes.

6. Primitive Circulatory Tubes or Blood Vessels

Accompanying the tubulations of the epidermal, neural, entodermal, and the two mesodermal areas on either side of the notochord, is the formation of a delicate system of ve.ssels which function for the transport of the circulatory fluid or blood. The formation of these blood vessels begins below the forming entodermal tube as two, subenteric (subintestinal) tubes or capillaries. These capillaries grow forward below the anterior portion of the forming digestive tube. Near the anterior end of the latter, they separate and pass upward on either side around the gut tube to the dorsal area, where they come together again below the notochord and join to form the rudiments of the dorsal aortae. The latter are two delicate supraenteric capillaries which extend from the forming head area caudally toward the trunk region. In the latter region, each rudiment of the dorsal aorta sends a small, vitelline blood vessel laterally into that portion of the gut tube or yolk area containing the yolk or other nutritional source. In the yolk area, each joins a plexus of small capillaries extending over the surface of the yolk substance. These capillaries in turn connect with other capillaries which join ultimately each of the original subintestinal blood capillaries. Below the anterior or foregut portion of the entodermal tube, the two subintestinal blood vessels fuse and thus form the beginnings of the future heart (figs. 234-237; 332). The further development of this system of primitive vessels is described in Chapter 17.

Fig. 221. Transverse sections through early neural fold embryo of the frog as shown in fig. 220A and B. (A-J) Sections are indicated in fig. 220B by lines A-J, respectively. Observe that the dorsal arching (dorsal upgrowth) movement of the dorsally situated tissues accompanies neural tube formation.

Fig. 222. Neural crest cells in Amhystoma punctatum. (A and B from Johnston: Nervous System of Vertebrates, Philadelphia, Blakiston, '06; C-F from Stone: J. Exper. Zool., '35.) (A) Transverse section of early neural tube of Am by stoma, neural crest cells located dorsally and darkly shaded. (B) Later stage than (A), showing relation of neural crest cells, epidermis, and neural tube. (C-F) Neural crest cells stippled, placodes of special lateral line sense organs and cranial nerve ganglia shown in black. The neural crest cells arise from dorsal portion of neural tube at points of fusion of neural folds and migrate extensively. A considerable portion of neural crest cells descends upon the mesoderm of visceral arches as indicated in (D-F) and contributes mesodermal cells to these arches, where they later form cartilaginous tissue.

7. Extra-embryonic Membranes

Associated with the development of body form and tubulation of the major, organ-forming areas, is the elaboration of the very important extra-embryonic membranes. As the essential purpose at this time is to gain knowledge of the changes concerned with tubulation of the major organ-forming areas and the development of primitive body form, consideration of these membranes is deferred until Chapter 22. The latter chapter is concerned with various activities relating to the care and nutrition of developing embryos of various vertebrate species.

B. Tubulation of the Neural, Epidermal, Entodermal, and Mesodermal, Organ-forming Areas in the Vertebrate Group

1. Neuralization or the Tubulation of the Neural Plate Area

a. Definition

The separation of the neural plate material from the skin ectoderm, its migration inward, and its formation into a hollow tube, together with the segregation of the accompanying neural crest cells, is called neuralization.

b. Neuralizative Processes in the Vertebrata

Neuralization is effected by two general procedures in the vertebrate subphylum.

1) Thickened Keel Method. In tcleost, ganoid, and cyclostomatous fishes, the neural plate material becomes aggregated in the form of a thickened, elongated ridge or keel along the middorsal axis of the embryo (figs. 21 OF; 218C). This keel separates from, and sinks below, the overlying skin ectoderm (fig. 218A). Eventually the keel of neural cells develops a lumen within its central area and thus gradually becomes transformed into an elongated tube, coincident with the tubulations of the other major organ-forming areas (fig. 218B). In the cyclostomatous fish, Petromyzon planeri, although neuralization closely resembles the condition in teleost fishes, in certain respects the behavior of the neuralizative changes represents an intermediate condition between the keel method of the teleost and neural fold method of other vertebrates described below (Selys-Longchamp.s, '10).

Fig. 223. Early neural tube stage of the frog, Rana pipiens, 2Vi to 3 mm. in length. (A) Dorsal view, (B) Midsagittal section of embryo similar to (A). (C) Same as (B) , showing organ-forming areas. Abbreviations: V. HD. = ventral hindgut divertic ulum; D. HD, = dorsal hindgut diverticulum; PHAR. ~ pharyngeal diverticulum of foregut. (D) Later view of (A). (E) Sec fig. 224.

2) Neural Fold Method* In the majority of vertebrates, the neural (medullary) plate area folds inward (i.e., downward) to form a neural groove. This neural groove formation is associated with an upward and median movement of the epidermal layers, attached to the lateral margins of the neural plate, as these margins fold inward to form the neural folds. A change of position in the mesoderm also occurs at this time, for the upper part which forms the somites shijts late rad from the notochordal area to a position between the forming neural tube and the outside epidermis. This mesodermal migration permits the neural tube to invaginate downward to contact the notochordal area. Also, this change in position of the somitic mesoderm is a most important factor in neuralization and neural tube development as mentioned at the end of this chapter. {Note: In this stage of development, the embryo is often described as a neurula, especially in the Amphibia. However, in the bird and the mammal, the embryo during this period is described in terms of the number of somitic pairs present, and this stage in these embryos is referred to as the somite stage.) Each lateral neural fold continues to move dorsad and mesad until it meets the corresponding fold from the other side. When the two neural folds meet, they fuse to form the hollow neural tube and also complete the middorsal area of the epidermal tube (cf. figs. 221, 224, 233, 234, 236, 237, 242, 245A). As a general rule, the two neural folds begin to fuse in the anterior trunk and caudal hindbrain area. The fusion spreads anteriad and posteriad from this point (figs. 223, 229, 233, 235, 242, 245A). It is important to observe that there are two aspects to the middorsal fusion process:

(a) The lateral edges of the neural plate fuse to form the neural tube; and

(b) the epidermal layer from either side fuses to complete the epidermal layer above the newly formed neural tube.

Fig. 225. Structure of 3'/2- to 4-mm. embryo of Rana pipiens (about eight pairs of somites are present). (See fig. 226A and B for comparable external views of lateral and ventral aspects of 5-mm., .yy/v6i//ca embryo.) (A) External dorsal view. (B) Midsagittal view. (C) Same, showing major organ-forming areas.

Associated with the fusion phenomena of the epidermis and of the neural tube, neural crest cells are given off or segregated on either side of the neural tube at the point where the neural tube ectoderm separates from the skin ectoderm (figs. 221C-E; 23 4B; 236B). The neural crest material forms a longitudinal strip of cells lying along either side of the dorsal portion of the neural tube. As such, it forms the neural or ganglionic crest. In some vertebrate embryos, as in the elasmobranch fish, Torpedo, and in the urodele, Ambystoma, the cells of the neural crest are derived from the middorsal part of the neural tube immediately after the tube has separated from the skin ectoderm (epidermis). (See fig. 222A, B.) In other vertebrates, such as the frog, chick, and human, the neural crest material arises from the general area of junction of neural plate and skin ectoderm as fusion of the neural folds is consummated (fig. 234B).

The neural crest gives origin to ganglionic cells of the dorsal root ganglia of the spinal nerves and the ganglia of cranial or cephalic nerves as described in Chapter 19. Pigment cells also arise from neural crest material and migrate extensively within the body, particularly to the forming derma or skin, peritoneal cavity, etc., as set forth in Chapter 12. A considerable part of the mesoderm of the head and branchial area arises from neural crest material (fig. 222C-F). (See Chapters 11 and 15.)

As the neural plate becomes transformed into the neural tube, it undergoes extension and growth. Anteriorly, it grows forward into the cephalic outgrowth, in the trunk region it elongates coincident with the developing trunk, while posteriorly it increases in length and forms a part of the tail outgrowth.

f. Closure of the Blastopore in Rounded Gastrulae, such as that of the Frog

Neuralization and the infolding of the neural plate cells begins in the frog and other amphibia before the last vestiges of the entoderm and mesoderm have completed their migration to the inside. As mentioned above, the neural folds begin, and fusion of the neural tube is initiated in the anterior trunk region. From this point, completion of the neural tube continues anteriad and posteriad. As the neural tube proceeds in its development caudally, it reaches ultimately the dorsal lip of the now very small blastopore. As the neural tube sinks inward at the dorsal blastoporal lip, the epidermal attachments to the sides of the infolding neural tube fuse in a fashion similar to the fusion of the edges of the neural tube to complete the dorsal epidermal roof. Associated with this epidermal fusion at the dorsal lip of the blastopore is the fusion of the epidermal edges of the very small blastopore. The extreme caudal end of the archenteron or blastoporal canal in this manner is closed off from the outside (fig. 220D), and the posterior end of the archenteron (the future hindgut area), instead of opening to the outside through the blastoporal canal, now opens into the caudal end of the neural tube. In this way, a canal is formed connecting the caudal end of the future hindgut with the neural tube. This neurenteric union is known as the neurenteric canal.

Fig. 226. External views of embryos of Rana sylvatica and Rana pi pie ns. (A to J after Pollister and Moore: Anat. Rec., 68; K and L after Shumway: Anat. Rec., 78.) (A, B) Lateral and ventral views of 5-mm. stage. Muscular movement is evident at this stage, expressed by simple unilateral flexure; tail is about one-fifth body length. (Pollister and Moore, stage 18.) (C, D) Lateral and ventral views of 6-rnm. stage. Primitive heart has developed and begins to beat; tail equals one-third length of body. (Pollister and Moore, stage 19.) (E, F) Similar views of 7-mm. stage. Gill circulation is established; hatches; swims; tail equals one-half length of body. (Pollister and Moore, stage 20.) (G, H) Ten-mm. stage, lateral and dorsal views. Gills elongate; tail fin is well developed and circulation is established within; trunk is asymmetrical coincident with posterior bend in the gut tube; cornea of eyes is transparent; epidermis is becoming transparent. (Pollister and Moore, stage 22.) (1, J) Eleven-mm. stage, true tadpole shape. Oper cular fold is beginning to develop and gradually growing back over gills. (K, L) Eleven-mm. stage of R. pipiens embryo. Observe that opercular folds have grown back over external gills and developing limb buds; opercular chamber opens on left side of body only. Indicated in fig. 257B.

It is to be observed in connection with the closure of the blastopore and the formation of the neurenteric canal that two important changes occur in the future hindgut area of the archenteron at this time, namely, the posterior dorsal end of the archenteron projects dorso-caudally to unite with the neural tube (fig. 220D), while the posterior ventral end of the archenteron moves ventrad toward the epidermis where it meets the epidermal invagination, the proctodaeum (fig. 220D).

d. Anterior and Posterior Neuropores; Neurenteric Canal

The fusion of the neural folds in the middorsal area proceeds anteriad and posteriad from the anterior somitic and hindbrain region as described above. At the anterior end of the forebrain when fusion is still incomplete, an opening from the exterior to the inside of the neural canal is present; it forms the anterior neuropore (figs. 229D; 23 IL; 235B; 242E-G; 245B). When fusion is complete, this opening is obliterated. The caudal end of the neural tube closes m a similar manner, and a posterior neuropore is formed (figs. 242E, G; 245). In the chick, as in the mammal, the posterior neuropore at first is a wide, rhomboidal-shaped trough, known as the rhomboidal sinus. The anterior end of the primitive streak is included within the floor of this sinus rhomboidalis (fig. 235A, B). The point of posterior neuroporal closure is at the base of the future tail in most vertebrates (fig. 245B), but, in the elasmobranch fishes, this closure is effected after the tail rudiments have grown caudally for some distance (fig. 229B-E).

The vertebrate tail arises from a mass of tissue, known variously as the tail bud, caudal bud, or end bud, and the posterior end of the neural tube comes to lie in the end-bud tissues (figs. 225, 238C). The end bud grows caudally and progressively gives origin to the tail. It consists of the following:

(a) the epidermal tube (i.e., the ectodermal covering of the end bud); within this epidermal layer are

(b) the caudal end of the neural tube;

(c) the caudal end of the notochord;

(d) mesoderm in the form of a mass of rather compact mesenchyme surrounding the growing caudal ends of the notochord and neural tube; and

(e) a caudal growth from the primitive intestine or gut.

This extension of the gut tube into the tail is called, variously, the tail gut, caudal gut or post-anal gut. It varies in length and extent of development in embryos of different vertebrate species. In some species it is joined to the neural tube; in others it is not so united. For example, the tail gut is as long as the trunk portion of the gut in the young shark embryo of 8 to 10 mm. in length, and at the caudal extremity it is confluent with the neural tube (figs. 21 7A; 229F). The confluent terminal portions of the neural and gut tubes form the neurenteric canal. This well-developed neurenteric canal extends around the caudal end or base of the notochord. In the developing frog on the other hand, the confluence between the neural and gut tubes is present only during the initial stages of tail formation, and it thus represents a transient relationship (fig. 223B, C). Consequently, as the tail bud in the frog embryo grows caudally, the neurenteric connection is obliterated and the tail gut disappears. On the other hand, in the European frog, Bombinator, the condition is intermediate between frog and shark embryos (fig. 228). True neurenteric canals within the developing tail are never formed in the reptile, chick, or mammal, although a tail or post-anal gut, much abbreviated, develops in these forms. (See paragraph below.) In teleost fishes, Kupffer's vesicle possibly represents a small and transient attempt to form a neurenteric canal (fig. 210G). However, the tail gut here, with the exception of the terminally placed Kupffer's vesicle, is a solid mass of cells. Thus, the shark and Bomhinator embryos, on the one hand, and the frog, chick, or mammal embryo, on the other, represent two extremes in the development of the tail gut in the vertebrate group.

Fig. 227. Stages of normal development of Necturus maculosus. (Slightly modified from Eycleshymer and Wilson, aided by C. O. Whitman; Chap. 11 in Entwicklungsgeschichte cl. Wirheltiere, by F. Keibel, '10.) (A) Stage 15, 14 days, 19 hours after fertilization. Blastopore is circular and reduced; neural groove is indicated in center of figure. (B) Stage 18, 17 days, 2 hours old. Blastopore is an elongated, narrow aperture between caudal ends of neural folds; neural folds prominent and neural groove is deeper. (C) Stage 21, 18 days, 15 hours old, 3 or 4 pairs of somites. Neural folds are widely separated in head region, narrower in trunk, and coalesced in tail area. (D) Stage 22, 20 days, 10 hours, 6 pairs of somites, length about 6 mm. Observe head has three longitudinal ridges, the middle one represents developing brain, while lateral ones are common anlagen of optic vesicles and branchial arches. (E) Stage 23, 21 days, 2 hours, 10 to 12 pairs of somites, 7 mm. long. Head projects forward slightly above egg contour; end of tail is prominent; large optic vesicles protrude laterally from head area; branchial arch region is caudal to optic vesicle enlargement; anus is below tip of tail. (F) Stage 24, 22 days, 17 hours, 16 to 18 pairs of somites, 8 mm. long. Anterior half of head is free from egg contour; optic vesicles and mandibular visceral arch are well defined. (G) Stage 25, 23 days, 10 hours, 20 to 22 pairs of somites, 9 mm. long. Head is free from egg surface; tail outgrowth is becoming free; mandibular, hyoid, first branchial and common rudiment of second and third branchial arches are visible. Otic vesicle lies above hyoid arch and cleft between hyoid and first branchial arches. (H) Stage 26, 24 days, 22 hours, 23 to 24 pairs of somites, length-^10 mm. Head and caudal outgrowths are free from egg surface; heart rudiment is shown as darkened area below branchial arches; cephalic flexure of brain is prominent. (1) Stage 27, 26 days, 26 to 27 myotomes, length - 11 mm. Outline of body is straighter; nasal pits and mouth are well defined, mandibular arches are long; heart is prominent below branchial arches; anterior limb buds are indicated; faint outlines of posterior limb buds are evident. (J) Stage 28, 30 days, 8 hours, 30 to 31 myotomes, length - 13 mm. Trunk of embryo is straight, head and tail are depressed; surface of yolk is covered by dense network of capillaries; vitelline veins are prominent; pigment appears below epidermis; anterior limb bud projects dorsally; nuchal or neck flexure is prominent above heart and limb-bud area. (K) Stage 29, 36 days, 16 hours, 36 to 38 myotomes, length - 16 mm. Mandibular arches are forming lower jaw; nuchal and tail flexures are straightening; eye and lens are well defined; anlagen of gill filament are present on gill bars; pigment cells are evident on head areas; vitelline veins are prominent; yolk-laden, ventro-lateral portion of trunk is becoming elongated and contracted toward dorsal region of embryo. (L) Stage 30, 40 days, 20 hours, 44 to 46 myotomes, length - 18 mm. Fore and hind limb buds are prominent; nasal openings are small. (M) Stage 31, larva 49 days, 21 mm. (N) Stage 32, larva 61 days, 25 mm. (O) Stage 33, larva 70 days, 28 mm. (P) Stage 34, larva 97 days, 34 mm. (Q) Stage 35, young adult form, 126 days, 39 mm.

Fig. 228. Sagittal section, showing organ-forming areas of Bomhinator embryo. (After O. Hertwig: Lehrhuch der Entwicklungsgeschkhte des Menschen und der Wirbeltiere. 1890. Jena, G. Fischer.) Observe elongated tail gut.

In the reptiles, also in some birds, such as the duck, in the human embryo, and certain other mammals, a transient notochordal-neural canal is present which connects the enteron or gut tube with the caudal area of the forming neural tube (figs. 200B, E; 207B; 23 IG-K). This canal is occasionally referred to as a neurenteric canal. However, it is best to view this condition as a special type of development within the above group, for it is not strictly comparable to the neurenteric canal formed in the developing tail of the embryos of the frog, shark, etc., where the neurenteric canal is formed by a definite union between neural and taihgut tubes as they project caudalward into the tail rudiment.

2. Epidermal Tubulation

The formation of the external, epidermal, tubular layer of the vertebrate body is a complex procedure. Its development differs considerably in the rounded type of gastrula of the Amphibia from that in the flattened gastrula of the chick or mammal.

a. Development of the Epidermal Tube in Amphibia

In the frog and other Amphibia, tubulation of the epidermal area of the blastula begins during gastrulation. At the end of gastrulation, the changes involved in epiboly have transformed the ectodermal area of the blastula into an oval-shaped structure, surrounding the internally placed mesoderm and entoderm (fig. 219). The neural plate material occupies the middorsal area of this oval-shaped, ectodermal layer, while the future epidermal area forms the remainder. Following gastrulation, the anterior end of this oval-shaped structure, in harmony with the forming neural tube, begins to elongate and grows forward as the head outgrowth (figs. 220, 223, 225). A cylindrical, epidermal covering for the entire head, in this manner, is produced as the cranial or brain portion of the neural plate folds inward (invaginates). A similar outgrowth in the tail area proceeds posteriorly, although here the neural tube grows caudally by proliferative activity within the epidermal tube instead of folding into the epidermal tube as it does in the cephalic outgrowth (figs. 223, 225). Coincident with these two outgrowths, the trunk area, with its ventral, yolk-filled, entodermal cells, elongates antero-posteriorly as the neural plate folds inward. It also grows larger in harmony with the head and tail outgrowths. VAs these activities continue, yolk substance is used up, and the ventro-lateral region of the trunk is retracted. A cylindrical shape of the trunk region thus is established, bringing the trunk area into harmony with the head and tail outgrowths. (Study particularly fig. 227.) The epidermal area of the late gastrula thus becomes converted into an elongated, epidermal tube which forms the external covering or primitive skin (see Chap. 12) for the developing body. In Amphibia, this primitive epidermal tube is two layered, consisting of an outer epidermal ectoderm and an inner neural ectoderm (figs. 221, 224). (See Chap. 12.) In the newly hatched larva, the epidermis is extensively ciliated in all anuran and urodele Amphibia.

Fig. 229. Early stages of tubulation of neural and epidermal organ-forming areas with resultant body-form development in the shark, Squaliis acanthias (drawn from prepared slides). Neural area shown in black; epidermal area is stippled white; neural folds are outlined in white around edges of black area. (Consult also fig. 230.) (A) Embryonic area is raised upward; neural plate is flattened; bilateral tail outgrowths are indicated. (B) Embryo is considerably elevated from extra-embryonic blastoderm; brain area is much expanded; trunk region of neural groove is pronounced. (C) Neuralization is considerably advanced; tail rudiments are converging. (D) Neural and epidermal areas are well tubulated; tail rudiments are fusing. (E) Young Squalus embryo, lying on left side; tail rudiments are fused into single caudal outgrowth. The body now consists of a flexed cephalic outgrowth, trunk region, and tail outgrowth. (F) Squalus embryo of about 10 mm. in length.

b. Tubulation of the Epidermal Area in Flat Blastoderms

In the flat blastoderms of the elasmobranch fish, chick, reptile, and mammal, the formation of the external body tube involves processes more complicated than that of the frog type. The following steps are involved:

1. A head fold produces a cephalic epidermal extension above the general tissues of the blastoderm. This rudimentary fold of the epidermis contains within it a similar fold of the entodermal layer, together with the invaginating, neural plate material. The notochordal rod lies between the forming entodermal fold and developing neural tube (figs. 213F; 230A; 232I-L; 242B, C). Shortly, the primitive head fold becomes converted into a cylindrical head outgrowth of the epidermal and entodermal layers, associated with the forming neural tube and notochord (figs. 229C, D; 230C; 233). The general process is similar to that in the frog, but it is more complicated in that the head rudiment first must fold or project itself up above the extra-embryonic areas, before initiating the outgrowth process.
2. A second procedure involved in epidermal tubulation in flattened blastoderms is the dorsal upgrowth movement of epidermal, mesodermal, and entodermal tissues. This activity lifts the trunk region of the embryo up above the general blastodermic tissues (figs. 213H-J; 234B; 241 ). In some forms, such as the chick, the dorsal upgrowth movement is more pronounced in the anterior trunk area at first, gradually extending caudad to the trunk region later (figs. 233, 235). However, in the pig, human, and shark embryos, the dorsal elevation extends along the entire trunk area, coincident with the head outgrowth, and thus quickly lifts the embryonic body as a whole up above the extra-embryonic tissues (figs. 229, 230, 242, 245).
3. The tail outgrowth, in reptiles, birds, and mammals, begins in a manner similar to that of the head region, and a tail fold first is developed which later becomes a cylindrical projection, bounded externally with epidermal cells, within which are found the notochord, tail mesoderm, and tail portions of neural and gut tubes (figs. 238C; 239K, L; 245B). direction of the notochord is much more pronounced in the flattened blastoderms than in the rounded blastoderms of the frog, salamander, etc. (cf. figs. 224; 237). {Note: Associated with the dorsal invagination of the roof of the midgut in the frog, is the detachment of a median rod of entodermal cells from the middorsal area of the gut. This median rod of cells comes to lie between the notochord and the roof of the midgut. It is known as the subnotochordal rod (fig. 225C). (See Chapter 15.)

Fig. 230. Sagittal sections of early elasmobranch embryos. (Slightly modified from Scammon. See Chap. 12 in Entwicklungsgesclnchte d. Wirheltiere, by F. Keibel. ) (A) Graphic reconstruction from sagittal sections of embryo of 2 mm., seen from left side (condition roughly comparable to stage between fig. 229A and B). Observe that neural plate is broad and flattened with slight elevation of neural folds. (B) Reconstruction of embryo of 2.7 mm., viewed from left side, showing mesoderm, forming gut, neural tubes, etc. (Consult (C) below.) (C) Same as (B) with mesoderm removed. Observe primitive gut and neural tubes. Note: (B) and (C) are comparable to stage shown in surface view in fig. 229C. (D, E) Same as (B) and (C), embryo 3.5 mm. in length. (This embryo is comparable to fig. 229D.) (F) Same as (D) with mesoderm removed, showing primitive vascular tubes and neural crest cells.

Fig. 231. Series of diagrams, showing stages in the development of the turtle. (A~F) Cleavage stages after Agassiz. (G J ) Stages of gastrulation, drawn from slide preparations. (K T) Stages during development of body fiirm. (P, Q, T from Agassiz; the others are original.) (See L. Agassiz, 1857, Cont. Nat. Hist, of U. S. A., Vol. 11.)

Fig. 232. Early post-gastrular development in the chick. (A-H represent a late head-process stage- - stage 5 of Hamburger and Hamilton, '51. Compare with figure 203D I-L show the beginnings of the head fold - intermediate condition between stages 7 and 8 of Hamburger and Hamilton, '51.) (A) Surface view, showing primitive streak, neural plate, and epidermal areas. (B-F) Cross sections of A at levels indicated on G. (G) Median sagittal section of (A). (H) Same, showing presumptive, organ forming areas of entoderm notochord, pre-chordal plate, neural plate, and primitive-streak mesoderm. (1) Surface view, demonstrating a marked antero-posterior extension of the neural plate area and beginnings of neural folds. Observe shortening of primitive streak. (J) Drawing of stained specimen. (K) Median sagittal section of (J). (L) Same, showing major organ-forming areas. In (G) and (H) the entoderm, notochord, and overlying neural ectoderm are drawn as separate layers. Actually, however, at this stage, the three layers are intimately associated.

Fig. 233, Early body-form development in chick of 3 to 4 pairs of somites. (Approximately comparable to Hamburger and Hamilton, '51, stage 8, 26 to 29 hours of incubation.) (A) Surface view, unstained specimen. (B) Stained, transparent preparation. Observe blood islands in caudal part of blastoderm. (C) Median sagittal section. (D) Same as (C), showing organ-forming layers.

The development of the rudimentary hindgut is consummated by caudal growth and extension of the posterior or tail region of the primitive archenteron of the late gastrula. These changes result in an extension of the archenteron in the direction of the developing tail and the area ventral to the tail (compare fig. 220B-D with figs. 223B, C; 225B, C).

Three general areas of the primitive gut are thus established:

(a) a tubular enlargement and outgrowth into the developing head, the primitive foregut,

(b) a tubular extension and growth in the caudal region toward the tail, the primitive hindgut, and

(c) a midgut area whose ventral wall is filled with yolk substance, while its roof or dorsal wall assumes a trough-like form extending below the notochord (figs. 223, 224, 225).

The foregut and hindgut areas at this time present the following special features:

1. Two terminal diverticula or evaginations evolve at the extreme anterior portion of the foregut; and
2. at the extreme caudal end of the hindgut, similar evaginations occur.

In the foregut region, one of these evaginations projects toward the brain and anterior end of the notochord, while the second diverticulum, more pronounced than the dorsal evagination, moves ventrad toward the epidermis underlying the developing brain. The dorsal evagination represents the preoral or head gut. In the frog it is much abbreviated (figs. 220B, C; 225B, C). On the other hand, the antero-ventrally directed, oral, or pharyngeal, evagination is relatively large and projects toward the ectoderm underlying the brain where it forms the future pharyngeal area of the foregut (figs. 220; 223; 225B, C). Ultimately an invagination from the epidermis, the stomodaeum, becomes intimately associated with the anterior end of the pharyngeal evagination (see Chap. 13). In the hindgut region, the diverticulum which projects dorsally into the tail is the tail gut, whereas the ventral evagination toward the epidermis below the tail represents the future rectal and cloacal areas of the hindgut (figs. 220; 223; 225B, C). It shortly becomes associated with an invagination of the epidermis, the proctodaeum (fig. 223B, C). As previously mentioned, the tail gut may be well developed, as in the European frog, Bombinator (fig. 228), or quite reduced, as in the frog, Rana (fig. 225).

c. Formation of the Tubular Metenteron in Flat Blastoderms

The development of the cylindrical gut tube in those vertebrate embryos which possess flattened gastrulae is an involved, complicated affair. The developmental mechanics are not clearly understood. For example, it is not clear whether the embryonic layers, lying in front of the head fold in figure 23 2G and H, are folded slightly backward in figures 232K and L and still farther caudad in figure 23 3C and D by autonomous activities within this tissue, or whether the actively growing head outgrowth proceeds so rapidly that it mechanically causes the area in front of the head fold to rotate backward under the developing foregut and thus contribute to the foregut floor. It is obvious, however, that the entodermal material, lying in front of the head fold of the embryo, is folded backward, at least slightly, and thus becomes a part of the floor of the foregut. The extent, however, varies considerably in different species. It appears to be greater in the mammal (fig. 242C) than in the chick. Another example suggesting the integration of different movements of cellular layers is presented in the formation of the floor of the hindgut of the developing pig embryo. In figure 242C, the rudiments of the fore gut and hindgut areas are established. However, in figure 242G, it is difficult to evaluate how much of the floor of the hindgut in this figure is formed by actual ingrowth forward from point “a” and to what extent the floor is formed by the rapid extension of tissues and backward growth of the caudal region of the embryo as a whole, including the allantoic diverticulum.

Fig. 234. Transverse sections of chick embryo with five pairs of somites. (This embryo is slightly older than that shown in fig. 233; a topographical sketch of this developmental stage is shown at the bottom of the figure with level of sections indicated.) Observe that a dorsal arching (dorsal upgrowth) movement of the dorsal tissues is associated with neural tube formation. See A and B.

Special processes also aid the formation of foregut and hindgut in many instances. For example, in the chick, the floor of the foregut is established in part by a medial or inward growth and fusion of the entodermal folds along the sides of the anterior intestinal portal, as indicated by the arrows in figure 234C. A similar ingrowth of entoderm occurs in the shark embryo (fig. 213J). although here the entoderm grows in as a solid layer from either side and is not present in the form of a lateral fold, as in the chick. However, it should be observed that the formation of the hindgut in the shark embryo arises by a most interesting and extraordinary method. In the flattened gastrulae of reptiles, birds, and mammals, the hindgut is established by the formation of tail folds, involving entodermal and epidermal layers. In the shark embryo, on the other hand, an enteric groove with enteric folds is formed, and the folds eventually move ventrad and fuse to form a hollow tube beneath the notochord of the developing tail.

Though the rudimentary foregut and hindgut areas of the metenteron arise almost simultaneously in mammalian embryos, such as in the pig and human embryos, in the chick a different sequence of procedure is present. In the latter species the foregut begins its development immediately following gastrulation when the first pairs of somites are present (fig. 233). The hindgut, on the other hand, begins its development at a considerably later period when the embryo has attained many pairs of somites (fig. 238).

Once the rudimentary, pouch-like, foregut and hindgut areas have been established in embryos developing from flattened gastrulae, their further development assumes morphogenetic features similar to those in the frog embryo. For example, the foregut possesses an antero-dorsal prolongation toward the brain, the pre-oral or head gut, while slightly posterior to the pre-oral gut, the future pharyngeal area makes contact ventrally with the stomodaeal invagination from the epidermal (ectodermal) tube (fig. 242G). Similarly, the caudal region of the hindgut rudiment contacts the proctodaeal invagination of the epidermal tube, while a tail gut extension continues into the tail (fig. 217).

The formation of definitive walls of the midgut area in embryos developing from the flattened gastrular condition (including the higher mammals which do not possess large amounts of yolk substance) occurs as follows:

1. Where the entoderm of the midgut terminates on either side of the notochord at the end of gastrulation, it grows mesad from either side below the notochord to complete the roof of the midgut (figs. 201 D; 209C; 21 OF; 213). This process is similar to that which occurs in the Amphibia (cf. fig. 219D).
2. A dorsal arching or evagination of the entoderm toward the notochordal area, comparable to that found in the frog and other Amphibia, is present also. A study of figures 213H-J; 217G; 23 4B; 237E-G; 241B-D demonstrates the marked dorsal upgrowth of all the forming body layers in the trunk area. {Note: In the elasmobranch fishes, a subnotochordah rod of cells of entodermal origin is formed similar to that in the frog and other Amphibia.)
3. The ventro-lateral walls of the midgut area, in contrast to those found in the frog, are established largely by actual ingrowth of the entoderm, mesoderm, and ectoderm with subsequent fusion in the median line in elasmobranch fishes, reptiles, birds, and mammals. This process involves the formation of lateral body folds which fold mesially toward the median plane. (Study fig. 241 A-D.) In teleost fishes the process is different, for in this group the entoderm and mesoderm grow outward beneath the primitive epidermis (ectoderm) and soon envelop the yolk. Thus, the end result in teleosts is much the same as in the frog and Necturus, It is well to observe, at this point, that a complete retraction of the ventro-lateral walls of the midgut and body-wall tissues surrounding the yolk or yolk-sac area, as in the frog and Necturus (fig. 227), does not occur in the higher vertebrates, although in the elasmobranch and teleost fishes such retraction does occur.

Fig. 235. Chick embryo of 9 to 10 pairs of somites. (Approximating Hamburger and Hamilton, '51, stage 10; 33 to 38 hours of incubation.) (A) Surface view, unstained. (B) Stained preparation. (C) Median sagittal section. Observe the following: heart is bent slightly to the right; three primary brain vesicles are indicated; foregut touches infundibular outgrowth of prosencephalon; first indication of downward bending of the head outgrowth, i.e., the cephalic (cranial) flexure is evident. (D) Same, showing major organ-forming areas.

Fig. 236. Transverse sections through chick embryo of about 12 to 13 pairs of somites, about 38 hours of incubation. (Approximately between stages 10 to 11 of Hamburger and Hamilton, '51, slightly older than that shown in fig. 235.) Observe that the optic vesicles are constricting at their bases; heart is bent slightly to the right; anterior neuropore is evident. (A) Optic vesicles. (B) Stomodaeal area. (C) Anterior end of developing heart. (D) Caudal extremity of forming heart. (E) Anterior intestinal portal and forming caudal portion of the heart. (F) Well-developed somites. (G) Open neural groove.

Fig. 237. Chick embryo of 17 to 19 pairs of somites. (Approximating Hamburger and Hamilton, '51, stage 13, 48 to 52 hours of incubation, sections indicated on outline drawing.) Head lies partly on left side; auditory pits are deep; cervical flexure is evident in region of rhombencephalon; cephalic flexure is marked; stomodaeum is a deep indentation touching foregut between the first pair of aortal arches; head fold of amnion reaches back to anterior part of rhombencephalon (hindbrain). (A) Anterior (telencephalic) portion of prosencephalon, showing closed neuropore; amnion is indicated. (B) Optic vesicles. (C) Anterior end of foregut, showing anterior extremity of stomodaeal invagination and first (mandibular) pair of aortal arches; notochord ends and pre -chordal plate area begins at about this section. (D) Anterior end of heart (ventral aorta); observe thin roof plate of neural tube, characteristic of the later myelencephalic (medulla) portion of rhombencephalon or hindbrain. (E) Otic (auditory) pits and anterior region of ventricular portion of heart. (F) Caudal limits of forming heart, dorsal mesocardium, neural crest cells. (G) Caudal end of heart, showing converging (vitelline) veins of the heart, sclerotome given off to notochordal area, lateral mesocardium forming. (H) Anterior trunk area, showing differentiation of somite and typically flattened condition of ectoderm, mesoderm, and entoderm. (I) Caudal trunk area, showing undifferentiated somite (epimeric mesoderm), intermediate mesoderm (mesomere), and lateral plate mesoderm (hypomere). (J) Similar to (I). (K) Caudal trunk region, showing closing neural tube. (L) Area of Hensen's node. (M) Primitive streak.

Fig. 238. Chick embryo of about 27 to 28 pairs of somites. (Corresponding approximately to Hamburger and Hamilton, '51, stage 16, 51 to 56 hours of incubation.) Forebrain (prosencephalon) is divided into telencephalon and diencephalon; epiphysis is appearing on roof of diencephalon; cephalic and cervical flexures are pronounced; tail bud is short; anterior part of body is rotated to the left back to about the thirteenth pair of somites; amnion now covers anterior three fifths of body; heart shows strong ventricular loop; three pairs of aortal arches can be seen. (A) External view. (B) Transparent wholemount." (C) Sagittal section, diagrammatic.

Fig. 239. Sections through chick embryo of age indicated in fig. 238. Level of sections is shown on diagram. (See Chap. 22.) In the elasmobranch fishes, this retraction of tissues contributes little to the formation of the wall of the enteron or to that of the body. However, in teleosts such contribution is considerable.

At this point reference should be made to figures 23 8C on the chick, 242C and G on the pig, and 245 B on the early human embryo to gain a visual image of the developing foregut, midgut, and hindgut areas of the primitive metenteron. Compare with the frog (fig. 225C).

4. Tubulation (Coelom Formation) and Other Features Involved in the Early Differentiation of the Mesodermal Areas

The differentiation of the mesodermal areas is an all-important feature of embryonic development, for the mesoderm contributes much to the substance of the developing body. (See Chaps. 11 and 15.) While the neural, enteric, and epidermal tubes are being established, radical changes occur within the two mesodermal layers on either side of the notochord as follows:

a. Early Changes in the Mesodermal Areas

1) Epimere; Formation of the Somites. The longitudinal mass of paraxial mesoderm which lies along the side of the notochord forms the epimere (figs. 221F, G; 234E, F). The two epimeres, one on either side of the notochord, represent the future somitic mesoderm of the trunk area. In the early post-gastrula, the epimeric mesoderm, together with the notochord, lies immediately below the neural plate. However, as neuralization is effected^ the area of the anterior trunk and posterior hindbrain region of the embryo. In the chick embryo (see Patterson, '07), the most anterior segment forms first, and later segmentation progresses in a caudal direction. This probably holds true for most other vertebrates. However, in elasmobranch fishes, segmentation of the epimeric mesoderm also extends forward from the hindbrain area into the head region presenting a continuous series of somites from the eye region caudally into the tail (fig. 217D). (Study figs. 217D, 230D.) Segmentation of epimeric mesoderm appears in the head region of Amphibia. In many higher vertebrates, three pairs of somitic condensations appear in the area just caudal to the eye but at a slightly later period of development than that of the elasmobranch fishes (fig. 217D-F).

Fig. 240. Chick embryo of about 72 to 75 hours of incubation, about stage 20 of Hamburger and Hamilton, '51.

2 ) Mesomere. The narrow longitudinal band of mesoderm, adjoining the lateral border of the epimere, is the mesomere (figs. 22 IF, G; 230D; 234E, F) . This mesoderm ultimately gives origin to much of the excretory (kidney) tissue and ducts and to certain of the reproductive ducts of many vertebrates. (See Chap. 18.) Because of the origin of nephric tissue from its substance, this longitudinal band of mesoderm generally is referred to as the urogenital or nephrotomic mesoderm. Synonymous terms often used are intermediate mesoderm or intermediate cell mass. The mesomere undergoes a segmentation similar to the epimeric area in its more anterior portion where the pronephric kidney develops in higher vertebrates, while in lower vertebrates, such as the shark embryo, it may be more extensively segmented.

3) Hypomere. The remainder of the mesoderm which extends lateroventrally from the mesomere forms the hypomere or hypomeric mesoderm. It also is called the lateral plate mesoderm or lateral plate mesoblast. This portion of the mesoderm does not become segmented in present-day vertebrates. (Compare with the condition in Amphioxus described on p. 505.)

b. Tabulation of the Mesodermal Areas

Coincident with the formation of the somites, a cavity begins to appear within the mesoderm. This cavity or primitive coelomic space separates the mesoderm into two layers, an outer layer near the ectoderm and an inner layer close to the neural, notochordal, and entodermal cells. This hollowing process within the mesodermal layer is known as coelom formation or tubulation of the mesoderm. In many embryos of the lower vertebrates, there is a strong tendency for the coelomic space to form throughout the entire lateral mass of mesoderm from the epimeric area ventrad into the lateral plate mesoderm. For example, in elasmobranch (shark) embryos of about 3 to 4 mm. in length and also in many early post-gastrular amphibia, the following features of the primitive coelom are found in the trunk region of each mesodermal mass:

(1) The mesoderm possesses a cavity, continuous dorso^ventrally from the epimere into the lateral plate (figs. 217G, H; 221 E). When the epimere (and to some extent the nephrotomic region as well) undergoes segmentation, the coelomic space within these areas becomes segregated within the segments and, thus, is present in a discontinuous condition.

(2) The early coelomic cavity in the shark and amphibian embryo, therefore, may be divided into three parts: (a) the myocoelic portion within the epimeric mesoderm, (b) the nephrocoel within the nephrotomic mesoderm, and (c) the splanchnocoel contained within the hypomeric or lateral plate mesoderm. While the myocoelic and nephrocoelic regions of the primitive coelom may become segmented and discontinuous, that within the splanchnocoel is continuous antero-posteriorly in the trunk region.

The coelomic cavities contained within the somites of the shark and amphibian embryo are soon lost. The coelomic cavity or nephrocoel within the nephrotome is concerned with the development of the lumen within the tubules and ducts of the excretory (urinary) system, while the splanchnocoels give origin to the coelomic cavity proper of the adult. The lateral wall of the splanchnocoel near the primitive epidermis is known as the somatopleural mesoderm, and the inner or medial wall associated with the gut tube and developing heart tissues constitutes the splanchnopleural layer. The epidermis and somatopleural mesoderm together form the somatopleure, while the entoderm and splanchnopleural mesoderm form the splanchnopleure.

In the embryos of higher vertebrates, the coelomic space of the somitic portion of the primitive coelom (i.e., the myocoels) is less pronounced and appears somewhat later in development than in the shark and amphibian embryo, but it does tend to appear. This is true also of the nephrocoel or coelomic cavity within the nephrotome. (See Chap. 18.) The coelomic condition or splanchnocoel within the hypomere forms similarly in all vertebrates. These matters will be described more in detail in Chapter 20.

C. Notochordal Area

The notochord is the elongated, median band of cells of the gastrula which lies between the two mesodermal areas. The notochord thus may be regarded as a specialized, median portion of the middle germ layer of mesodermal tissue. During gastrulation and shortly after, there may be a tendency for the notochordal material in certain forms to canalize or tubulate. Later, the notochordal material becomes converted into a definite rod of notochordal cells which represents the primitive skeletal axis of the embryo. The notochord and its relation to the early skeletal system are discussed in Chapter 15.

D. Lateral Constrictive Movements

While the neural, epidermal, and entodermal tabulations are in progress, a lateral constriction or invagination of the body wall occurs on either side in all vertebrate embryos from the fishes to the mammals. These constrictions are effected at the level of the notochord and lower margin of the somitic area from the anterior trunk region caudally into the tail. As a result, a transverse section of the early vertebrate body appears pyriform or pear shaped, with the neck of the pear directed dorsally (fig. 241 C). The constriction line is shown typically in the developing embryo of Necturus (fig. 227 ) where it extends from the lower aspect of the head outgrowth along the lower boundary of the somitic area to the base of the tail. A line, drawn across the body from the general area of the two lateral constrictions and passing through the notochord, divides the embryonic body into an upper or epaxial (epiaxial) region above the level of the notochord and a lower or hypaxial (hypoaxial) region below the level of the notochord.

E. Tubulation of the Neural, Epidermal, Entodermal, and Mesodermal, Organ-forming Areas in Amphioxus

1. Comparison of the Problems of Tubulation in the Embryo OF Amphioxus with that of the Embryos in the Sub Phylum Vertebrata

a. End-bud Growth

In Amphioxus, the procedures involved in tubulation of the major organforming areas and development of primitive body form differ from those in the vertebrate group. For example, in the latter group, the basic rudiments of the head, pharyngeal, trunk, and tail regions appear to be well established at the end of gastrulation. During tubulation of the major organ-forming areas, these subregions become extended in an antero-posterior direction and the rudiments of specific structures begin to express themselves. This is especially true of the head, pharyngeal, and trunk regions. The vertebrate tail, however, arises from an end-bud tissue which progressively lays down the various parts of the tail by means of a proliferative growth in the caudal direction. On the other hand, in Amphioxus, only a small portion of the anterior end of the future body is laid down during gastrulation. Further development of the epidermal, neural, enteric, and mesodermal cellular areas together with the notochord are dependent upon cell proliferation at the caudal end of the late gastrula and later embryo. Much of the body of Amphioxus, therefore, is formed by a caudal proliferative growth of end-bud cells, somewhat comparable to the end-bud growth of the tail in the vertebrate group.

b. Position Occupied by the Notochord and Mesoderm at the End of Gastrulation

A second feature of difference in the developing embryo of Amphioxus from that of the vertebrate embryo lies in the arrangement of the notochordmesoderm complex of cells in the late gastrula. In the late gastrula of Amphioxus, this potential, third germ layer forms a part of the entodermal roof, although the studies of Conklin ('32) have demonstrated that notochord and mesoderm are distinct cellular entities even in the blastula. In contrast to this condition, the notochord and the mesoderm already are segregated as a middle germ layer between the ectoderm and the entoderm in the late vertebrate gastrula. The gastrula of Amphioxus, therefore, has the added problem of segregating the notochordal and mesodermal cells from the entoderm during tubulation of the major organ-forming areas.

2. Neuralization and the Closure of the Blastopore

In the late gastrula of Amphioxus, a longitudinal middorsal plate of cells, the neural plate, elaborated by cell division and extension during gastrulation, represents the future central nervous system (fig. 247E). As the period of gastrulation comes to its end, the blastopore decreases greatly in size (fig. 247A-D). The archenteric opening also moves dorsally, coincident with a shifting of the caudal end of the archenteron in such a way that it projects in a dorso-caudal direction (figs. 189G, H; 247H). This movement of the archenteron is associated with the migration of the mass of mesodermal cells from the two lateral areas of the blastoporal lips (fig. 247A, B) to the dorsomedial portion of the blastopore (fig. 247C), where the mesoderm comes to lie on either side of the notochord below the neural plate (fig. 247C). As these changes occur, the dorsal area of the gastrula near the blastopore becomes flattened with a subsequent depression of the neural plate (fig. 247C, D). In sagittal section, the gastrula now appears oval in shape and considerably elongated in the antero-posterior direction (fig. 189G, H); in transverse view, it is triangular, especially at the caudal end (fig. 247D).

As the above changes are brought about, the ectoderm of the ventral lip of the blastopore grows dorsad, while that of the lateral lips grows mediad. In this way, the opening of the blastopore is closed by the coming together and fusion of these ectodermal (epidermal) growths (fig. 247D-F). However, the archenteron does not lose its connection, at this time, with the outside environment of the embryo for two reasons:

1. As observed above, the caudal end of the archenteron previously had shifted in such a manner that it now projects dorso-caudally; and
2. synchronized with the epidermal growth which closes the blastoporal opening (fig. 248A), the neural plate sinks downward, becoming detached along its margin from the epidermal area (fig. 248B-D).

The downward sinking of the neural plate and its detachment from the epidermal layer begins at the dorsal lip of the blastopore and spreads anteriad. (Compare fig. 248D with 248B and C.) Consequently, as the epidermal growth along the lateral lips of the blastopore reaches the area of the sinking neural plate in the region of the dorsal blastoporal lip, it continues forward along the epidermal margins of the insinking neural plate, growing mesad and fusing in the midline over the neural plate (fig. 247E-G). In this way, the epidermal growth forms a covering for the neural plate. It follows, therefore, that the posterior end of the archenteron will now open into the space between the neural plate and its epidermal covering. This new passageway between the epidermalneural plate cavity and the archenteron is the beginning of the neurenteric canal (figs. 247H; 248A).

Fig. 242. Early development of the pig embryo (B, C, and G from Patten: Embryology of the Pig, Philadelphia, Blakiston; A is from Streeter: Carnegie Inst. Publ. No. 380; Contrib. to Embryol. 100; D, E, and F from Heuser and Streeter: Carnegie Inst. Publ. No. 394, Contrib. to Embryol. 109. All figures have been modified), (A) Early, neural groove stage. Neural area is shown in black; amnion is cut away as indicated. (B) Four-somite stage. (C) Median sagittal section, approximating the stage of development shown in (B). Observe foregut, midgut, and hindgut areas. (D) Embryo of about six pairs of somites. (E) Embryo of about 7 to 8 pairs of somites. (F) Eighteen pairs of somites. (G) Sagittal .sectional diagram of embryo slightly younger than (F), showing neural and gut tubes, amnion, allantois, and forming heart.

The flattened neural plate, canopied by the epidermal overgrowth, then begins to fold itself into the form of a tube. In doing so, its lateral edges swing gradually toward the middorsal line, as shown in figure 195. The actual grooving and tubulation of the neural plate starts at a point about midway along the embryo at the stage of development shown in figure 247F. It proceeds anteriorly and posteriorly from this point. At its extreme anterior end, the neural tube remains open to the surface as the anterior neuropore (figs. 247H; 249A-D). Eventually the caudal end of the neural plate becomes tubulated, and a definite canal is formed, connecting neural and enteric tubes. This canal is the neurenteric canal. The neurenteric canal disappears between the stage of development shown in figure 249C and that shown in figure 249D. The continued caudal growth of the neural tube is accomplished by cell proliferation from the posterior end of the tube and neurenteric canal area.

Fig. 243. Sections of pig embryo of about stage shown in fig. 242 (B) and (C). (Modified from Patten: Embryology of the Pig, 3d Ed., Philadelphia, Blakiston, '48.) (A) Line 1, fig. 242C. (B) Line 2, fig. 242C. (C) Line 3, fig. 242C. (D) Line 4, fig. 242C.

Fig. 244. Development of body form in the pig embryo. (A and B from Keibel: Normentafel zur Entwicklungsgeschichte des Schweines (Sus scrofa domesticus). 1897. Jena, G. Fischer. C, D, and E slightly modified from Keibel, previous reference, and from Minot: A Laboratory Text-book of Embryology. 1903. Philadelphia, P. Blakiston's Son & Co.) (A) About 4 to 5 mm. (B) About 6 mm. (C) Ten mm. (crown-rump measurement). (D) Fifteen mm. (E) Twenty mm.

3. Epidermal Tubulation

After the neural plate sinks downward and becomes separated from the outside epidermis, the medial growth of the epidermis over the neural plate completes the middorsal area of the primitive epidermal tube (fig. 247 E-H). It then comes to enclose the entire complex of growing and elongating neural, mesodermal, and entodermal tubes and with them it continues to grow in length principally by rapid cell proliferation at the caudal end of the embryo.

Fig. 245. Human embryo of ten somites. (After G. W. Corner: Contrib. to Embryol Carnegie Inst., Washington, Publ. No. 394, 112.) (A) Dorsal view. (B) Median sagittal section of model.

Fig. 246. Development of body form in human embryo. (C from Keibel and Mall: Manual of Human Embryology, Vol. I, 1910. Philadelphia and London, Lippincott. A, B, D, and E from Keibel and Elze: Normentafel zur Entwicklungsgeschichte des Menschen. Jena, 1908. G. Fischer.) (A) Early neural fold stage. Somites are beginning to form; notochordal canal is evident. (B) About nine pairs of somites. (C) His's embryo M. (D) About 23 pairs of somites, 4-5 mm. long. (E) About 35 pairs of trunk somites, 12 mm. long.

4. Tubulation of the Entodermal Area

The primitive metenteron of Amphioxus is derived from the archenteron of the late gastrula as follows.

a. Segregation of the Entoderm from the Chordamesoderm and the Formation of the Primitive Metenteric Tube

The mesoderm and notochord which occupy the roof of the archenteron of the gastrula evaginate dorsally at the anterior end of the embryo and, thus, become separated from the entoderm. (Compare fig. 195 with fig. 250A.) This separation of notochord and mesoderm by dorsal evagination from the entoderm continues slowly in a caudal direction from the anterior end until an embryonic condition is reached approximating about 13 to 14 pairs of mesodermal segments. At this level, the notochord and mesoderm become completely separated from the entoderm. As a result, the enteric or gut tube from this point in its growth posteriad is a separate entity. (See tabulation of mesoderm on p. 505. Anterior to the fourteenth somite, after the notochord and mesoderm separate from the entoderm, the latter grows medially from either side to complete the entodermal roof below the evaginated notochord and mesoderm (fig. 250A). A primitive metenteric tube thus is formed, as shown in figure 249C, whose only opening is that which leads by way of the neurenteric canal (fig. 249 A, C) into the neurocoel of the neural tube and from thence to the outside through the anterior neuropore.

Fig. 247. Closure of the blastopore and epidermal overgrowth of neural plate in Amphioxus (original diagrams, based on data supplied by Conklin, '32). (A) Vegetal pole view of early stage of gastrulation, showing general areas occupied by notochordal, entodermal, and mesodermal cells. (B) Same view of gastrula, one hour later, showing triangular form of blastopore, (C) Posterior view of late gastrula. Blastopore is now ovoid in shape and dorsally placed. Gastrula is triangular in transverse section with dorsal surface flattened. (D) Same view, later. Slight epidermal upgrowth, indicated by arrows (a and a') merges with ingrowing epidermal edges along lateral lips of blastopore (b and b') which spreads along epidermal edges of neural plate. (E) Dorsal view a brief period later than (D). Epidermal ingrowth from lateral blastoporal lips is now closing the blastoporal opening, shown in broken lines, and also is proceeding craniad along edges of sinking neural plate. (See fig. 248.) (F, G) Later stages of epidermal overgrowth of neural plate. (H) Sagittal section of (G).

Fig. 248. Sinking of neural plate and epidermal overgrowth of neural plate in Amphioxus. (Slightly modified from Conklin, '32.) (A) Sagittal section of embryo comparable to that shown in fig. 247F. (B, C, D) Sections through embryo as shown by lines B, C, D, respectively, on (A). Observe that the neural plate begins to sink downward from region of closed blastopore and proceeds forward from this point.

b. Formation of the Mouth, Anus, and Other Specialized Structures of the Metenteron

At the anterior end of the metenteron, a broad, dorsal outgrowth occurs which continues up on either side of the notochord and becomes divided into right and left dorsal diverticula (fig. 249B, H). The left diverticulum remains small and thick-walled and later fuses with an ectodermal invagination to form the pre-oral pit, described as a sense organ. The right diverticulum, however, increases greatly in size, becomes thin-walled, and gives origin to the so-called head cavity.

The mouth develops at a time when the larva acquires about 16 to 18 pairs of mesodermal segments or somites (fig. 249D). It appears when the overlying epidermis about halfway up on the left side of the body fuses with the entoderm, a fusion which occurs just posterior to the forming pre-oral pit (left diverticulum). (See black oval fig. 249D, and fig. 249F.)

At the time that the mouth forms, the entoderm opposite the first pair of somites pushes ventrally and fuses with the ectoderm. This area of fusion finally perforates and forms the first gill slit. The gill slit, once formed, moves up on the right side of the body (fig. 249E). The entodermal area from which the first and later gill slits make their appearance is known as the branchial rudiment (fig. 249D).

At the caudal end of the larva, following the degeneration of the neurenteric canal, a small area of entoderm fuses with the ectoderm and forms the anal opening. The anus is first ventral in position, but later moves up to the left side as the caudal fin develops (fig. 249E, G).

5. Tubulation of the Mesoderm

Tubulation of the mesoderm and the formation of a continuous anteroposterior coelom in Amphioxus differs considerably from that found in the subphylum Vertebrata. This fact becomes evident in tracing the history of the mesoderm from the time of its segregation from the entoderm of the late gastrula and later embryo to the stage where a continuous antero-posterior coelomic space is formed, comparable to that found in the vertebrates.

Fig. 249. Various stages of development of Amphioxus. (A from Kellicott, '13, and Conklin, '32; B from Kellicott, '13, slightly modified; C-I, slightly modified from Conklin, '32.) (A) Six -somite stage, comparable to fig. 247G and H. The animal hatches about the time that two pairs of somites are present. (B) Nine-somite stage. The larva at this stage swims by means of cilia which clothe the entire ectodermal surface. (C) About fourteen pairs of somites are present at this stage. Neurenteric canal is still patent. (D) About 16 to 18 pairs of somites. Neurenteric canal is degenerating; mouth is formed. (E) About 20 to 22 pairs of somites. Anal opening is established between this stage and that shown in (D). (F) Transverse section, showing oral opening, looking from anterior end of animal. (G) Same through anal area. (H) Frontal section of a 24-hour larva near dorsal side showing notochord, somites (S-1, S-8, etc.) and undifferentiated tissue at caudal end. Neural tube shown at anterior end. Nine pairs of somites are present. (I) Frontal section of a 38-hour larva at the level of the notochord showing section through the neural tube at the anterior and posterior ends, i.e., in region where larva bends ventralwards. Thirteen pairs of somites are present with muscle fibrillae along the mesial borders of the somites.

The mesoderm of the late gastrula of Amphioxus is present as a dorsomedian band of cells on either side of the notochord, and together with the notochord, occupies the dorsal area or roof of the archenteron as mentioned previously. In the region of the blastopore, the two mesodermal bands diverge ventrally and occupy the inner aspect of the lateral walls of the blastopore (fig. 190F, G; 247B). At about the time of blastoporal closure, the two mesodermal masses of cells, located along the lateral lips of the blastopore, are retracted dorsally, where they come to lie on either side of the notochord (fig. 247C). In this position the two bands of mesoderm and the notochord continue to form the dorsal region or roof of the archenteron until approximately the time when the embryo is composed of 13 to 14 pairs of mesodermal segments or somites (fig. 249C). (See Hatschek, 1893, pp. 131, 132; Willey, 1894, p. 115; Conklin, '32, p. 106.) When the embryo reaches a stage of development wherein 15 to 16 pairs of somites are present, the notochord and mesoderm have separated entirely from the entoderm (fig. 249D). At about this period the neurenteric canal between the metenteron and the neural tube disappears (fig. 249C, D).

Fig. 250. Differentiation of somites in Amphioxus. (A and B from Conklin, '32; C, E, and F after Hatschek, 1888 and 1893; D from MacBride, 1898; all figures are modified.) (A) Somites shortly after separation from entoderm. (B) Later stage, the somites grow ventrally. (C) Semitic wall begins to differentiate into a thickened, dorsal, myotomic area, located near notochord and neural tube, and thinner somatic and visceral areas. (D) Horizontal septum formed which separates dorso-myotomic portion of somite from splanchnocoelic area below. (E, F) Later stages in differentiation of myotome and myocoelic diverticulum. (See text.)

The formation of a continuous, antero-posterior, coelomic cavity in Amphioxus may be described as follows. The mesodermal bands on either side of the notochord of the post-gastrular embryo become converted into mesodermal grooves as each mesodermal band folds inwards or evaginates into the residual blastocoelic space between the archenteron and the outside ectoderm (fig. 195). Beginning at the anterior end, these longitudinal grooves of mesoderm soon become divided into distinct segments or somites by the appearance of transverse divisions (fig. 249 A, B, H). The first and second pairs of somites are formed at the anterior ends of the mesodermal grooves at about the time that the embryo hatches and swims about by means of ciliary action.

Eventually each somite becomes entirely constricted from the notochord and entoderm. In this segregated condition the somite forms a rounded structure retaining within itself a portion of the original archenteric cavity (fig. 250A). Hence, the cavity within the somite is called an enterocoel and represents the beginnings of the coelomic cavity of later development, at least in the anterior 13 or 14 pairs of somites. (Note: It is to be observed in this connection that the primitive somite in Amphioxus is not comparable to the primitive somite of the vertebrate embryo. In the latter, the somite represents merely a segment of the epimeric mesoderm, whereas in Amphioxus it is the entire mesoderm in each half of a particular segment of the embryo.)

After hatching, the mesodermal bands continue to form into grooves as the embryo elongates, and, synchronously, successive pairs of somites are formed. At about the time 8 to 10 pairs of somites are present (fig. 249B, H), the enterocoels of the first two pairs of somites have become entirely separated from the archenteron. The enterocoels of the following six pairs of somites are small and are not as evident at first as those of the first two pairs. Ultimately a definite enterocoel is found, however, in each somite.

Posterior to the eighth or ninth pairs of somites, the forming mesodermal grooves do not show the enterocoelic pouches as plainly as the more anterior somites. Slit-like mesodermal grooves tend to be present, however, and, when the somite is entirely free from the archenteron, this slit-like cavity expands into the enterocoelic space of the somite. As the region of the fourteenth pair of somites is approached, the slit-like mesodermal groove becomes more and more indefinite. Posterior to the fourteenth or fifteenth pair of somites, the somites originate from a solid mesodermal band on either side of the notochord. An enterocoelic origin of the cavity within each somite, therefore, is not possible caudal to this area, and the coelomic space arises by a hollowing-out process similar to coelomic cavity formation in the vertebrate group.

At about the time when eight pairs of somites are established, a shift of the mesoderm on either side of the embryo produces a condition wherein the somites of either side may be slightly intersegmental in relation to the somites on the other side (fig. 249H).

During its later development, each somite grows ventrally (fig. 250B). That portion of the somite contiguous to the notochord and neural tube thickens and forms the myotome. The region of the somite near the epidermal ectoderm is called the somatic or parietal mesoderm, while that associated with the entoderm forms the visceral or splanchnic mesoderm (fig. 25 OC).

As the myotome enlarges, the coelomic space becomes more and more displaced ventrally, and most of it comes to lie on either side of the enteron (metenteron). (See fig. 250D.) This ventral coelomic space forms the splanchnocoel, while the dorsal space, lateral to the myotome, is known as the myocoel. Eventually, the splanchnocoels of each pair of somites push ventrally to the lower portion of the enteron, where they ultimately fuse (fig. 250D-F). Gradually the splanchnocoels of each segment fuse anteroposteriorly and in this way a continuous, antero-posterior, splanchnocoelic space below and around the gut tube is formed. Tubulation or the formation of a continuous, antero-posterior, coelomic cavity thus is effected by fusion of the splanchnocoels of the respective somites on either side (fig. 25 OF). A horizontal septum, the intercoelomic membrane also appears, separating the myocoels above from the splanchnocoelic cavity below (fig. 250D).

6. Later Differentiation of the Myotomic (Dorsal) Area of the Somite

While the above events are taking place in the ventral portion of the somite, the upper, myotomic region undergoes profound modification.

As shown in figure 250D, the myotomic portion of the somite has two unequally developed areas;

1. a medial muscular portion, the myotome and
2. the laterally placed, thin-walled, parietal part which surrounds the coelomic space, or myocoel.

The muscular portion enlarges rapidly and, as seen in figure 25 OE and F, forms the muscle plate or myotome of the adult. These muscle plates very early assume the typical > shape characteristic of the adult. On the other hand, the myocoelic portion contributes important connective or skeletal tissue to the framework of the body. In each segment, the wall of the myocoel gives origin to three diverticula as follows:

(a) a lower sclerotomic diverticulum,

(b) a ventral diverticulum, and

(c) a dorsal sclerotomic diverticulum.

The lower sclerotomic diverticulum (fig. 250D, E) extends up between the myotome and the medially placed notochord and nerve cord, as diagrammed in figure 250F. Its walls differentiate into two parts:

1. an inner layer which, together with a similar contribution from the somite on the opposite side, wraps around the notochord and nerve cord and, subsequently, gives origin to a skeletogenous sheath of connective tissue which enswathes these structures; and
2. an outer layer which covers the mesial (inner) aspect of the myotome with a fascia or connective tissue covering.

The outer surface of the myotome does not have a covering of fascia.

The ventral diverticulum extends between the lateral wall of the splanchnococl and the epidermal layer of the body wall (fig. 250E, F) and separates the parietal wall of the splanchnocoel from the epidermal wall (fig. 250F). This ventral diverticulum or dermatomic fold, together with the external or parietal wall of the myocoel above, forms the dermatome. The inner and outer layers of the ventral diverticulum gradually fuse to form the cutis or dermal layer of the integument or skin in the ventro-lateral portion of the body, whereas the parietal wall of the myocoel above gives origin to the same dermal layer in the body region lateral to the myotome. The dorsal sclerotomic diverticula form the fin-ray cavities in the dorsal fin. These cavities become entirely isolated from the rest of the myocoelic spaces. Several fin-ray cavities occupy the breadth of a single myotome. The dorsal myotomic portion of the somite thus differentiates into three main structural parts:

(a) the muscular myotome,

(b) the mesial sclerotome or skeletogenous tissue, and

(c) the latero- ventral dermatome or dermal tissue of the skin.

7. Notochord

The notochord arises as a middorsal evagination of the primitive archenteron up to about the stage of about 13 to 14 pairs of somites (fig. 195). Posterior to this region it takes its origin by proliferative growth from a separate mass of notochordal tissue, lying above the gut and between the two mesodermal masses of cells. Its origin posterior to the general area of the thirteenth to fourteenth body segments, therefore, has no relation to the entoderm. It rapidly develops into a conspicuous skeletal rod, lying below the neural tube and between the mesodermal somites and resting in a slight depression along the dorsal aspect of the metenteron or entodermal tubulation (fig. 249E, H). It continues forward in the head region, anterior to the brain portion of the neural tube (fig. 249E).

(The student is referred to the following references for further details relative to the early development of Amphioxus: Cerfontaine, '06; Conklin, '32; Hatschek, 1893; Kellicott, '13; MacBride, 1898, '00, '10; Morgan and Hazen, '00; and Willey, 1894.)

F. Early Development of the Rudiments of Vertebrate Paired Appendages

Two pairs of appendages, placed at the anterior (pectoral) and posterior (pelvic) extremities of the trunk, are common to all vertebrate groups. However, all vertebrates do not possess two pairs of paired appendages. Certain lizards of the genera Pygopus and Pseudopus have only a posterior pair of appendages, while in certain other vertebrates the opposite condition is found, the anterior pair being present without posterior appendages. The latter condition is found in certain teleost and ganoid fishes; the amphibian. Siren lacertina; the lizard, Chirotes; and among the mammals, the Sirenia and Cetacea. Again, some vertebrates are entirely apodal, e.g., cyclostomatous fishes and most snakes, although the boa constrictors and pythons possess a pair of rudimentary posterior appendages embedded in the skin and body wall. Some have rudimentary appendages only in the embryo, as the legless amphibians of the order Gymnophiona, and certain lizards. Consequently, the presence of embryonic rudiments of the paired appendages is a variable feature when the entire group of vertebrates is considered.

The rudiments of the paired appendages also are variable, relative to the time of their appearance in the vertebrate group as a whole. They are more constant in the Amniota, i.e., reptiles, birds, and mammals, in time of appearance than in the Anarnniota, i.e., fishes and amphibia. In the reptiles, birds, and mammals, the limb buds arise when primitive body form is being evolved. In the anuran amphibia, the anterior rudiments may appear and go on to a high degree of differentiation before the appearance of the posterior pair of appendages. For example, in the frog, Rana pipiens, the posterior limb buds first make their appearance a brief period before the beginning of metamorphosis of the tadpole into the adult form. However, the anterior limb buds differentiate earlier but remain concealed beneath the operculum until they become visible during the later stages of metamorphosis. In urodele amphibia, the fore limb bud is not covered by an operculum, and it is visible at the time of its initial appearance which occurs before the hind limb rudiment arises (fig. 227J-L).

In the majority of vertebrates, the limb rudiment first makes its appearance as an elongated, dorso-ventrally flattened fold of the epidermis, containing a mass of mesodermal cells within, as shown, for example, in the chick and mammalian embryos (figs. 240, 244, 246). The contained mesodermal cells may be in the form of epithelial muscle buds derived directly from the myotomes (e.g., sharks) or as a mass of mesenchyme (chick, pig, human). (See Chap. 16.) The early limb-bud fold may be greatly exaggerated in certain elasmobranch fishes, as in the rays, where the anterior and posterior fin folds fuse together for a time, forming one continuous lateral body fold. On the other hand, in the lungfishes (the Dipnoi) and in amphibia (the Anwa and Urodela), the appendage makes its first appearance, not as an elongated fold of the lateral body wall, flattened dorso-ventrally, but as a rounded, knob-like projection of the lateral body surface (fig. 227K-M).

G. The Limb Bud as an Illustration of the Field Concept of Development in Relation to the Gastrula and the Tubulated Embryo

In Chapter 9 it was observed that the major presumptive organ-forming areas are subdivided into many local, organ-forming areas at the end of gastrulation. In the neural and epidermal areas, this subdivision occurs during gastrulation through influences associated with local inductive action. At the end of the gastrular period, therefore, each local area within the major organforming area possesses the tendency to give origin to a specific organ or a part of an organ. The restricted, localized areas within each major organforming area represent the individual, or specific, organ-forming fields. During tubulation, the major organ-forming areas with their individuated, organ-forming fields are molded into tubes, and, thus, the individual fields become arranged along each tube. Consequently, each tube possesses a series of individual, organ-forming areas or fields, distributed antero-posteriorly along the tube.

As a result of the close association of cells and substances during gastrulation and tubulation, many specific organ-forming fields are related to more than one of the body tubes. Specific organ-forming fields, therefore, may have intertubular relationships. For example, the lens field is located in the epidermal tube, but, in many species, its origin as a lens field is dependent upon influences emanating from the optic vesicle of the neural tube (see Chap. 19). Another example of an association between the parts of two contiguous tubes is the limb-bud field in the urodele, Amby stoma punctatum. As the limb-bud field in this species illustrates various aspects and properties of an organ-forming field, it will be described below in some detail.

The presumptive anterior limb disc or limb field of Amby stoma is determined as a specific limb-forming area in the middle gastrular stage (Detwiler, '29, '33). Later on in the embryo, it occupies a circular-shaped area within trunk segments three to six. According to Harrison ('18) and Swett ('23), its properties as a field mainly are resident in the cells of the somatic layer of the mesoderm in this area. If, for example, the somatic layer of mesoderm in this area is transplanted to another area, a well-developed limb will result. Also, the mesoderm of the dorsal half of the field forms a greater part of the limb than the other parts, with the anterior half of the limb disc next in importance. It appears, therefore, that the limb-forming potencies are greatest in the dorso-anterior half of the limb field and become less postero-ventrally. Moreover, not “all of the cells which are potentially limb forming go into the limb” (Swett, '23). As demonstrated by Harrison ('18) half discs (half fields), left intact in the developing embryo or removed and transplanted to other areas, develop into normal limbs.

The above experiments of Harrison, together with those of Detwiler ('29, '33) suggest that while the limb field is irreversibly determined at an early stage to form limb tissue, the exact determination of the various parts within the field is absent at the earlier phases of development. One kind of precise determination is present, however, for the first digit-radial aspect (i.e., the pre-axial aspect) of the limb appears to arise only from the anterior end of the field, whether the field is allowed to develop intact or is split into two parts. That is, if it is split into two portions, the anterior extremity of the posterior portion, as well as the original anterior part of the limb field, develops the pre-axial aspect of the limb. This antero-posterior polarization is present from the first period of field determination. On the other hand, the dorso-ventral polarity is not so determined; for if the transplanted limb disc is rotated 180 degrees (i.e., if it is removed and reimplanted in its normal place dorsal side down) it will develop a limb with the dorsal side up but with the antero-posterior axis reversed (Harrison, '21). In these cases the first digit-radial aspect will appear ventral in position. This result indicates that the pre-axial aspect of the limb becomes oriented always toward the ventral aspect of the limb. However, the experiments of Swett ('37, '39, '41) tend to show that the reversal of the dorso-ventral axis occurs only when implanted below the myotome s; for when the rotated limb field is implanted in the somitic (myotomic) area, it will remain inverted. Factors other than those resident within the limb field itself, probably factors in the flank area, appear thus to induce the normal dorso-ventral axis when the limb disc is implanted in its normal site.

In the descriptions given above, the importance of the somatic layer of mesoderm as the seat of the limb-forming factors is emphasized. It is obvious, however, that the epidermal covering of the limbs derived from the epidermal tubulation also is important in limb formation. For example, epidermal importance is suggested by the experiments of Saunders ('49) on the developing limb bud of the chick wherein it was found that the apical ridge of ectoderm, located at the apex of the early limb bud, is essential for normal limb development.

Individual, or specific, organ-forming fields which appear in the gastrula and early tubulated embryo thus are generalized areas determined to form individual organs. As development proceeds, two main limitations are imposed upon the field:

1. The cellular contribution of the field actually entering into the organ becomes restricted; and
2. specific parts of the field become progressively determined to form specific parts of the organ.

It is obvious, therefore, that the fields of influence which govern the development of specific organs may be much more extensive in cellular area than the actual cellular contributions which take part in the formation of the specific organ structures. Experiments on the forming limb of Amby stoma also have demonstrated that a particular area of the field is stronger in its limb-forming potencies than other regions of the field. This property probably is true of other fields as well.

(For a detailed discussion of the field concept in embryonic development, reference should be made to Huxley and DeBeer, '34, Chaps. 8 and 9; Weiss, '39, p. 289 ff.)

H. Cephalic Flexion and General Body Bending and Rotation in Vertebrate Embryos

The anterior end of the neural tubulation is prone to assume a bent or flexed contour whereby the anterior end of the neural tube is directed downward toward the ventral aspect of the embryo. This general behavior pattern is strong in vertebrate embryos with the exception of the teleost fishes. In teleost fishes this bending habit is slight. As the later development of the head progresses in other vertebrate embryos, the neural tube shows a pronounced cephalic (cranial) flexure in the region of the midbrain, in some species more than in others. (See Chap. 19.) An additional bending occurs in the posterior hindbrain area. The latter flexure is the cervical or nuchal flexure (figs. 231, 238, 240, 244, 246).

Aside from the acute bending which takes place in the formation of the cephalic and the nuchal flexures, there is a definite tendency for many vertebrate embryos to undergo a general body bending, with the result that the anterior part of the body and the caudal portion of the trunk and tail may be depressed in a ventral direction (figs. 222C-E; 227; 229F; 238; 240; 244; 246). In the frog embryo, at hatching, the opposite tendency may prevail for a brief period, and the dorsal trunk region may appear sagging or hollowed (fig. 226A, C).

In addition to these bending movements, in the embryos of higher vertebrates, a rotation or twisting (torsion) of the developing body along the antero-posterior axis is evident. In the chick embryo, for example, the head region begins to rotate toward the right at about 38 hours of incubation. Gradually this torsion continues caudally (figs. 237, 238, 239, 260). At about 70 to 75 hours, the rotational movement reaches the tail region, and the embryo then comes to lie on its left side throughout its length (fig. 240). In exceptional embryos, the rotational movement is toward the left, and the embryo comes to lie on its right side. Similar movements occur in the pig and other mammals.

This rotational movement is advantageous, particularly in long-bodied Amniota, such as the snakes, where it permits the developing embryo to coil in spiral form within the extra-embryonic membranes. The coiling tendency, however, is not alone confined to the snake group, for the habits of general body bending, referred to above, essentially is a coiling tendency. Viewed thus, the rotation or torsion of the developing body along its median axis is a generalized behavior pattern which permits and aids the coiling habit so prevalent among the embryos of higher vertebrates. It may be observed further that the coiling behavior is a common attitude during rest not only among snakes but also among the adults of many higher vertebrates.

I. Influences Which Play a Part in Tubulation and Organization of Body Form

In Chapter 9, it was pointed out that the pre-chordal plate material, that is, organizer material which invaginates first during gastrulation and which comes to lie in the future head region, induces the organization of certain head structures and itself may form a part of the pharyngeal wall and give origin to head mesoderm, etc. On the other hand, the trunk-organizer material (notochord and somitic mesoderm) which moves to the inside, following the pre-chordal plate material, organizes the trunk region. The following series of experiments based upon work by Spemann, '31, sets forth the inductive properties of these two cellular areas:

Experiment

1. Head-organizer material, taken from one embryo and placed at head level of a host embryo, will induce a secondary head, having eyes and ear vesicles
2. Head-organizer material, transplanted to trunk and tail levels in host embryos, induces a complete secondary embryo, including head
3. Trunk-organizer material (i.e., notochord and somitic mesoderm), placed at head level in host embryo, induces a complete secondary embryo, including the head structures
4. Trunk-organizer material, placed at future trunk or tail levels in host embryos, induces trunk and tail structures only

The many influences which play a part in the organization of the vertebrate head and body constitute an involved and an unsolved problem. The extreme difficulty of this general problem has long been recognized. (See Kingsbury and Adelmann, '24.) The above-mentioned work of Spemann represents a beginning attempt to analyze this aspect of development and to understand the factors involved. It demonstrates that the organization of the neural tube and other axial areas is dependent upon specific cellular areas which migrate inward during gastrulation. However, this is but one aspect of the problem. As observed in the series of experiments above, trunk-organizer material is able to organize a complete secondary embryo, including the head, when placed at head level in the host but can only organize trunk and tail structures when placed in trunk and tail areas of the host. In other words, there exists a mutual relationship between the level of the host tissues and the transplanted organizer material of the trunk organizer in effecting tlie formation of a head at the head level.

Fig. 251. Dependency of neural tube formation upon surrounding tissues. (A) Effect of notochord without myotomes. (B) Effect of myotomes without notochord. (C) Absence of notochord and myotomes.

Another forceful example of the interrelationship of developing parts and formative expression of body structures is shown by the work of Holtfreter ('33) on the development of the neural tube. This work demonstrates that the form of the neural tube is dependent upon influences in its environment, as shown in figure 251. The presence of the later developing notochord determines a thin ventral floor of the neural canal, whereas the contiguous myotome determines a thick wall of the neural tube. Normally, in development, the notochord lies below the neural tube, while the somites with their myotomie parts come to lie lateral to the tube. That is to say, the normal bilateral symmetry of the neural tube is dependent upon the relationship, in their normal positions, of the notochord and the myotomes.

The behavior of the developing neural tube, relative to the notochord and the myotomes, demonstrates the importance of the' migration of the somitic mesoderm from a position contiguous and lateral to the notochord at the beginning of neuralization to one which is lateral to the forming neural tube as neuralization and differentiation of the neural tube progresses.

A further illustration of the probable influence of the notochordal area in morphogenesis and organization of body form is the behavior of the developing metenteron or enteric tube. As observed previously, the gut tubulation tends to invaginate or arch upward toward the notochord not only in embryos developing from flattened gastrulae but also in amphibia. The movement of the entoderm toward the notochord strikingly resembles the behavior of the neural plate ectoderm during the formation of the neural tube. This comparison becomes more striking when one considers the manner of enteron formation in the tail and hindgut regions in the shark embryo, Squalus acanthias, already mentioned, p. 484. In this species the entoderm of the developing tail actually invaginates dorsad and closes in a manner similar to the forming neural tube. That is to say, in the developing tail of the shark, two invaginations toward the notochord are evident, one from the dorsal side, which involves the formation of the neural tube, and the other from the ventral side, effecting the developing enteric tube.

The above facts suggest, therefore, that one of the main organizing influences at work during tubulation and primitive body formation emanates from the pre-chordal plate area, the notochord, and the epimeric portion of the mesoderm. From this general area or center, a chain of acting and interacting influences extends outward, one part acting upon another, to effect the formative expression of the various parts of the developing body.

J. Basic Similarity of Body-form Development in the Vertebrate Group of Chordate Animals

In the earlier portion of this chapter, differences in the general procedures concerned with tubulation and primitive body formation in round and flattened gastrulae were emphasized. However, basically all vertebrate embryos show the same tendency of the developing body to project itself upward and forward in the head region, dorsally in the trunk area and dorso-posteriad in the tail region. Literally, the embryonic body tends to lift itself up out of, and above, the area which contains the yolk and extra-embryonic tissues. This proneness to move upward and to protrude its developing head end forward and its caudal end backward is shown beautifully in the development of the embryos of the shark (figs. 229, 230), the mud puppy (fig. 227), the chick (fig. 235C), and the pig (fig. 242). The embryo struggles to be free from its bed of yolk and extra-embryonic tissue, as it were, and it reminds one of the superb imagery employed by the poet, John Milton, in his immortal poem, Paradise Lost, where he describes the development of the lion thus:

The grassy clods now calv'd; now half appear'd

The tawny lion, pawing to get free

His hinder parts, then springs as broke from bonds,

And rampant shakes his brinded mane.

In summary, therefore, although it appears that rounded and flattened gastrulae in the vertebrate group may have slightly different substrative conditions from which to start, they ail employ essentially similar processes in effecting tubulation of the respective, major organ-forming areas and in the development of primitive body form.

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