The peripheral surface of a fertilized, uncleaved egg is subdivided through cleavage and is allotted to constituent cells. This is called the primary surface. In an early morula a constituent cell has two kinds of surfaces: the primary surface, and the secondary surface, which does not participate in forming the periphery of the embryo. Electron-microscopic observations showed structural differences between the two surfaces.

When the dorsal marginal zone of an early gastrula of Hynobius nebulosus is excised and immersed in Feldman’s solution, the piece can easily be separated into two layers: the outer layer, whose constituent cells are given a share of the primary surface, and the inner layer, whose constituent cells are completely covered only by the secondary surface.

Both an explanted piece of the outer layer and an intact double-layered piece show three kinds of movement: spreading, convergence followed by stretching, and spherical thickening. The inner layer is kinetically very inert, showing slight spreading and thickening.

An explanted piece of the outer layer differentiates into axial mesodermal structures, while the inner layer does not.

When a piece of either the inner or the outer layer is implanted in the blastocoel of another gastrula, it induces deuterencephalic and spino-caudal structures and seems to differentiate into axial mesodermal structures.

Differences of kinetic properties and differentiation are considered to result from the fact that the outer layer has the primary surface, while the inner layer does not.

Functional effects of the primary surface on the movement of tissues and differentiation are discussed.

It is well known that during gastrulation the dorsal marginal zone of an early amphibian gastrula executes conspicuous movements of convergence towards the median line and of stretching in the antero-posterior direction (Vogt, 1922, 1929). A similar pattern of movements is also shown in this area even when it is isolated from a gastrula (Ikushima, 1958, 1961). It has been inferred that these movements of tissues occur owing to reshuffling or rearrangement of constituent cells (Willier, Weiss & Hamburger, 1955; Waddington, 1962); but the mechanism of the reshuffling and the rearrangement has not yet been clearly demonstrated.

During experiments on dissociation of embryonic tissues, we found that a piece taken from the dorsal marginal zone of an early gastrula of Hynobius nebulosus could easily be separated into outer and inner layers after a short treatment in dissociating agents. Moreover, we found that in the inner layer movement and differentiation of the tissue was not necessarily similar to that in the outer layer. The present experiments were attempted to investigate differences of developmental tendencies of the two layers.

MATERIALS AND METHODS

Embryos of Hynobius nebulosus and Triturus pyrrhogaster were used. The separation of the dorsal marginal zone into two layers was possible only in the embryos of the former. The tissues were separated or dissociated in Feldman’s solution (Feldman, 1955). A square piece was isolated from the dorsal marginal zone of an early gastrula (Harrison’s stage 10, 1952). When the piece was immersed in Feldman’s solution the cells began to disaggregate, especially at the corners of the piece, within 5–10 min. In these pieces the outer cell layer could easily be peeled off from the inner cell layer with a hair loop. Generally, the former seemed to be composed of a single layer of cells and the latter of two or more layers of cells. Immediately after the separation the two layers were removed from Feldman’s solution to full strength Holtfreter’s solution.

In order to investigate the kinetic properties of the tissue the following explantation experiments were performed. A square piece taken from the dorsal marginal zone was cut into two parts along its median line. The two parts were immediately combined again into one in which their inner surfaces were placed in close contact with each other (Fig. 1). Within 12 h the two parts were completely fused together, resulting in a single rectangular-shaped explant. In a similar way rectangular explants were prepared from square pieces of the outer and inner cell layers respectively. The kinetic properties of the tissues were investigated by observing the change in configuration of these rectangular explants during the first 48 h of cultivation.

Fig. 1.

Schematic representation of the method of explantation to investigate the kinetic properties of the dorsal marginal zone. (A, B) Excision of a square piece from the dorsal marginal zone of an early gastrula. (C) The piece is cut into two parts along the median line. (D) The two parts are recombined.

Fig. 1.

Schematic representation of the method of explantation to investigate the kinetic properties of the dorsal marginal zone. (A, B) Excision of a square piece from the dorsal marginal zone of an early gastrula. (C) The piece is cut into two parts along the median line. (D) The two parts are recombined.

To investigate the differentiating capacity, the intact square piece, and the square pieces of the outer layer and of the inner layer, were cultured in full strength Holtfreter’s solution. To investigate the inducing capacity, they were implanted into the blastocoel of another gastrula of the same age. Operations and cultivation of these embryonic tissues were always performed in glass dishes lined with 1·5% agar-agar. After cultivation for 3–4 weeks tissues were fixed with Bouin’s fixative, embedded in paraffin wax, sectioned at 10 μ and then stained with Mayer’s haemalaum and eosin. In some cases they were fixed with OsO4 after cultivation for 24 h, embedded in Epon, sectioned at 10μ on an LKB Ultrotome and stained with toluidine blue (Trump, Smuckler & Benditt, 1961).

For electron-microscopic observations, specimens were fixed for 30 min in ice-cold 1 % OsO4 in phosphate buffer at pH 8·0. After fixation they were dehydrated rapidly in a graded series of ethanol and embedded in methacrylate. Thin sections were cut with an LKB Ultrotome. Observations were carried out with a Hitachi H-7 microscope, and micrographs were taken at an original magnification of 2000–8000.

1. Preliminary experiments

When morulae of H. nebulosas and T. pyrrhogaster were immersed in Feldman’s solution they dissociated into discrete cells within 30–60 min. As shown in Fig. 2B the surface of a cell derived from the animal polar region is composed of two parts -a heavily pigmented part and the remaining paler part. The pigmented part is undoubtedly identical to the peripheral surface of the fertilized, uncleaved egg. In the present report this part of the surface is provisionally named ‘primary surface’, and the remaining part, the paler part in the case of the animal polar cells, is named ‘secondary surface’. As a result of cleavage the primary surface is subdivided and allotted to every one of the constituent cells of a morula. In the dorsal marginal zone of an early gastrula, light-microscopic observations showed that the surface of a cell in the outer layer seemed to be composed of both primary and secondary surfaces, whereas in the inner layer the surface seemed to be composed exclusively of secondary surface.

Fig. 2.

(A) An intact morula of Hynobius nebulosus. (B) A morula dissociated into discrete cells 45 min after it was immersed in Feldman’s solution.

Fig. 2.

(A) An intact morula of Hynobius nebulosus. (B) A morula dissociated into discrete cells 45 min after it was immersed in Feldman’s solution.

During the present dissociation experiments, it was frequently observed that the border of the primary surface began to constrict a cell (Fig. 3) or the primary surface area shrank suddenly. These effects were most clearly shown in discrete cells derived from a morula, and were observed not only in Feldman’s solution but also after they were removed into Holtfreter’s solution. Although the factors which make the primary surface area shrink have not yet been clearly demonstrated, these observations seem to indicate that structural differences exist between the two surfaces. In order to examine this possibility, discrete cells from a morula of T. pyrrhogaster were investigated electron-microscopically. In Fig. 4 a cross-section of the surface area of a discrete cell in which the primary surface shrank markedly is shown. At the periphery of the primary surface area a specific stratum is found just beneath the deeply folded plasma membrane. This stratum shows a fine alveolar structure, and is mostly about 0·5μ thick; at the margin the thickness reaches 1μ tor more. Just inside this stratum there is a cytoplasmic stratum about 5 μ thick containing an abundance of pigment granules and hardly any yolk platelets. These two kinds of strata do not seem to shrink independently but to be combined, forming a unitary layer. This layer very probably represents the macroscopically named ‘primary surface’, and is presumably identical to the ‘cortex’ defined by Pasteels (1964) and to the ‘cortical layer’ of Balinsky (1965). On the other hand, in the secondary surface these two kinds of strata are hardly recognizable electron-microscopically. From these results it seems beyond doubt that structural differences exist between the primary and secondary surfaces.

Fig. 3.

Discrete cells which are constricted at the border of the primary surface. The cells are derived from a morula of Triturus pyrrhogaster.

Fig. 3.

Discrete cells which are constricted at the border of the primary surface. The cells are derived from a morula of Triturus pyrrhogaster.

Fig. 4.

Electron micrograph of a section of the surface area of a discrete cell from a morula of T. pyrrhogaster.

Fig. 4.

Electron micrograph of a section of the surface area of a discrete cell from a morula of T. pyrrhogaster.

2. Kinetic properties

(a) Combined explant of both the outer and the inner layers (Fig. 1)

The cross-section of this explant 12 h after combination (Fig. 5) reveals that a continuous cell layer encircles an empty lumen. From this figure it may be considered that the fusion of the two parts is finally accomplished through lateral adhesion of the cut edges to each other and not by adhesion of the inner surface.

Fig. 5.

A cross-section of a combined explant of both the outer and the inner layer 12 h after the two parts were combined into one. An epoxy section stained with toluidine blue, n, Nucleus.

Fig. 5.

A cross-section of a combined explant of both the outer and the inner layer 12 h after the two parts were combined into one. An epoxy section stained with toluidine blue, n, Nucleus.

Within the first 24 h of cultivation it could easily be seen that the lower one-third of the explant became thick and spherical, and the middle one-third became narrow in a lateral direction and stretched along the antero-posterior direction of the original embryo. The upper one-third remained unchanged in some specimens and wrinkled or spread out in the others. In the following 24 h these three kinds of movements of tissues became more and more marked.

(b) Combined explant of the outer layer

Immediately after the explants were removed from the dissociating to the culture medium the constituent cells became closely aggregated and the explant diminished slightly in size. Within the first 24 h of cultivation the upper part became wrinkled and spread out, the middle part narrowed and stretched and the lower part became thick and spherical. In the following 24 h the spreading of the upper part and the narrowing and the stretching of the middle part became more and more marked, but the spherical thickening of the lower part hardly increased its thickness. Consequently, it may be stated that the change of shape in these explants is quite similar to that in the preceding case (a).

(c) Combined explant of the inner layer

When the explants were removed from the dissociating to the culture medium they diminished slightly in size, and their corners became rounded. During the first 24 h of cultivation the lower part became slightly thicker, the upper part remained almost unchanged and the middle part did not narrow or stretch at all or show any other type of movement. In the following 24 h the explants spread out to some extent. The spreading occurred not only in the upper part but also in the middle and the lower part. These are shown in Fig. 6. The narrowing and the accompanying stretching were never observed in any region of the explant during the first 48 h or in the following days of cultivation.

Fig. 6.

Change of configuration of the combined explant of the inner layer. (A) 3 h after the two parts were combined; (B) 24 h after combination; (C) 48 h after combination. The line in the figure represents 100 μ.

Fig. 6.

Change of configuration of the combined explant of the inner layer. (A) 3 h after the two parts were combined; (B) 24 h after combination; (C) 48 h after combination. The line in the figure represents 100 μ.

3. Differentiating capacity

(a) Differentiation of the unseparated explants

Immediately after the beginning of cultivation they began to curl up, so that the original peripheral surface of the pieces, presumably identical to the primary surface, completely covered the mass of cells. After 4 weeks’ cultivation fifteen specimens were available. Among them eleven remained a solid mass till the end of cultivation, while the other four finally developed into large vesicles. Among the four vesicular explants only a small mass of neural tissue and mesenchyme were found in one specimen, and a small amount of muscle, pronephric tubules and lateral plate occurred without neural tissue in the other three. In the eleven specimens remaining as solid masses, notochord, muscle, deuterencephalon and spinal cord were always found (Fig. 7), and in one specimen an archencephalic structure with an eye was also found.

Fig. 7.

A section of an unseparated explant after 4 weeks’ cultivation.

Fig. 7.

A section of an unseparated explant after 4 weeks’ cultivation.

(b) Differentiation of the explants of the outer layer

As soon as the explants were removed from the dissociating to the culture medium they diminished slightly in size and began to curl up, so that the cell mass was completely covered by the primary surface. After 4 weeks’ cultivation fourteen specimens were available. Combinations of differentiated tissues appearing in these explants are summarized in Table 1. In six specimens notochord, muscle, deuterencephalon and spinal cord were found (Fig. 8). In two specimens an abundance of muscle and ear vesicles occurred without notochord. In the remaining cases neural or sensory structures were not found; in four specimens notochord and muscle occurred without neural tissue and in the other two only mesenchyme and mesenteric tissue were found in a large epithelial vesicle.

Table 1.

Combination of tissues differentiated in explants of the outer layer and of the inner layer

Combination of tissues differentiated in explants of the outer layer and of the inner layer
Combination of tissues differentiated in explants of the outer layer and of the inner layer
Fig. 8.

A section of an explant of the outer layer after 4 weeks’ cultivation.

Fig. 8.

A section of an explant of the outer layer after 4 weeks’ cultivation.

(c) Differentiation of the explants of the inner layer

When the explants were removed from the dissociating to the culture medium they diminished in size and became slightly rounded. The curling up of the pieces was not so marked as in the previous two cases. After 4 weeks’ cultivation thirteen specimens were available. Among them three specimens developed into large vesicles. They were composed of a very small mass of neural tissue and mesenchyme, and cartilage in one specimen (Fig. 9). The remaining ten were solid masses composed of a large amount of undifferentiated tissue and a small amount of neural tissue (Fig. 10); cartilage was found in two of them. These results are summarized in Table 1.

Fig. 9.

A section of an explant of the inner layer after 4 weeks’ cultivation.

Fig. 9.

A section of an explant of the inner layer after 4 weeks’ cultivation.

Fig. 10.

A section of another explant of the inner layer after 4 weeks’ cultivation.

Fig. 10.

A section of another explant of the inner layer after 4 weeks’ cultivation.

4. Inducing capacity

(a) Implantation of the outer layer

During 3–4 weeks’ cultivation of the hosts, secondary embryonic structures were produced generally in their mid-ventral region. Microscopic observation revealed that in sixteen specimens out of the twenty available deuterencephalon and spinal cord were always found accompanied by well-differentiated notochord and muscle in the secondary embryos (Fig. 11). In one specimen a large amount of neural tissue and four ear vesicles were found without mesodermal organs. In two specimens only a small amount of muscle and an abundance of mesenchyme were found without notochord and neural tissue, and in the remaining specimen only a small quantity of muscle and a large number of blood cells were found.

Fig. 11.

A section of an embryo in which a secondary embryo is induced by the implanted piece of the outer layer.

Fig. 11.

A section of an embryo in which a secondary embryo is induced by the implanted piece of the outer layer.

(b) Implantation of the inner layer

During 3–4 weeks’ cultivation the secondary embryonic structures were found generally in the mid-ventral region of the hosts. Microscopic observations revealed that in twelve specimens out of the seventeen available, deuterencephalon and spinal cord occurred with well-differentiated notochord and muscle (Fig. 12). In three specimens, only mesenchyme and a small amount of muscle were found without notochord and neural tissue. In the remaining two specimens archencephalic structures with eyes occurred, one formed antero-ventrally and the other mid-ventrally.

Fig. 12.

A section of an embryo in which a secondary embryo is induced by the implanted piece of the inner layer.

Fig. 12.

A section of an embryo in which a secondary embryo is induced by the implanted piece of the inner layer.

The results of the implantation experiments reveal that the implanted pieces of the outer and the inner layer both induce deuterencephalic and spino-caudal structures in the hosts in the majority of cases. Although archencephalic structures were induced only by the inner layer, they occurred in only two specimens out of seventeen. From these results it seems difficult to point out distinct differences in inducing capacity between the two layers.

On the other hand, it must be noted that well-differentiated notochord and muscle are found in the secondary embryos which are induced by the pieces of the inner layer. These pieces were not able to differentiate into axial mesodermal structures in the explantation experiments. However, in the case of implantation experiments it seems probable that axial mesodermal structures in the secondary embryos are derived from the implanted piece of the inner layer.

The role of the primary surface on the development of the dorsal marginal zone

The present experiments show that when explanted pieces of the inner layer are cultured in saline, they do not differentiate into axial mesodermal structures, while pieces of the outer layer do. At first, this fact seems to indicate that the intrinsic potential for development of the inner layer is quite different from that of the outer layer. However, the results obtained in the implantation experiments show that, like the outer layer, the inner layer can also differentiate into axial mesodermal structures and can induce deuterencephalic and spinocaudal structures. These results do not necessarily support the above view; another interpretation may be possible.

In the present explantation experiments the intact square explants and the explants of the outer layer both begin to curl up immediately after the beginning of cultivation. In consequence of the curling up, the mass of cells is completely covered by the primary surface. In the case of the implantation experiments there is no doubt that the implanted piece develops in the blastocoel which is the interior of the primary surface. In contrast, the cells of the inner layer in the explantation experiments are cultured without a covering of the primary surface. The failure of notochord and muscle formation occurs exclusively in this experimental situation. Looking through these results it seems likely that the cells of the dorsal marginal zone of the early gastrula require a specific environment in order to differentiate into axial mesodermal structures. This environment is presumably produced when the aggregate of cells is covered by the primary surface.

Next there arises the question of what sort of influence the covering of the primary surface exerts upon the interior mass of cells. On this question a clear solution cannot yet be given. However, as pointed out by Holtfreter (1943) and ascertained by Loeffler & Johnston (1964), there seems no doubt that the outer surface of the amphibian embryo has a specific trait which prevents or reduces the entrance of water, electrolytes and other substances. From this, it may be presumed that there is a specific environment within the permeability barrier, caused by the protection from unfavourable external factors, or by the prevention of the escape of necessary substances from cells to the surrounding medium, or by both. It seems likely that the cells of the dorsal marginal zone can only realize their potency to differentiate into axial mesodermal tissue in this specific environment, although its character has not yet been clearly demonstrated.

In this report it is also shown that there are differences in kinetic properties between the outer and the inner layer of the dorsal marginal zone. Generally speaking, the outer layer seems to be kinetically active and the inner layer inert. The most conspicuous difference is that the middle part of the former shows marked convergence and stretching, while the latter does not. At the same time, it must also be noted that an intact piece composed of both the outer and inner layers shows marked stretching as in the case of the outer layer alone. From these facts it seems clear on the one hand that the outer layer plays a leading role in the movement of the dorsal marginal zone, and on the other hand that the inner layer, although seemingly inert kinetically, is not rigid but stretches, conforming itself to the outer layer where the two layers are in direct contact.

How do the cells of the outer layer play a leading role in the movement of the dorsal marginal zone? In this report it is shown that the most marked morphological difference between the two layers is that the cells of the outer layer have a share of the primary surface, while those of the inner layer have not. Consequently it seems possible to suppose that the primary surface is responsible for the active movement of the dorsal marginal zone.

In order to verify this supposition it seems necessary to demonstrate first that the primary surface has intrinsic tendencies to contract, to expand or to stretch. On this point the results obtained by Curtis (1960) are relevant. He demonstrates that the cortical material possesses morphogenetic properties, and that these properties may be transferred with the cortical material even when it is grafted. The cortical material used in his grafting experiments is composed of a true cell surface at the periphery and a thin hyaline layer containing mitochondria and pigment granules. Its thickness varies from 0·5 to 3·0 μ. The primary surface in the present experiments also contains a true cell surface, a thin alveolar and a cytoplasmic stratum and pigment granules. Its thickness as shown in Fig. 4 is about 5·6 μ. However, since this is undoubtedly abnormal thickening owing to contraction, it seems reasonable to state that the primary surface in the present report is identical to the cortical material of Curtis’s experiments. Consequently, it seems reasonable to consider that the primary surface has intrinsic morphogenetic properties. Moreover, as shown by Holtfreter (1943), when the viscosity of the surface layer is reduced by various agents, the mass movements of gastrulation and neurulation are prevented or altered and exogastrulae and other malformations are produced. This fact may also support our present hypothesis.

On the problem of whether the primary surface has a syncytial nature

As discussed above, it seems conceivable that the primary surface has intrinsic morphogenetic properties. However, it seems also true that, in as much as contraction or stretching of the primary surface occurs independently in individual cells, these movements do not necessarily result in reshuffling or rearrangement of cells in embryonic tissues. Thus it is difficult to understand how independent movements of the primary surface in individual cells inevitably result in a change of configuration of an embryonic tissue. The leading role of the primary surface in movements of tissues could be most easily understood if it is verified that the primary surfaces of individual cells are integrated into a supercellular syncytial unit.

On this problem, Holtfreter (1943, 1948) has indicated that a special surface layer named ‘surface coat’ exists at the periphery of amphibian eggs. According to him, the spherical black pigment granules which are found at the surface of the egg are contained in this coat, and this coat is divided up by the process of cell division, but re-establishes a continuous envelope cementing together the peripheral part of the blastomeres. However, recent electron-microscopic observations do not necessarily support the existence of such a syncytial envelope. These show that the pigment granules undoubtedly occur in the cytoplasm which lies just inside the plasma membrane, and that no extracellular cortical structures which correspond to Holtfreter’s surface coat are to be found (Wartenberg & Schmidt, 1961 ; Balinsky, 1965). Our present observations also agree with these findings: that is, the primary surface which contains the pigment granules is not an extracellular, but an intracellular structure. So far as the primary surface is an intracellular structure, it must inevitably be considered that the primary surface is not a syncytium but is composed of discrete structures separated from each other by plasma membranes.

Thus there remains the difficult question of how the movements of discrete primary surfaces give rise to mass movement of cells.

In our time-lapse microcinematography (N. Ikushima & S. Maruyama, unpublished observations) it can be seen, as Holtfreter has pointed out, that in cleaving eggs the pigmented surface which has once been divided by a cleavage furrow behaves as if it re-establishes a continuous envelope. Moreover, it is observed that the primary surface does not adhere to the primary or to the secondary surface. Only the secondary surface adheres to that of another cell. When dissociated discrete cells come in contact, adhesion always begins and proceeds exclusively between their secondary surfaces. At the same time, it seems worthy of notice that the adhesion of cells appears to be stabilized as soon as the borders of the primary surfaces of respective cells come in contact with each other. In other words, it appears that the border of the primary surface of a cell has a particular adhesiveness to that of an adjacent cell. In relation to this point, electron-microscopic studies in amphibian gastrulae show that the cells composing the outer surface adhere to each other very closely just at their distal ends and are probably joined by some cement substance which is not easily broken (Balinsky, 1965). If it is true that cells of an early embryo are connected with each other by cement substances which are secreted outside cells just at the border of the primary surface, it may consequently be conceivable that the intracellular, discrete primary surfaces are connected with each other by cement substances across the plasma membranes. Of course this does not mean that the primary surfaces are morphologically integrated into a true syncytial layer, but it does indicate the possibility that discrete primary surfaces are functionally organized into a syncytial unit. On this point, however, further studies would be necessary.

Aufbau und Entwicklungstendenz der dorsalen Randzone in der frühen Gastrula von Amphibien

Die Oberfläche, die ein befruchtetes und ungeteiltes Ei umfasst, wird durch Zellteilung abgeteilt und in die einzelnen Zellen ausgeteilt. Diese nennen wir eine primare Oberfläche. In einer frühen Morula findet man die zweiartigen Oberflächen in einzelnen Zellen; die eine ist die primare Oberfläche, und die andere, die sich nicht an dem Umfang des Keimes beteiligt, ist die sekundäre Oberfläche. Die strukturelle Verschiedenheit ist elektronenmikroskopisch zwischen zwei Oberflächen gezeigt.

Wenn ein Stück aus der dorsalen Randzone isoliert und in die Feldmansche Ldsung getaucht wird, kann es in die zwei Schichten geteilt werden. In der äusseren Schicht hat jede Zelle die primare und sekundäre Oberfläche. In der inneren Schicht hat jede Zelle nur die sekundäre Oberfläche.

Das Explantat der äusseren Schicht stellt die dreiartigen Gestaltungsbewegungen dar; Ausbreitung, Konvergenz mit Streckung und sich Kugelung. Dieselbe Bewegungen finden sich in gleicher Weise in dem normalen Explantat mit der äusseren und der inneren Schicht. Das Explantat der inneren Schicht ist kinetisch sehr träge und stellt nur schwache Ausbreitung und sich Kugelung dar.

In dem Explantat der äusseren Schicht werden die axialen, mesodermalen Strukturen gebildet, aber gar nicht in dem Explantat der inneren Schicht.

Wenn ein Stück der inneren Schicht ins Blastocdl eingesteckt wird, induziert das Implantät die deuterencephalen und spinalen Strukturen. Das Implantät wird gleichzeitig Chorda und Somiten. Dieselbe Induktion und Differenzierung finden sich in gleicher Weise in der Implantation der äusseren Schicht.

Die Verschiedenheit der Gestaltungsbewegungen und der Differenzierungsfähigkeiten zwischen den äusseren und den inneren Schichten mag aus dem Grund geschehen, daB die äussere Schichte die primäre Oberfläche, aber die innere Schicht sie nicht habe.

Funktion der primäre Oberfläche über die Gestaltungsbewegungen und die Differenzierungsfähigkeiten der dorsalen Randzone werden erörtert.

     
  • ASt

    alveolar stratum

  •  
  • bl

    borderline of the primary surface

  •  
  • PSr

    primary surface

  •  
  • PSt

    cytoplasmic stratum containing pigment granules

  •  
  • SSr

    secondary surface

  •  
  • ca

    Cartilage

  •  
  • e

    ear vesicle

  •  
  • m

    muscle

  •  
  • ne

    neural tissue

  •  
  • nt

    notochord

  •  
  • und

    a mass of undifferentiated tissue

The line in each figure represents 200 μ.

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