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Primitive streak being only present in caudal half of embryo affecting gastrulation?

Primitive streak being only present in caudal half of embryo affecting gastrulation?



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It is my understanding that in gastrulation epiblast cells migrate to the primitive streak where they start to form the three germ layers; does this mean that gastrulation only occurs at the caudal half of the developing embryo


It would help a little in answering your question to know more about what organisms you are interested in, or the specific source that you are referencing, with e.g. the relevant quote.

For example, the primitive streak is specific to amniotes: avians, mammals, and reptiles. In other common model organisms such as flies or amphibians gastrulation happens differently.

Another thing to keep in mind is that gastrulation is a very large and structurally distending process, so it can't be really said to happen "only on one end" of the embryo. The whole embryo is going through gastrulation. We can however talk about the placement of certain gross morphological structures on the embryo. But really each cell is affected by gastrulation, whether it's immediately obvious under a microscope or not.

Looking at the Amniote section of the wiki page on gastrulation, it appears that the main morphological changes in gastrulation do occur on the caudal side - for amniotes.

However, in Drosophila, gastrulation involves the formation of an approximately homologous structure called the ventral furrow, which is obviously on the ventral side, extending most of the anterior-posterior axis. However the invagination event does actually happen on the dorsal/caudal side.

So, it's complicated, and it depends a lot on which organism you're talking about.

Update following response

Humans are amniotes, they follow more or less the normal amniote path in gastrulation according to the wiki link above.

An important note missing in my previous answer above is that gastrulation itself defines bilateral symmetry in embryos; prior to this establishment animal-vegetal is the only obvious axis. So we are kind of defining the rostral-caudal axis according to the formation of such structures as the primitive streak, which are formed during gastrulation.

So it's a little circular to say that gastrulation happens on caudal end only; as gastrulation includes the step that establishes the rostral-caudal axis. Without gastrulation that axis just doesn't exist.

Again, gastrulation is a process that happens to the entire embryo. It doesn't just happen in one area. Structures such as the primitive streak are just obvious clues that the process is happening. It might be proper to say that the primitive streak begins forming caudally (I don't know of exceptions, though I'm not an expert), but it would not be proper to say that gastrulation is a caudal process.


Reassembling gastrulation

Gastrulation leads to stereotypical germ layer progenitor specification and organization.

Gastrulation relies on tight interactions between signaling pathways and embryonic morphology.

In culture, embryonic substructures can recapitulate certain aspects of gastrulation.

In vitro stem cell models show a high capacity to trigger germ layer formation also de novo.

Such models allow to dissect the specific contributions of molecular, cellular and biophysical mechanisms.


RESULTS

Using in situ hybridization essentially as described by Nieto and colleagues (Nieto et al., 1996 ), except that the proteinase K, hydrogen peroxide, and RNase A steps were omitted, we characterized the expression patterns of thirteen genes (Table 1) at stages XII-4. The expression patterns of 11 of the 13 genes were previously described during gastrulation (usually individually and not at the full range of stages presented here) the expression patterns of 2 of the 13 genes (Slug and Cripto) had not been previously described during stages of formation and early progression of the primitive streak.

Gene (chicken) Reference Accession no.
Wnt8c Hume and Dodd ( 1993 ) U02097
Slug Nieto et al. ( 1994 ) X77572
Vg1 Saleiro et al. ( 1996 ) U73003
Nodal Levin et al. ( 1995 )
Fgf8 Crossley et al. ( 1996 ) U41467
Brachyury Knezevic et al. ( 1997 ) U67086
Cripto Colas and Schoenwolf ( 2000 ) AF228760
cNot1 Stein and Kessel ( 1995 ) X82575
Shh Riddle et al. ( 1993 ) L28099
Hnf3β Ruiz i Altaba et al. ( 1995 ) L38904
Chordin Streit et al. ( 1998 ) AF031230
Goosecoid (GSC) Izpisúa-Belmonte et al. ( 1993 ) X70471
Crescent Pfeffer et al. ( 1997 ) AF006508

Group 1 Genes

Group 1 genes comprised Wnt8c, Slug, Vg1, Nodal, Fgf8, Brachyury and Cripto (Figs. 1–5). At stage XII/XIII (i.e., prior to the appearance of the primitive streak), the expression domains of Wnt 8c, Slug, Vg1, and Nodal (group 1A genes) in the epiblast were confined to the portion composing the middle two-thirds of Köller's sickle (Fig. 1a–e), whereas Fgf8, Brachyury, and Cripto (group 1B genes) were ubiquitously expressed in most of the epiblast of the epiblast of the area pellucida (Fig. 1f–h). Thereafter, between stages 2 and 3a/b, all group 1 genes were up-regulated in the primitive streak but down-regulated in the epiblast (Figs. 2a–g, 3a–g, 4a–g, 5a–g), except Cripto, which maintained its strong epiblast expression up to stage 3d/4 (Figs. 1h, 2g, 3g, 4g, 5g). Also, from stage 3a/b onwards, Slug expression at the tip of the primitive streak was down-regulated. By stage 3c, the expression of group 1 genes in the primitive streak had extended variably to the ingressed lateral mesoderm (Fig. 4a–g). Also at this stage, expression of Nodal at the tip of the primitive streak was lost (Fig. 4d). Further differences in the extent of group 1 gene expression within the primitive streak became more apparent at stage 3d/4. Whereas Wnt8c, Slug, and Nodal were expressed in the middle three-fourths of the primitive streak (Fig. 5a, b, d), thus excluding Hensen's node, here Vg1, Brachyury, and Cripto were expressed in the rostral three-fourths of the primitive streak including Hensen's node (Fig. 5c, f, g). In contrast to the above, the expression domain of Fgf8 was confined to the upper one-third of the primitive streak, excluding Hensen's node (Fig. 5e).

Expression of group 1 genes, Wnt8c, Slug, Vg1, Nodal, Fgf8, Brachyury, and Cripto, during formation and progression of the chick primitive streak. Whole mounts (viewed dorsally) and a transverse section of stages XII-4 chick embryos. Fig. 1. a–h: Epiblast expression domains at stage XII/XIII. Figs. 2a–g: 3a–g: Expression within the primitive streak at stages 2 and 3a/b. Fig. 4a–g: Primitive streak and lateral mesodermal expression at stage 3c. Fig. 5a–g: Variable expression within the epiblast, primitive streak, lateral mesoderm and Hensen's node at stage 3d/4. Arrows, position of Hensen's node. Scale bars (1a, 1c–h, 2a–g, 3a–g, 4a–g, 5a–g) = 100 μm, (1b) = 300 μm.

Group 2 Genes

Group 2 genes comprised cNot 1, Sonic hedgehog (Shh), Hnf3β, and Chordin (Figs. 6–10). Expression of groups 2 genes becomes largely confined to Hensen's node. At stage XII/XIII, only cNot1 and Chordin had expression domains in the epiblast (Fig. 6a, d), with the domain of cNot1 located in most of the epiblast of the area pellucida, and that of Chordin confined to the midline epiblast just rostral to Köller's sickle. At stage 2, cNot1 expression in the epiblast became restricted to the rostral half of the area pellucida (Fig. 7a). Also at this stage, the expression of both Hnf3β and Chordin was evident at the rostral end of the primitive streak (Fig. 7c, d). By stage 3a/b, all four group 2 genes had expression domains at the rostral end of the primitive streak, and this pattern continued through stage 3c (Fig. 9a–d) to stage 3d/4, when expression was confined principally to Hensen's node (Fig. 10a–d).

Expression of group 2 genes, cNot1, Sonic hedgehog (Shh), Hnf3β and Chordin, during formation and progression of the chick primitive streak. Whole mounts (viewed dorsally) of chick embryos at stages XII-4. 6a–d: Epiblast expression at stage XII/XIII. 7a–d: Expression in epiblast and rostral primitive streak at stage 2. 8a–d, 9a–d: Expression in rostral primitive streak at stage 3c. 10a–d: Expression in Hensen's node at stage 3d/4. Scale bar (6a–d, 7a–d, 8a–d, 9a–d, 10a–d) = 100 μm. Expression of group 3 genes, Crescent and Goosecoid (GSC), during formation and progression of the chick primitive streak. Whole mounts (viewed ventrally) of chick embryos at stages XII-4. 11a, 12a, b, 13a, 14a, 15a, 16a: Expression of Goosecoid (GSC) in hypoblast, rostral end of primitive streak and Hensen's node at stages XII-4. 11b, 12c, 13b, 14b, 15b, 16b: Expression of Crescent in hypoblast at stages XII-4. Scale bar (11a,b, 12a,c, 13a,b, 14a,b, 15a,b, 16a,b) = 100 μm, (12b) = 300 μm.

Group 3 Genes

Group 3 genes, Goosecoid (GSC) and Crescent, were expressed mostly in the caudal half of the hypoblast at stage XII/XIII (Figs. 11–16). However, at stage XIV, the expression domains of both genes extended rostrally, thereby including the rostral half of the hypoblast as well (Fig. 12a–c). Additionally, Goosecoid (GSC) was expressed by midline epiblast cells located rostral to Köller's sickle (Fig. 12b). Thereafter, a striking difference in the expression of the two genes became evident. Goosecoid (GSC) gradually decreased in the hypoblast layer, almost disappearing by stage 3d/4, but its expression remained strong at the tip of the primitive streak and in Hensen's node (Figs. 13a, 14a, 15a, 16a). Crescent, in contrast, maintained its strong hypoblast expression as the latter was displaced rostrally, but its expression remained absent in the primitive streak and Hensen's node (Figs. 13b, 14b, 15b, 16b).


RESULTS

Scanning Electron Microscopy

Stages X–XII.

The blastoderm during Stages X–XII consists of two parts: a central disc-like region, the area pellucida, and a peripheral ring-like area, the area opaca. In the area pellucida, the blastoderm comprises two layers, a superficial epiblast layer and a deep, incomplete hypoblast layer. The epiblast has a basal lamina on its ventral surface (not shown, but see below: “Distribution of fibronectin and laminin”), that masks cell morphology. The hypoblast at these stages is a non-epithelialized layer consisting of numerous globular cells scattered on the ventral surface of the epiblast (Fig. 1a,b). Most of the hypoblast cells are grouped into clusters. Previous studies have shown that they are derived from the epiblast and undergo polyingression (Weinberger and Brick, 1982a ,b Penner and Brick, 1984 Weinberger et al., 1984 Stern and Canning, 1990 Harrisson et al., 1991 ). Additional evidence of this is the presence of rounded holes in the ventral surface of the epiblast, with hypoblast cells exhibiting processes extending through the holes into the epiblast layer (Fig. 1b).

a: SEM of the ventral surface of a stage XI/XII chick embryo. Area opaca (AO) area pellucida (AP). Scale bar = 400 μm. b: enlargement of the central part of a showing a non-epithelialized hypoblast layer (H) consisting of numerous rounded cells. Arrows point to gaps in the epiblast layer (E), through which the hypoblast cells (H) are polyingressing. Scale bar = 40 μm.

Stages XIII–XIV.

By Stages XIII–XIV, the hypoblast consists of a complete layer of cells ventral to the epiblast throughout the entire area pellucida (Fig. 2a,b). Hypoblast cells are large and irregularly shaped, with some having bulging surfaces and others, flattened surfaces. Most hypoblast cells have small globular projections on their ventral surface. Epithelialization of the hypoblast seems to progress rostrally from the caudal end, as in some embryos at this stage of development, the rostral portion of the area pellucida is devoid of this layer and scattered non-epithelialized hypoblast cells are still present there. Surrounding the margin of the epithelialized hypoblast layer at the junction between the area pellucida and area opaca is a layer of cells that constitutes the marginal zone (MZ Fig. 2a). An extracellular matrix covers the ventral surfaces of the MZ cells, giving them a flattened appearance (Fig. 2c).

a: SEM of the ventral surface of a stage XIII chick embryo showing an epithelialized hypoblast layer (H) and surrounding marginal zone (MZ) area opaca (AO). Scale bar = 500 μm. b: Enlargement the hypoblast layer showing both flattened cells and cells with bulging surfaces. Scale bar = 500 μm. c: Enlargement of the marginal zone showing cells with numerous lamellipodia. Scale bar = 40 μm.

The basal surface of the epiblast can be examined after removal of the epithelialized hypoblast layer. The position of the incipient primitive streak is often indicated in the caudal half of the basal surface of the epiblast by a midline ridge (Fig. 3a). Higher magnification of this ridge reveals a line of cells emerging from the ventral surface of the epiblast (Fig. 3b) this line constitutes the first crop of primitive streak cells (and perhaps a few attached hypoblast or endodermal cells). A transverse section through the caudal half of the area pellucida shows that the epiblast and hypoblast consist of simple columnar and flattened epithelial layers, respectively, and that the midline epiblast at the level of the forming primitive streak is thicker than the more lateral epiblast (Fig. 3c).

a: SEM of a Stage XIV chick embryo in which the hypoblast was removed revealing the ventral (basal) surface of the epiblast (E). Arrows indicate the caudal midline of the epiblast where the primitive streak is forming. Area opaca (AO) marginal zone (MZ). Scale bar = 60 μm. b: Enlargement of the forming primitive streak showing cells emerging from the ventral (basal) surface of the epiblast. The lightest-appearing cells may represent attached hypoblast or endodermal cells. Scale bar = 40 μm. c: SEM of a transverse slice through the primitive streak-forming area of a stage XIV chick embryo. Note the increased thickness of the epiblast (E) in the midline. Arrows point to cells between the epiblast and the epithelialized hypoblast (H) layers these cells are probably non-epithelialized hypoblast cells not yet integrated into the hypoblast. Scale bar = 100 μm.

Stage 2.

At Stage 2, the initial primitive-streak stage, the epithelialized hypoblast seems similar to that at Stages XIII–XIV. Overt formation of the primitive streak commences at this stage. The initial primitive streak is triangular in shape, with its apex pointing rostrally (Fig. 4a), and it occupies the caudal one-third of the length of the area pellucida. On its ventral surface, the basal lamina of the epiblast is disrupted in the entire region occupied by the primitive streak, thus exposing the surfaces of the cells forming the streak (Fig. 4b). Within the primitive streak, remnants of the disrupted basal lamina are identified in patches on some of the cells. Elsewhere on the epiblast, the basal lamina of the epiblast is intact (Fig. 4c). In comparison with the flanking epiblast, the primitive streak is about twice the thickness, with a greater cell density (Fig. 5a,b). Also, whereas the cells of the primitive streak are mostly globular in shape, those of the epiblast are columnar. Near the dorsal part of the primitive streak, a few bottle-shaped cells can be identified. At this stage, no apparent distinguishing features are present at the dorsal surface of the epiblast between the primitive streak cells and those of the rest of the epiblast.

a: SEM of a stage 2 chick embryo in which the hypoblast was removed revealing the ventral (basal) surface of the epiblast (E) and the initial primitive streak (PS), which has a triangular shape. Scale bar = 60 μm. b: Enlargement of the primitive streak (PS). A basal lamina is absent ventral to the primitive streak cells, allowing the identification of individual cells. Scale bar = 100 μm. c: Enlargement of the more lateral epiblast (E). A basal lamina is present ventral to the more lateral epiblast, obscuring cell borders. Arrow points to the area opaca. Scale bar = 100 μm.

a: SEM of a transverse slice through a Stage 2 chick embryo showing the primitive streak in the midline. Scale bar = 180 μm. b: Enlargement of the primitive streak and adjacent epiblast (E). The former consists of principally globular cells, whereas the latter, of columnar cells. Hypoblast (H). Scale bar = 20 μm.

Stages 3a/b.

With formation of the linear primitive streak, the definitive (gut) endoderm begins to appear ventral to the primitive streak (not shown), where it is surrounded peripherally by the hypoblast. The definitive endoderm is derived from two sources: the rostral portion is from the epiblast the caudal portion is from the deep layer of the caudal MZ (Vakaet, 1970 Stern and Ireland, 1981 Eyal-Giladi et al., 1992 Bachvarova et al., 1998 ). The primitive streak occupies the midline of about the caudal half of the area pellucida (Fig. 6a). It is more elongated than that at Stage 2, and its caudal end is usually broader than its middle and rostral portions (in early Stage 3a embryos, the caudal end of the primitive streak is often triangular in shape, as is seen in Stage 2 embryos). Along the entire length of the primitive streak, cell surfaces are covered by remnants of the basal lamina formerly underlying the epiblast (Fig. 6a–d). Rostral to the rostral end of the primitive streak, cells seem to emerge from the ventral surface of the epiblast (Fig. 6b). Because they are not in continuity with the rostral tip of the primitive streak, this gives the impression that lengthening of the primitive streak between Stages 2 and 3a/b probably occurs by successive rostral “induction” of the primitive streak from midline epiblast cells. Although what tissue might provide such a signal is currently unknown, two candidates seem likely: the primitive streak itself, and the hypoblast or ingressed endoderm underlying the epiblast. Migration of mesodermal cells away from the primitive streak is in progress at its lateral margins along its entire length (Fig. 6a–d). Such migrating cells are interconnected by their processes as they move on the ectodermal basal lamina. At the rostral end of the primitive streak, however, a rostral migration of mesodermal cells is not always apparent. Unlike the cells within the primitive streak, the surfaces of migrating mesoderm cells are devoid of basal lamina material. A transverse section through the middle portion of the primitive streak reveals a predominance of globular primitive streak cells, as in Stage 2 embryos, with only a few columnar cells near the dorsal surface (Fig. 6e).

a: SEM of the ventral surface of a Stage 3a/b chick embryo in which the hypoblast and definitive endoderm was removed, revealing the ventral (basal) surface of the epiblast and the linear primitive streak (the blastoderm curled during processing). Scale bar = 400 μm. b: Enlargement of the rostral end of the primitive streak. Cells (arrows) are emerging from the ventral (basal) surface of the epiblast. Scale bar = 100 μm. c: Enlargement of the mid-streak level. Mesoderm cells are migrating from the lateral margins of the primitive streak (arrows). Scale bar = 100 μm. d: Enlargement of the caudal end of the primitive streak. Primitive streak cells are densely packed at the caudal end of the primitive streak. Scale bar = 100 μm. e: SEM of a transverse slice through the mid-primitive streak of a Stage 3a/b chick embryo primitive streak cells have predominantly globular shapes. Scale bar = 65 μm.

Stage 3c.

The area pellucida is overtly pear shaped by Stage 3c, with a broader rostral portion and a narrower caudal end. The endodermal layer has spread to cover most of the area pellucida, displacing the hypoblast with which it is in contact, toward the germ cell crescent at the rostral end of the blastoderm. At Stage 3c, the primitive streak is more elongated than that at Stage 3a/b, with its rostral end reaching the level of the widest diameter of the area pellucida (Fig. 7a). It is also narrower transversely than that at Stage 3a/b. Its rostral portion consists of a “ball” of concentrically arranged cells, indicating the position of Hensen's node (Fig. 7b). Remnants of basal lamina material are often observed on the surfaces of the cells forming the ball. Disruption of the basal lamina of the epiblast is no longer apparent rostral to the ball of cells. Mesodermal cell migration from the lateral margins of the primitive streak is more advanced than at earlier stages (cf. Figs. 6a and 7a), reaching about midway between the midline and the lateral margin of the area pellucida. Rostrally, the lateral mesodermal front on each side of the streak is caudal to the rostral end of the streak (Fig. 7a). Another difference between the primitive streaks of embryos at Stages 3c and 3a/b is a marked increase in cell density at Stage 3c (cf. Figs. 6c and 7c). At Stage 3c, ingression of cells through the primitive streak is well underway as evidenced by the presence of a midline groove marking the midline of the primitive streak on the dorsal surface of the epiblast (Fig. 7d) additionally, many elongated cells are present within the primitive streak (Fig. 7e).

a: SEM of the ventral surface of the blastoderm of a stage 3c chick embryo in which the definitive endoderm and hypoblast were removed revealing the ventral (basal) surface of the epiblast (E), primitive streak, and ingressing mesoderm. Arrows point to the mesodermal front. Scale bar = 500 μm. b,c: Enlargements of the rostral and mid-primitive streak, respectively. Note that cells in the rostral streak (i.e., Hensen's node) collectively form a ball-like structure. Scale bar = 100 μm. d: SEM of the dorsal surface of the epiblast of Stage 3c chick embryo. The primitive groove (arrows) has formed along the length of the primitive streak. The arrowhead points to Hensens node. Scale bar = 170 μm. e: SEM of a transverse slice through the level of the mid-streak of a Stage 3c chick embryo. Arrows point to ingressing cells. Newly formed mesoderm (M). Scale bar = 50 μm.

Stages 3d/4.

During Stage 3d/4, the hypoblast is concentrated in the germ cell crescent (Fig. 8a), and the primitive streak has reached its maximum length (Fig. 8b) its rostral end is rostral to the level of the widest diameter of the area pellucida. A coating appears on the surfaces of the cells along the entire extent of the primitive streak from this stage onwards it progressively obscures cellular outlines. This coating is also present on the migrating mesodermal cells caudally and laterally (Fig. 8c,d) and it is similar in appearance to the basal lamina of the epiblast. Laterally, the mesoderm cell front is at the level of the rostral end of the primitive streak, whereas in the midline, mesodermal cells extend beyond the rostral end of the primitive streak (beyond Hensen's node).

a: SEM of the ventral surface of a Stage 3d chick embryo the endoderm and hypoblast have been left in place arrows point to the position of the primitive streak arrowhead points to the position of Hensen's node. Germ cell crescent (G). Scale bar = 250 μm. b: SEM of the ventral surface of a stage 4 chick embryo in which the endoderm was removed. Arrows point to the mesodermal front arrowhead points to Hensen's node C and D mark the positions enlarged in 8c and d, respectively. Primitive streak (PS). Scale bar = 375 μm. c: Primitive streak cells from the area marked C in b. Cell boundaries are not easily discernible. Scale bar = 10 μm. d: Mesodermal cells from the area marked D in b. Unlike the primitive streak cells shown in c, individual cell outlines are clear. Arrows point to mesodermal cell processes. Epiblast (E). Scale bar = 20 μm.

Stages 5 and 6.

The primitive streak initiates regression during Stages 5 and 6. Laterally, the mesodermal front extends beyond the level of the rostral end of the streak, reaching almost the rostral end of the notochord by Stage 6 (Fig. 9, 10). Ingression of cells through the primitive streak continues as the primitive streak undergoes regression (Fig. 11).

SEM of the ventral surface of a Stage 5 chick embryo in which the endoderm was removed. The front of the ingressing mesoderm (arrows) has advanced rostrally as compared with earlier stages. Primitive streak (PS). Scale bar = 250 μm.

SEM of the ventral surface of a Stage 6 chick embryo in which the endoderm was removed. Asterisk marks the head fold of the body and the incipient cranial intestinal portal primitive streak (PS) notochord (N). Scale bar = 300 μm.

SEM of a transverse slice of a Stage 5 chick embryo through the mid-streak level showing ingressing cells. Scale bar = 50 μm.

Changes in the Shape of the Primitive Streak

The length of the primitive streak and its width at two rostrocaudal levels were calculated in three groups of living embryos over an 8-hr period. In the first group, measurements were made beginning at Stage 2, in the second group, beginning at Stage 3a/b, and in the third group, beginning at Stage 3c. By the end of the 8-hr period, embryos reached Stages 3c–4.

As shown in Table 1, the length of the primitive streak progressively increased in each of the three groups over the 8-hr period, with embryos from the first group almost doubling the length of their primitive streaks. In addition, the width of the rostral primitive streak increased in the first two groups, with formation of Hensen's node. In contrast, as the length of the primitive streak increased, the width of the mid-streak decreased. In the first two groups, where the greatest change occurred in the length of the primitive streak, a concomitant decrease occurred in the width of the mid-streak. In particular, note in the first group that as the length of the primitive streak approximately doubled (770–1475 μm), the width of the mid-streak approximately halved (325–145 μm). This relationship provides evidence that coordinated convergent-extension movements occur during elongation of the primitive streak.

Stage 0 Hr 2 Hr 4 Hr 6 Hr 8 Hr % Increase or decrease
Length of primitive streak
2 770 (44) 985 (109) 1170 (72) 1290 (68) 1475 (187) 91
3a/b 1075 (130) 1323 (129) 1465 (137) 1655 (89) 1815 (106) 69
3c 1235 (19) 1390 (21) 1600 (51) 1765 (77) 1995 (77) 62
Width of rostral primitive streak
2 115 (6) 145 (6) 170 (12) 175 (18) 190 (22) 65
3a/b 193 (33) 235 (31) 235 (12) 233 (15) 230 (12) 19
3c 175 (28) 195 (22) 225 (12) 165 (24) 175 (7) 0
Width of mid-primitive streak
2 325 (19) 295 (77) 200 (39) 145 (28) 145 (12) −55
3a/b 233 (30) 190 (24) 148 (24) 120 (12) 135 (12) −42
3c 130 (26) 130 (23) 115 (13) 110 (16) 120 (6) −8
  • * Values represent mean lengths or widths (μm) for five embryos in each cell. The standard error of the mean is given in parentheses. Negative values are percentage decrease.

Distribution of Fibronectin and Laminin

The distribution patterns of both fibronectin and laminin were similar in appearance and, therefore, are described together. Both glycoproteins are present in the basal lamina before the appearance of the primitive streak (Fig. 12a). With the appearance of the streak, however, fibronectin and laminin become depleted in the region it occupies (Fig. 12b,c). Elsewhere beneath the epiblast, the glycoproteins continue to be expressed (Fig. 12b,c). As ingression occurs, remnants of the disrupted glycoproteins are observed within the streak (Fig. 12d).

Transverse sections of chick embryos labeled with anti-fibronectin (a, b, d) or anti-laminin (c) antibodies. The epithelialized hypoblast or endoderm have been removed to ensure antibody penetration. a: Stage XIII. The ventral surface of the entire pre-streak epiblast is labeled. b: Stage 2. Labeling of the ventral surface of the initial primitive streak is patchy. Arrows point to gaps in labeling. c: Stage 3a/b. As the primitive streak elongates, its entire ventral surface becomes devoid of label. Arrow points to the junction between epiblast and primitive streak on one side. Note that the ventral side of the epiblast is labeled in contrast to the ventral side of the primitive streak. d: Stage 3c. With ingression well underway, remnants of labeled basement membrane (arrow) can be identified at the ventral side of individual ingressing epiblast cells. Scale bar = 100 μm.

Fate Mapping Experiments

Injections of the primitive streak.

To document early mesodermal and endodermal cell migration from the primitive streak and the timing of this migration in relation to progression of the primitive streak, dye was injected into the primitive streaks of embryos at Stages 2, 3a/b, and 3c at three levels (i.e., rostral, middle, and caudal, designated, respectively, as Sites 1, 2, and 3 Fig. 13a). At each injection site when examined immediately after injection, the dye labeled a small group of dorsal surface cells in the midline of the primitive streak together with ingressing cells in the midline (Fig. 13b). After 4 hours of re-incubation, Stage 2 embryos had developed to Stage 3a/b, and Stages 3a/b and 3c embryos, to Stages 3c and 3d/4, respectively. In embryos injected at Stage 2, cell migration from Sites 1, 2, and 3 was generally away from the midline in a lateral direction (Fig. 13c). Additionally, a few labeled cells had migrated rostrally from the rostral end of the streak (Fig. 13d). A majority of the mesodermal cells together with the underlying endoderm was labeled at all injection sites (Fig. 13e). Between injection sites, however, the mesodermal cells that were labeled were only those that were close to the endodermal layer (Fig. 13f). In the dorsal midline of the primitive streak, a small group of surface cells located mainly rostral and caudal to injection Site 2, as well as within injection Site 2, were labeled (Fig. 13c,e,f).

Injections of the primitive streak at three levels (1, 2, 3) in chick embryos at Stage 2 (processed for peroxidase immunocytochemistry using anti-rhodamine primary antibody). a: Whole mount (viewed dorsally) at time zero, the dye is confined to cells at the three injection sites. Scale bar = 750 μm. b: A transverse section through level B (at the original injection Site 2) in a shows labeling of cells in the midline of the primitive streak throughout its entire dorsoventral extent. Scale bar (shown in a) = 100 μm. c: Whole mount (viewed dorsally) of an embryo similar to that shown in a after 4 hours of re-incubation after injection at three sites. Movement of labeled cells from the primitive streak has occurred at all three levels such movement from all levels is bilateral, with some rostral migration also occurring from original injection Site 1. Scale bar (shown in a) = 500 μm. d: A transverse section at level D (rostral to original injection Site 1) in c shows that the mesendoderm is labeled at a level that is rostral to the labeling of Hensens node (hence, the epiblast is unlabeled at level D). Scale bar (shown in a) = 100 μm. e,f: Transverse sections at levels E (at original injection Site 2) and F (rostral to original injection Site 3), respectively, of c shows that the midline dorsal surface cells (arrowheads) are labeled, as are the deeper cells of the primitive streak, but not the intermediate cells. Scale bar (shown in a) = 100 μm.

Injections in embryos at Stages 3a/b and 3c showed similar features, so they are described together. The labeling characteristics of cells at the injection sites for 0 hours and 4 hours of reincubation were similar to those for Stage 2 embryos. Cell migration from Site 1 occurred in a rostral direction, but the ingressed cells spread laterally as they advanced (Fig. 14a). Most cells rostral to the node were labeled, apart from a few at the extreme lateral sides (Fig. 14b), which presumably were derived from more caudal levels of the primitive streak. Movement of cells from Site 2 was often similar to that at Stage 2, but sometimes, it resembled that at Site 3, which tended to occur rostrolaterally, with the cells forming v-shaped streams. Close to injection Sites 2 and 3, most of the mesodermal cells within and close to the primitive streak were labeled (Fig. 14c), but more rostral or caudal to these injection sites, only mesodermal cells close to the endoderm and those lateral to the streak were labeled (Fig. 14d). At these stages of development, movement of the midline group of dorsal surface cells in the primitive streak occurred both rostrally and caudally from Site 2, but mainly rostrally from Site 3 sometimes, these joined together in the dorsal midline (Fig. 14e).

Injections of the primitive streak at three levels (1, 2, 3) in a chick embryo at Stage 3c (processed for peroxidase immunocytochemistry using anti-rhodamine primary antibody). a: Whole mount (viewed ventrally) 4 hours postinjection. e: Whole mount (viewed dorsally) of a similar embryo as in a 4 hours postinjection. Note the spread of cells in the dorsal midline between original injection Sites 1 and 2, and 2 and 3. b: Ingressed mesoderm cells rostral to the node at level B (rostral to original injection Site 1) in a are labeled except for those at the extreme lateral edges. The definitive endoderm cells at this level are mostly unlabeled. c: At level C (at original injection Site 2) in a ingressed cells within and adjacent to the primitive streak are labeled. d: More caudally at level D (rostral to original injection Site 3 and just caudal to original injection Site 2) in a only the midline dorsal surface cells (arrowhead), deeper primitive-streak cells, and those ingressed cells lateral to the streak are labeled. Scale bar (shown in 13a) for a, e = 500 μm b–d = 100 μm.

To establish whether the labeling of the midline group of dorsal surface cells of the primitive streak was real or an artifact of endogenous peroxidase activity, control embryos lacking dye injections were processed in a manner identical to that used for injected embryos. No labeling was detected anywhere in the blastoderm (Fig. 15a,b), demonstrating the lack of endogenous peroxidase activity.

A control Stage 3a/b chick embryo in which dye was not injected (processed for peroxidase immunocytochemistry using anti-rhodamine primary antibody). a,b: Background labeling, for example, due to endogenous peroxidase activity, is completely lacking in both whole mounts (a viewed dorsally) and transverse sections (b through level B in a). Note in particular that the existence of labeled midline dorsal surface cells in injected embryos cannot be explained by an artifact of immunocytochemistry. Scale bar (shown in 13a) for a = 500 μm b = 100 μm.

To determine whether the labeled midline group of primitive streak cells were complete cells or cell fragments, embryos at Stages 2–3c were examined with transmission electron microscopy. The results revealed that the cells in the dorsal midline of the primitive streak had nuclei (Fig. 16) consequently, such cells were not cell fragments, such as left-behind apical cytoplasmic processes of previously ingressed cells.

TEM of a transverse section at the mid-primitive streak level showing the midline dorsal surface cells in a Stage 3a/b chick embryo. Arrows point to those midline dorsal surface cells whose nuclei fall within the plane of the section and are located near the dorsal surface of the epiblast, showing that the midline dorsal surface cells are truly complete cells and not merely cell processes. Mitotic figures also occur in the dorsal midline, suggesting that midline dorsal surface cells undergo mitosis. Scale bar = 6 μm.

Injections of the parastreak epiblast.

To determine the timing and pattern of ingression of epiblast cells through the primitive streak, dye was injected on each side of the primitive streak in embryos at Stages 2–3c of development. At 0 hr, the dye labeled the entire thickness of the epiblast at the injection site (Fig. 17a,b). After 5 hours of re-incubation, although the labeled cells in the epiblast had moved closer to the midline, none had ingressed through the streak. The migration of the labeled epiblast cells was in a rostromedial direction at mid and caudal levels of the streak, but caudomedially at rostral streak levels (Fig. 17c,d). By 7 hours of re-incubation, the two bilaterally labeled epiblast patches had merged into one overlapping patch in the midline primitive streak, and labeled cells were now present within the mesodermal layer (Fig. 17e,f). Additionally, by 7 hours of re-incubation primitive streak cells from injection site 2 had spread both rostrally and caudally within the primitive streak such labeled cells occupied the entire thickness of the midline primitive streak, including much of its dorsal surface (Fig. 17e,f).

Bilateral injections of the epiblast at three levels (1, 2, 3) in chick embryos at Stage 3a/b (processed for peroxidase immunocytochemistry using anti-rhodamine primary antibody). a,c,e: Whole mounts (viewed dorsally) at 0 hours, 5 hours, and 7 hours post-injection. b,d,f: Transverse sections through level B, D, F in a, c, e, respectively. By 5 hours post-injection, labeled epiblast cells have migrated medially toward the primitive streak but have not yet ingressed (c, d). Ingression commences about 7 hours post-injection (e, f). Scale bars (shown in a, b) for a, c, e = 500 μm b, d, f = 50 μm.


DISCUSSION

The key findings of the present study are that: (1) the intra-embryonic region of the endodermal layer is generated mainly by cells from the rostral half of the primitive streak between stages 3a/b and 4 (2) more peripheral regions of the endodermal layer (extending peripherally to the area pellucida-area opaca border and beyond) are derived from principally more caudal levels of the primitive streak (with the exception of the rostral peripheral endoderm, which derives from the rostral end of the primitive streak) (3) epiblast cells fated to become endoderm undergo ingression through the primitive streak to contribute to the definitive endodermal layer of the area pellucida and (4) displacement of the hypoblast layer by the forming endoderm is accompanied by cell-cell intercalation between the two populations.

On the basis of our mapping experiments, we constructed prospective fate maps of the definitive endoderm (Fig. 7). As most of the intra-embryonic endoderm arises from the rostral end of the primitive streak as the latter forms and undergoes progression, we constructed prospective fate maps that show the rostrocaudal subdivisions of the definitive endoderm originating from the rostral end of the primitive streak (Fig. 7,to the left of the broken vertical line). In addition, as rostrocaudal position within the primitive streak ultimately translate into mediolateral position within the definitive endoderm, we constructed a prospective fate map that shows the mediolateral subdivisions of the definitive endoderm originating from the rostral, mid and caudal levels of the primitive streak(Fig. 7, to the right of the broken vertical line and above the broken horizontal line). Moreover, as mesodermal and endodermal regions arise in concert within the primitive streak and migrate in parallel after ingression, we constructed a prospective fate map comparing mesodermal and endodermal subdivisions(Fig. 7, to the right of the broken vertical line and below the broken horizontal line).

Prospective fate maps showing the primitive-streak origins of the definitive endoderm. The origins of the rostrocaudal subdivisions of the definitive endoderm are shown to the left of the broken vertical line in embryos at three pairs of stages: stage 3a/b to stages 7-8, stage 3c/d to stages 8-9, and stage 4 to stages 9-10. Only injections at the rostral end of the streak are shown, as more caudal injections (mid-streak and caudal streak)contributed endoderm only to the most caudal end of the embryo in a u-shaped configuration centered on the midline (more rostral injections labeled slightly more rostral cells, whereas more caudal injections labeled slightly more caudal cells). The origins of the mediolateral subdivisions of the definitive endoderm are summarized to the right of the broken vertical line,above the broken horizontal line. More rostral injections label more medial definitive endoderm, whereas more caudal injections label more lateral definitive endoderm. A comparison of the mesodermal and endodermal origins from the primitive streak is summarized in a diagrammatic cross-section to the right of the broken vertical line, below the broken horizontal line. Ectoderm is indicated in gray. Endoderm is indicated by different colors in the mediolateral plane and outlined in black it consists of only the lower layer. Mesoderm consists of the tissue below the ectoderm and above the endoderm and is also indicated by different colors in the mediolateral plane. The comparison of the origin of the mesoderm and endoderm from the primitive streak is based on the data in the current study, as well as those published previously (Garcia-Martinez et al.,1993 Lopez-Sanchez et al.,2001). Note: (1) that for both mesoderm and endoderm, rostrocaudal position within the primitive streak translates after ingression to mediolateral position within the blastoderm (that is, more rostral sites contribute to more medial tissues and more caudal sites contribute to more lateral tissues) and (2) endoderm contributions from a particular rostrocaudal site tend to be spread more laterally than do mesodermal contributions from the same site. ao, area opaca ap, area pellucida ps,primitive streak solid inverted u-shaped line, rostral border of neural plate/head broken inverted u-shaped line, location of the cranial intestinal portal vertical line in midline, notochord squares, somites.

Prospective fate maps showing the primitive-streak origins of the definitive endoderm. The origins of the rostrocaudal subdivisions of the definitive endoderm are shown to the left of the broken vertical line in embryos at three pairs of stages: stage 3a/b to stages 7-8, stage 3c/d to stages 8-9, and stage 4 to stages 9-10. Only injections at the rostral end of the streak are shown, as more caudal injections (mid-streak and caudal streak)contributed endoderm only to the most caudal end of the embryo in a u-shaped configuration centered on the midline (more rostral injections labeled slightly more rostral cells, whereas more caudal injections labeled slightly more caudal cells). The origins of the mediolateral subdivisions of the definitive endoderm are summarized to the right of the broken vertical line,above the broken horizontal line. More rostral injections label more medial definitive endoderm, whereas more caudal injections label more lateral definitive endoderm. A comparison of the mesodermal and endodermal origins from the primitive streak is summarized in a diagrammatic cross-section to the right of the broken vertical line, below the broken horizontal line. Ectoderm is indicated in gray. Endoderm is indicated by different colors in the mediolateral plane and outlined in black it consists of only the lower layer. Mesoderm consists of the tissue below the ectoderm and above the endoderm and is also indicated by different colors in the mediolateral plane. The comparison of the origin of the mesoderm and endoderm from the primitive streak is based on the data in the current study, as well as those published previously (Garcia-Martinez et al.,1993 Lopez-Sanchez et al.,2001). Note: (1) that for both mesoderm and endoderm, rostrocaudal position within the primitive streak translates after ingression to mediolateral position within the blastoderm (that is, more rostral sites contribute to more medial tissues and more caudal sites contribute to more lateral tissues) and (2) endoderm contributions from a particular rostrocaudal site tend to be spread more laterally than do mesodermal contributions from the same site. ao, area opaca ap, area pellucida ps,primitive streak solid inverted u-shaped line, rostral border of neural plate/head broken inverted u-shaped line, location of the cranial intestinal portal vertical line in midline, notochord squares, somites.

The early phase of endoderm formation

This phase of endoderm formation coincides with the pre- and incipient stages of primitive streak formation. During this period of development, cells fated to contribute to the endodermal layer are generally believed to originate from two sources. First, cells residing in the germ wall (i.e. the lower layer of the area opaca near its border with the area pellucida) are presumed to undergo a centrifugal movement to give rise to the junctional endoblast, defined as the endodermal layer at the periphery of the area pellucida (Vakaet, 1970 Stern and Ireland, 1981 Stern, 1990). Experimental evidence in support of the above mechanism of formation of the junctional endoblast comes from a study in which the original lower layer was ablated in pre- and incipient primitive streak embryos(Stern and Ireland, 1981). It was noted that after ablation, cells migrated from the germ wall to adjacent parts of the area pellucida. The observations of the present study on the origin of the junctional endoblast, however, are not in accordance with the idea that a junctional endoblast arises from the germ wall during normal development in the intact embryo rather, they demonstrate that the junctional endoblast in the intact embryo receives contributions from the primitive streak (and largely from its caudal region), instead of from the germ wall(Fig. 7: origins of mediolateral endodermal subdivisions). The migration of cells from the germ wall in Stern and Ireland's experiment(Stern and Ireland, 1981) is,therefore, likely to represent a response to an experimental manipulation of the lower layer, rather than an event that occurs normally during development. The second source of endodermal cells is believed to be the lower layer underlying Koller's sickle (Pasteels,1937 Bellairs,1953 Malan, 1953 Vakaet, 1970 Bachvarova et al., 1998). Studies suggest that cells from this location migrate rostrally in tandem with cells from the epiblast overlying Koller's sickle to contribute to the sickle endoblast and the primitive streak, respectively(Vakaet, 1970 Stern and Ireland, 1981 Callebaut and Van Nueten, 1994 Bachvarova et al., 1998 Foley et al., 2000 Lawson and Schoenwolf, 2001b). Both components of the early endoderm (the junctional and sickle endoblasts)are believed to be fated to become extra-embryonic(Rosenquist, 1971 Rosenquist, 1972 Fontaine and Le Douarin,1977), not contributing to the definitive structure of the embryo(Callebaut et al., 2000).

The intermediate phase of endoderm formation

This stage of endoderm formation takes place as the primitive streak lengthens and cells ingress through it. It is characterized by the formation of the definitive endodermal layer of the area pellucida. The demonstration in the present study that the primitive streak serves as a source of cells for the definitive endodermal layer confirms the findings of several earlier studies (Vakaet, 1970 Nicolet, 1971 Rosenquist, 1971 Rosenquist, 1972 Fontaine and Le Douarin, 1977 Stern and Ireland, 1981 Garcia-Martinez et al., 1993 Lopez-Sanchez et al., 2001 Bachvarova et al., 1998 Foley et al., 2000). By following the movements of labeled cells at different levels of the primitive streak between stages 3a/b and 3d, we demonstrated that the rostral part of the primitive streak generates most of the intraembryonic endoderm(Fig. 7: origins of rostrocaudal endodermal subdivisions). This confirms the findings of Rosenquist (Rosenquist, 1971),who used grafts labeled with tritiated thymidine followed by autoradiography. Additionally, age-related differences were observed in the contribution of labeled cells from the rostral end of the primitive streak to the definitive endodermal layer. Whereas at stage 3a/b labeled cells from the rostral streak populated most of the definitive endodermal layer, at stage 3d the contribution from the rostral primitive streak was restricted only to the region of the definitive endoderm rostral to Hensen's node. These findings,while demonstrating that formation of the definitive endoderm is spatially and temporally regulated, also provide insight into the nature of cell movement taking place at the rostral end of the primitive streak during its progression.

The late phase of endoderm formation

The late phase of endoderm formation occurs at stage 4 when the primitive streak has attained its maximum length and Hensen's node has formed. With the formation of Hensen's node, contributions of cells from the rostral streak were virtually restricted to the midline, probably because Hensen's node generates midline structures such as floor plate, notochord and dorsal midline endoderm (as well as the ventral midline endoderm of the foregut and outflow tract of the heart) (Kirby et al.,2003). Cells from the rostral end of the primitive streak also generate the prechordal plate, important for regionalizing the forebrain(Pera and Kessel, 1997), and the ventral midline endoderm of the foregut, a region well situated to form a ventral axis of the head (Kirby et al.,2003).

Comparison of the origin of the mesoderm and endoderm during avian gastrulation

Previous fate mapping studies from our laboratory have revealed the detailed origins and fates of prospective mesodermal cells in both the primitive streak and epiblast at the full range of elongating primitive streak stages (Garcia-Martinez and Schoenwolf,1993 Lopez-Sanchez et al.,2001). Although contributions of the primitive streak and the epiblast to the definitive endoderm were observed, they were not highlighted in these studies. In one study(Garcia-Martinez and Schoenwolf,1993), epiblast plugs of quail cells were transplanted isochronically and homotopically to chick hosts. Quail cells were detected in resulting chimeras by using Feulgen staining to label the quail nucleolus. However, this method is unreliable in our hands for detecting quail cells in highly squamous sheets of cells like the endoderm or endothelium thus,contributions to such layers are grossly underestimated. In the second study(Lopez-Sanchez et al., 2001),also based on the transplantation of quail cells to chick hosts, an anti-quail antibody was used to detect quail cells. This method is very effective in detecting quail cells regardless of their histological structure. However, our small epiblast grafts contained only a couple of hundred cells, and as the vast majority (certainly more than three-quarters) of epiblast cells contribute to mesoderm rather than endoderm, only small patches of endodermal cells were detected in the published study. By contrast, with the use of dyes,sites (especially within the primitive streak) contain reservoirs of label,such that labeled cells are generated within the site as cells move through it throughout the course of the experiment. Thus, many more cells are labeled,and the contributions of such sites to the endoderm can be analyzed readily. Although data on contributions to the endoderm are limited in our previous studies, when their results are compared with those of the present study it is clear that the results are consistent. Two general findings arise from these results (Fig. 7: cross section comparing the origin of mesoderm and endoderm from the primitive streak):contributions to the mesoderm and endoderm occur in concert from any particular site (i.e. primitive streak or epiblast), and contributions to the endoderm from any particular site tend to spread more laterally than those to the mesoderm from the same site. As reported previously, more rostral sites within the primitive streak or epiblast tend to contribute to more medial regions of the mesoderm, and more caudal sites tend to contribute to more lateral regions of the mesoderm. We show here that this same relationship holds for the endoderm, with more rostral sites tending to form more medial regions of the definitive endoderm and more caudal sites tending to form more lateral sites (see Fig. 7:origins of mediolateral endodermal subdivisions).

The striking relationship in the origins of prospective mesodermal and endodermal cells implies that signals for mesodermal patterning may be similar to those of the underlying endodermal layer, or that both signals may occur concurrently. Alternatively, each may be involved in the patterning of the other. Indeed, there is evidence to suggest that interactions between endoderm and adjacent mesodermal tissues allow the endoderm to acquire some positional identity and morphogenetic information(Cleaver et al., 2000 Cleaver and Krieg, 2001). In addition, specification of the prechordal mesoderm may occur via rostral endoderm-derived TGFβ family signaling(Vesque et al., 2000).


Acknowledgements

We thank A. Bolondi, R. Weigert and other members of the Meissner laboratory, M. Chan and D. Hnisz for discussions and advice, S. Otto for experimental support characterizing the EED-knockout mES cell line, M. Walter for support with embryo isolations, T. Ahsendorf for help with initial efforts to optimize our genotyping pipeline, and D. Andergassen for discussions on SNP typing. We are also grateful to F. Koch and the transgenic facility, including M. Peetz, and C. Giesecke-Thiel of the Flow Cytometry Facility for their feedback and support. We thank M. Saitou for the mVenus Prdm14 promoter sperm that were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan (Acc. No. CDB0461T). This work was funded by the National Institutes of Health (NIH 1P50HG006193, P01GM099117, 1R01HD078679 and 1DP3K111898) and the Max Planck Society.


Origin of Primitive Streak Formation

We address in this paragraph the question of why a streak will form. (In the next part we will address the question of ingression and streak retreat).

We start from the 𠇏low” of the epiblast, which is actually a step-by-step deformation under stress. We have a pulling force exerted by the 𠇏low” in the region of Kohler's sickle. If we isolate by gedanken experiment, an infinitesimal volume of matter in the sickle region, we see that this element of matter is divided into two components, the presumptive mesoderm, which is pulled forward towards Kohler's sickle, and the extra cellular matrix, which bears the traction which allows the presumptive mesoderm to move forward. This traction can be intuitively understood if one remembers that the cells move by filopodia traction. The dynamics of filopodia extension and contraction amounts to a solid tensile stress on the stiffer extra cellular matrix, by which the cells pull themselves forward, while the cell body deformation amounts to the strain component of the “Poiseuille” deformation ( Figs. 3 and ​ and4 4 ).

It is rarely realized that cell motility in one direction on a medium requires a traction on the medium in the opposite direction, by the principle of action and reaction (Newton's law). If one thinks of a cell moving on a Petri dish, the Petri dish is solid, and holds easily the tension exerted by the cell. However, if the Petri dish were very soft, then the motility activity would simply pull the substrate towards the cell, and the cell would not move. This is much like running a car: the road undergoes a very strong pull, by which the car moves forward if the road is soft (e.g., muddy), the car does not move forward: the soil is pulled under the wheels. To be honest, this fact has actually been used in this field for 20 years to detect and study cellular traction. Indeed, a now classical means to study the traction force exerted by cells on the extracellular medium consists in depositing and anchoring cells to very soft substrates, like very thin polymer silicon membranes. These thin films are so thin that they wrinkle under the traction exerted by the cells. Optical visualization of the wrinkles (e.g., by Nomarski contrast) enables one to count the wrinkles, and hence to measure the magnitude of the force exerted by the cells. Such techniques, and alternative ones, have been reviewed recently. 11

If we now turn to the 𠇏low” map we see that the traction force exerted on the tissue which allows the motion is simply opposed to the stress generated by the 𠇏low”. Therefore, in order to find the stress field, it suffices to find the displacement U, and then to form the stress field. Although these could be written analytically, they contain rational polynomials up to the 8 th power in x and y, this is why we simply give a numerical solution by Mathematica ( Fig. 9 ) which shows the map of stress components σ and σyy (the x axis being the vertical axis in the Fig. 9 ). In this figure, the color gives the magnitude of the component (black is tensile, white is compressive), but remember that the stress is a tensor, and therefore, at each point in Figure 9A , the quantity being considered is a vector oriented along x (σxx) while in 9B the quantity considered is a vector oriented along y (σyy). We see that along the �udal line” represented by the axis of left/right symmetry of the blastodisc, the stress is tensile, and oriented orthoradially. The stress map is formed of large oval lobes of high magnitude stress laying over this axis. In the perpendicular direction, the stress is compressive. Therefore, for a distribution of forces forming two dipoles which contract the tissue towards the caudal pole, the force on the extra cellular matrix actually stretches the mid line. If we assume that above a tensile stress σ* the extra cellular matrix cracks, a “primitive streak” will open which will nucleate at the center of traction, which is the intersection of Kohler's sickle with the AP axis, and will next propagate along the AP axis in the P𡤪 direction, and also in the caudal direction, if Kohler's sickle is not strictly caudal at the moment of cracking. This is analogous to a paper cracking perpendicularly to the edge, as fingers pull two parts of a sheet asunder this is more than a metaphore in that the blastodisc behaves as the paper sheet, and the traction centers as two hands pulling apart, in an actual piece of paper, the stress field is also probably like in Figure 9 .

It is not entirely clear to the author whether in the true world, the primitive streak propagates in the P𡤪 or A→P direction. At www.gastrulation.org one finds in display a movie movie15_3.avi, in which the primitive streak seems to open in the A→P direction, from Hensen's node (although Hensen's node progresses towards the anterior part, the mesoderm ingression starts first at Hensen's node, and eventually reaches the caudal pole). If this is true, then the center of the pattern in Figures 6 and ​ and9 9 is Hensen's node, and the apparent forward growth is in fact due to elongation of the caudal part. This center in Figures 6 and ​ and9 9 is characteristic in that it is the spot of highest stress in all directions : quite tensile in one direction, quite compressive in the other. In this view, Hensen's node is instructed by mechanical forces.


RESULTS

Initial activation of Hoxb1 and Hoxb8 in the primitive streak region

The spatiotemporal patterns of activation of the 3′ gene Hoxb1 and the 5′ gene Hoxb8 were compared. Expression of Hoxb1 began in the most caudal part of the primitive streak, at the junction between extra-embryonic and embryonic tissues, at the late midstreak (LMS) stage (E7.0) (Fig. 1A). Dynamic rostral expansion of the transcription domain followed. This domain spread along and lateral to the primitive streak(Fig. 1B-D) and later beyond the node (Fig. 1E,F). Transcription of Hoxb8 started about 12 hours later than Hoxb1 (late neural plate, E7.5, Fig. 1G). The spreading of the expression domains of either Hox gene from posterior to the node region took less than 8 hours. Hoxb1 and Hoxb8 transcripts reached the anterior end of the streak at the late streak early bud (LSEB)/neural plate(NP) stage (Fig. 1D), and early headfold/headfold (EHF/HF) stage, respectively(Fig. 1I). Variation between embryos in the time of arrival of the expression boundary at the node is shown in Table 1 (with Hoxb4included for comparison). During this phase, Hoxb8 transcripts remained mainly restricted to the primitive streak and nascent mesoderm(Fig. 1G-I), while Hoxb1 was transcribed more widely laterally and anteriorly in the mesodermal wings (Fig. 1D-F). This distribution was confirmed in histological sections(Frohman et al., 1990 Deschamps and Wijgerde, 1993). Therefore, late streak nascent lateral and paraxial mesoderm only expresses Hoxb1, while mesoderm born later at the EHF stage expresses both Hoxb1 and Hoxb8. Both ectoderm and mesoderm at the level of the node, but not the axial mesoderm or endoderm express Hoxb1 at the NP stage and both Hoxb1and Hoxb8 at the HF stage.

Hox gene activation in the mouse primitive streak. Expression of Hoxb1 (A-F) and Hoxb8 (G-J) at E7.0 to 8.0 assayed by in situ hybridisation. (A,G) Onset of expression in the proximal part of the primitive streak. (B-D,H,I) Rostral and lateral spreading of Hox expression domains towards the distal part of the streak (D,I), containing the organiser or node (n) and beyond (E,F,J). Arrowhead, rostral expression front. Lines in A and G, primitive streak. Late midstreak/late streak (A), late streak (B),late streak early bud (C), late streak early bud/neural plate (D), late neural plate (G), early headfold (E,H,I), headfold (F), late headfold (J). Anterior is towards the left, posterior towards the right. Scale bar: 100 μm.

Hox gene activation in the mouse primitive streak. Expression of Hoxb1 (A-F) and Hoxb8 (G-J) at E7.0 to 8.0 assayed by in situ hybridisation. (A,G) Onset of expression in the proximal part of the primitive streak. (B-D,H,I) Rostral and lateral spreading of Hox expression domains towards the distal part of the streak (D,I), containing the organiser or node (n) and beyond (E,F,J). Arrowhead, rostral expression front. Lines in A and G, primitive streak. Late midstreak/late streak (A), late streak (B),late streak early bud (C), late streak early bud/neural plate (D), late neural plate (G), early headfold (E,H,I), headfold (F), late headfold (J). Anterior is towards the left, posterior towards the right. Scale bar: 100 μm.

Passage of anterior boundary of Hox expression through the node region

. LSOB . LSEB . NP . EHF . HF . LHF .
Hoxb15 002 6 * 00 100 040 060 01
Hoxb4 1 000 310 050 07
Hoxb8 4 004 300 300 14
. LSOB . LSEB . NP . EHF . HF . LHF .
Hoxb15 002 6 * 00 100 040 060 01
Hoxb4 1 000 310 050 07
Hoxb8 4 004 300 300 14

Numbers of embryos at different stages are shown. Regular type, posterior to the node bold, at the node italic, anterior to the node.

Anterior expression boundary at posterior edge of node.

Upstream inducing interactions are set much earlier than actual Hox gene expression

Earlier work had made it clear that the spread of the Hox expression domains along the primitive streak was not by proliferative expansion of the initially expressing cell population(Deschamps and Wijgerde, 1993)and did not involve diffusion of inducers from posterior to anterior in the streak (Gaunt and Strachan,1994). In order to investigate whether inducing molecules were involved earlier than at the stages analysed by Gaunt and Strachan for Hoxb4, we analysed the autonomy of expression of one of the earliest Hox genes, Hoxb1, in explants of anterior and posterior streak regions.

We investigated whether Hoxb1 was activated autonomously in embryonic tissues at stages preceding initial gene expression. To do this, we cultured posterior streak region (PSR) and anterior streak region (ASR)(Fig. 2, upper panels)separately at different primitive streak stages, and examined the expression of Hoxb1 and Hoxb1-lacZ. In control experiments, explant culture conditions supported normal Hox and marker gene expression at the different stages, as shown by the maintenance of Hoxb1 and Hoxb8 expression in culture in 100% of PSR and ASR explants from headfold stage embryos (E7.5-7.75): such pieces already express the genes at the time of excision (data not shown). The 24-hour culture period was chosen because this time is sufficient for Hox gene expression to progress from the posterior to the anterior streak region both in vivo and in longitudinally bisected egg cylinders in vitro (Fig. 2, second row and data not shown).

PSR explants excised at different stages between early streak (ES) and late mid streak (LMS) stages expressed Hoxb1(Fig. 3A-D) and Hoxb1-lacZ (not shown) after culture, with the proportion of positive explants rising from 54% for ES explants to 80-100% from the early mid streak(EMS) to the LMS stages (Fig. 3O). Likewise, 80-100% median streak region (MSR) explants from mid streak (MS) and LMS stages had activated the Hoxb1 gene and transgene (Fig. 3E,F,O) after culture. By contrast, ES ASR explants failed to express Hoxb1 and Hoxb1-lacZ (Fig. 3G,O). The proportion of ASR explants expressing the gene rose with increasing age, from 18% at the EMS stage (with a low number of positive cells in this latter case) (Fig. 3H,O), to 36% at the MS stage(Fig. 3I,O), and to 57% for the LMS embryos (Fig. 3J,O). The absence of Hoxb1 expression in the youngest material was not due to inappropriate culture conditions because the explants expressed the Hox-independent genes brachyury (T)(Fig. 3K,L) and chordin(Fig. 3M,N). Hox gene expression could not be induced by increasing the culture period of ES ASR explants to 30-32 hours (data not shown), whereas 100% MS ASR explants were positive after a similar culture period (versus 36% after 24 hours culture,data not shown).

Hoxb1 and Hoxb1-lacZ activation in cultured embryonic explants. Explants dissected at different anteroposterior levels of the primitive streak (PSR, A-D MSR, E,F ASR, G-J) at different stages before the onset of Hoxb1 expression in vivo, were cultured for 24 hours. Expression of Hoxb1 was assayed by in situ hybridisation after culture. A representative selection of the data is shown, and a quantitative(total numbers above bars) representation of all data obtained with Hoxb1 and Hoxb1-lacZ is given in O. Results for gene and reporter transgene were similar. (K,L) Brachyury/T and (M,N) chordin expression in ES and EMS ASR. Scale bar: 100 μm.

Hoxb1 and Hoxb1-lacZ activation in cultured embryonic explants. Explants dissected at different anteroposterior levels of the primitive streak (PSR, A-D MSR, E,F ASR, G-J) at different stages before the onset of Hoxb1 expression in vivo, were cultured for 24 hours. Expression of Hoxb1 was assayed by in situ hybridisation after culture. A representative selection of the data is shown, and a quantitative(total numbers above bars) representation of all data obtained with Hoxb1 and Hoxb1-lacZ is given in O. Results for gene and reporter transgene were similar. (K,L) Brachyury/T and (M,N) chordin expression in ES and EMS ASR. Scale bar: 100 μm.

In summary, these results show that Hoxb1 expression can start autonomously in PSR explants cultured from the ES stage onwards. ASR explants were only able to activate Hoxb1 autonomously at the EMS/MS stage and later. This suggests that, early in gastrulation and more than 12 hours before the first Hoxb1 expression appears in the embryo, the underlying molecular genetic interactions have occurred in proximo-posterior embryonic tissues. Alternatively, cell interactions within the explants might set these instructions up in vitro.

The activation pattern of Hoxb8 in explants was also examined. A similar time period (about 12-16 hours) was found to separate permissiveness to `autonomous' activation in explanted tissues(Fig. 4), and effective activation in the intact embryo in vivo(Fig. 1). PSR explants can autonomously activate Hoxb8 expression after culture from the LMS stage onwards (E7.0) (Fig. 4A,B) but not earlier, and the MSR and ASR explants from the late streak early bud (LSEB) stage on (E7.25)(Fig. 4C-F), although Hoxb8 is only expressed in the embryo from late neural plate/head fold (LNP/HF) stages (E7.75) (Fig. 1G-J). The dynamics of activation of Hoxb1 and Hoxb8 in the explant system suggest that, like Hox expression itself,the process which anticipates this expression in the primitive streak takes place sequentially (for the 3′ genes earlier than the 5′ genes) in a proximal (posterior) to distal (anterior) sequence (compiled in Fig. 2, bottom panel).

Hoxb8 activation in cultured embryonic explants. Posterior streak region, PSR, blue anterior streak region, ASR, orange median streak region,MSR, green. Hoxb8 expression in PSR (A,B), MSR (C,D) and ASR (E,F)explants cut at two different stages preceding the onset of gene expression in vivo and cultured for 24 hours. The fraction of positive explants of each type is indicated in the top right-hand corner.

Hoxb8 activation in cultured embryonic explants. Posterior streak region, PSR, blue anterior streak region, ASR, orange median streak region,MSR, green. Hoxb8 expression in PSR (A,B), MSR (C,D) and ASR (E,F)explants cut at two different stages preceding the onset of gene expression in vivo and cultured for 24 hours. The fraction of positive explants of each type is indicated in the top right-hand corner.

The proximal posterior region of the primitive streak has Hox-inducing capacity

As the PSR appears to be instructed for Hoxb1 expression before the ASR, and very young ASRs were unable to activate Hoxb1autonomously, we asked whether a PSR explant would induce Hoxb1 in an ES ASR explant when recombined. We combined ASR explants from Hoxb1-lacZ ES embryos with PSR explants from non transgenic embryos at different stages (E6.5 to E7.5) and analysed the lacZ expression after 24 hours of co-culture (Fig. 5A).

Recombination with a PSR induces Hoxb1-lacZ expression in a non-expressing ASR from ES and EMS stages. Hoxb1-lacZ expression in ASR explants cut from transgenic embryos (drawn in blue) at ES or EMS stages(E6.5-6.7), cultured for 24 hours in combination with either a PSR (A) or another ASR (B) cut from wild-type embryos (E6.5, ES, EMS and MS E7.5, EHF). Combination with a PSR explant resulted in a strong induction of transgene expression in early ASR explants. ASRs, except for the oldest (HF stage), were unable to activate Hoxb1lacZ in transgenic ES ASRs. See Table 2 for explant numbers. Scale bar: 100 μm.

Recombination with a PSR induces Hoxb1-lacZ expression in a non-expressing ASR from ES and EMS stages. Hoxb1-lacZ expression in ASR explants cut from transgenic embryos (drawn in blue) at ES or EMS stages(E6.5-6.7), cultured for 24 hours in combination with either a PSR (A) or another ASR (B) cut from wild-type embryos (E6.5, ES, EMS and MS E7.5, EHF). Combination with a PSR explant resulted in a strong induction of transgene expression in early ASR explants. ASRs, except for the oldest (HF stage), were unable to activate Hoxb1lacZ in transgenic ES ASRs. See Table 2 for explant numbers. Scale bar: 100 μm.

Of the ES ASR/PSR recombinates containing PSR explants from ES to HF stage embryos, 92% (11/12) showed lacZ expression, which indicates that Hoxb1 expression was induced in the ASR explant tissues[Fig. 5A (early streak) and Table 2]. Combination with a PSR also strongly increased Hoxb1-lacZ expression in cells of ASR explants from EMS and MS embryos (78% and 100% of positives, respectively),compared with expression seen in ASR explants cultured alone (17% and 27%)(Table 2 and Fig. 3). However, PSR explants were not able to induce Hoxb1-lacZ expression in explants from the extra-embryonic part of ES/EMS embryos, a region that never expresses Hox genes during in vivo development (Table 2). In contrast to the induction observed in ASR/PSR recombinates,no lacZ expression was scored in ASR/ASR control recombinates when the non transgenic ASR was taken from ES to MS embryos (E6.5-E7.0)(Fig. 5B Table 2). Interestingly though,we observed Hoxb1-lacZ expression in 67% (2/3) of recombinates between transgenic ES ASR explants and the node region of E7.5 headfold stage embryos [Fig. 5B (HF) and Table 2], showing the emergence of a Hoxb1-inducing capacity in late ASRs. The Hox-inductive capacity therefore is present in the PSR early during gastrulation (ES stage, E6.5)until at least the HF stage (E7.5), a time when it can also be identified in the anterior streak region.

Hoxb 1-lacZ expression in recombinates between two explants from the primitive streak region of gastrulating embryos

Explants and embryonic stage of cutting * . Proportion of β-gal positive recombinates (number of positives/total number) † .
ES ASR Lac (from Hoxb1-lacZ embryos)0 (0/9)
ES ASRLac + ES PSR 100 (2/2)
ES ASRLac + EMS PSR 80 (4/5)
ES ASRLac + MS PSR 100 (1/1)
ES ASRLac + HF PSR 100 (4/4)
ES ASRLac + ES ASR 0 (0/4)
ES ASRLac + EMS ASR 0 (0/2)
ES ASRLac + MS ASR 0 (0/1)
ES ASRLac + HF ASR 67 (2/3)
EMS ASRLac17 (4/24)
EMS ASRLac + ES PSR 60 (3/5)
EMS ASRLac + EMS PSR 100 (3/3)
EMS ASRLac + MS PSR 100 (1/1)
EMS ASRLac + ES ASR 0 (0/4)
EMS ASRLac + EMS ASR 0 (0/6)
MS ASRLac27 (6/22)
MS ASRLac + EMS PSR 100 (3/3)
MS ASRLac + EMS ASR 33 (1/3)
ES ExtrEmbLac 0 (0/7)
ES ExtrEmbLac + ES PSR 0 (0/3)
Explants and embryonic stage of cutting * . Proportion of β-gal positive recombinates (number of positives/total number) † .
ES ASR Lac (from Hoxb1-lacZ embryos)0 (0/9)
ES ASRLac + ES PSR 100 (2/2)
ES ASRLac + EMS PSR 80 (4/5)
ES ASRLac + MS PSR 100 (1/1)
ES ASRLac + HF PSR 100 (4/4)
ES ASRLac + ES ASR 0 (0/4)
ES ASRLac + EMS ASR 0 (0/2)
ES ASRLac + MS ASR 0 (0/1)
ES ASRLac + HF ASR 67 (2/3)
EMS ASRLac17 (4/24)
EMS ASRLac + ES PSR 60 (3/5)
EMS ASRLac + EMS PSR 100 (3/3)
EMS ASRLac + MS PSR 100 (1/1)
EMS ASRLac + ES ASR 0 (0/4)
EMS ASRLac + EMS ASR 0 (0/6)
MS ASRLac27 (6/22)
MS ASRLac + EMS PSR 100 (3/3)
MS ASRLac + EMS ASR 33 (1/3)
ES ExtrEmbLac 0 (0/7)
ES ExtrEmbLac + ES PSR 0 (0/3)

See Fig. 5 for experimental procedure and legend to Fig. 2for abbreviations. ExtrEmb, explant of extra-embryonic tissues cut on the posterior side. Explants not designated as `Lac' are from non transgenic embryos.

Recombinates were cultured for 24 hours before fixation and X-gal staining procedure.

These results indicate that the posterior part of the primitive streak region is capable of producing signals leading to the induction of Hoxb1 expression in competent anterior streak tissues. Regardless of whether this anterior tissue still represents anterior streak, or is differentiating as neurectoderm and mesoderm, the results imply that rostral spreading of Hox expression from the posterior streak to the node region requires cell-cell interactions.

The spread of Hox expression domains rostral to the node is not lineage related

Rostral spread of Hox expression continues beyond the node(Fig. 1) during a period when the presumptive hindbrain and spinal cord territories are expanding anterior to the node (Tam, 1989) and the node `regresses' while generating spinal cord(Mathis and Nicolas, 2000). Given this coincidence, a plausible mechanism for the continued spread of the expression front along the AP axis is that a cell acquires a Hox code and positional specification while in the node region and its descendants retain it after leaving the node region (Deschamps and Wijgerde, 1993). Descendants remaining (temporarily) in the node region would acquire a new Hox code as the more 5′ genes are expressed there. If this hypothesis is valid, predictions about the final position of the most rostral descendants of cells at the Hox gene expression front at the node at different stages can be made on the basis of the later rostral expression limits of different Hox genes in neurectoderm and mesoderm at E8.5-9.5. Both a general prediction with regard to neurectoderm and mesoderm descendants, and specific predictions can be made.

The general prediction is that the anterior limit of mesodermal clones will be several somite lengths posterior to neurectodermal clones generated in the node region at the same stage (Gaunt et al., 1988 Frohman et al.,1990). Clones labelled with HRP were generated in epiblast at the node region from LS to HF stages (E7.0-E7.7), and the embryos cultured for 1 day (Fig. 6). Some clones were also generated in the axial epiblast anterior to the node at LSEB and older stages. Most (93%) of the clones generated at the node after the LS stage and contributing to neurectoderm were restricted to the ventral half of the neural tube 78% of the clones contributing to non axial mesoderm anterior to the node were in paraxial mesoderm. The anterior limit of neurectodermal descendants in clones generated at the node was progressively more posterior with advancing initial stage (LS versus LSEB, P<0.01 NP versus HF, P=0.01) with the exception that LSEB and NP did not differ significantly (Fig. 7, upper set). A similar progression was seen in the mesoderm (LS stages versus HF, P<0.01). This trend confirms the sequential addition of neural and mesodermal material from the node region. At no stage was the anterior limit reached by mesodermal clones generated at the node posterior to the anterior limit of neurectodermal clones (Wilkoxon rank test). At the HF stage, mesoderm clones were even slightly more rostral to neurectoderm ones (P=0.05). Clones initiated in the mesoderm layer did not differ from those initiated in the epiblast. Axial mesoderm, which does not express Hox genes in the mouse(Deschamps and Wijgerde, 1993),behaved differently: it remained associated with the node, and therefore relatively posterior (Fig.7). Therefore axial extension from the node progresses at similar speed in neurectoderm and paraxial mesoderm and the general prediction from the hypothesis was not fulfilled.

HRP-labelled clones in cultured embryos. (A) Seven somite embryo labelled in the node region at the NP stage. There are seven mesoderm descendants(arrow) (not all in focus) in the second somite and a further seven lightly labelled descendants in postnodal ectoderm (star) (not all visible), which were presumably derived from a sibling still in cytoplasmic connection with the labelled cell at the time of injection. (B) Four somite embryo labelled at the anterior edge of the node region at the LS stage. Twelve labelled neurectoderm descendants are distributed in small clusters from prospective r5/r6 (arrow) towards the node. Scale bars: 100 μm.

HRP-labelled clones in cultured embryos. (A) Seven somite embryo labelled in the node region at the NP stage. There are seven mesoderm descendants(arrow) (not all in focus) in the second somite and a further seven lightly labelled descendants in postnodal ectoderm (star) (not all visible), which were presumably derived from a sibling still in cytoplasmic connection with the labelled cell at the time of injection. (B) Four somite embryo labelled at the anterior edge of the node region at the LS stage. Twelve labelled neurectoderm descendants are distributed in small clusters from prospective r5/r6 (arrow) towards the node. Scale bars: 100 μm.

Anteroposterior clone distribution after labelling epiblast at different stages. The upper set of figures shows results after labelling at or near the node, the lower set after labelling anterior to the node. In each set, the upper figure shows the positions of the progenitor cells projected on a sagittal section of average dimensions. The extent of the primitive streak is indicated by a curved line. A part of the sagittal section to the right of LS,LSEB and HF shows the position of progenitors initially labelled in the mesoderm layer. The positions of the clone progenitors are divided by broken lines into subregions, comprising three or four subregions from anterior to posterior in the node region and, anterior to the node, anterior axial (within 55 μm of the midline) and paraxial. Filled blue circles, progenitors with only ectoderm descendants open blue circles, progenitors with ectoderm and mesoderm/endoderm descendants filled red circles and squares, progenitors with only mesoderm descendants open red circles and squares, progenitors with paraxial mesoderm and axial mesoderm or endoderm descendants filled brown circles and squares, progenitors with only axial mesoderm descendants. The lower figures in each set show the lineal AP distribution of each clone on a schematic representation of the neural tube the regions of the neural tube and the corresponding `segments' in the paraxial mesoderm are shown on the left. The position of the node is indicated by an asterisk. The presence of one of more labelled cells in a `segment' is indicated by a coloured line,absence of labelled cells by a broken line connecting the clone to the node. Blue, ectoderm red, paraxial mesoderm (unless otherwise indicated) brown,axial mesoderm (notochordal plate) yellow, endoderm. The clones generated by individual progenitors can be identified as follows: for the node region,anterior precede posterior subregions and, within subregions, proximal precedes distal. Anterior towards the node, the anterior axial subregion precedes the paraxial subregion, and anterior or proximal precedes posterior or distal within each subregion. The subregional divisions are indicated by broken vertical lines on the clonal distribution scheme. Clones initiated in mesoderm follow the last epiblast subregion. Anterior clonal limits predicted by the hypothesis that Hox codes are fixed in the node region and carried further by lineage transmission are indicated by blue (neurectoderm) and red(paraxial mesoderm) arrowheads at the appropriate stages for Hoxb1and Hoxb8. i.m., intermediate mesoderm l.p., lateral plate mesodermMes, mesencephalon Pros, prosencephalon r, rhombomere Rh, rhombencephalonS, somite SC, spinal cord Sm, somitomere(Meier and Tam, 1982) +,additional to paraxial mesoderm.

Anteroposterior clone distribution after labelling epiblast at different stages. The upper set of figures shows results after labelling at or near the node, the lower set after labelling anterior to the node. In each set, the upper figure shows the positions of the progenitor cells projected on a sagittal section of average dimensions. The extent of the primitive streak is indicated by a curved line. A part of the sagittal section to the right of LS,LSEB and HF shows the position of progenitors initially labelled in the mesoderm layer. The positions of the clone progenitors are divided by broken lines into subregions, comprising three or four subregions from anterior to posterior in the node region and, anterior to the node, anterior axial (within 55 μm of the midline) and paraxial. Filled blue circles, progenitors with only ectoderm descendants open blue circles, progenitors with ectoderm and mesoderm/endoderm descendants filled red circles and squares, progenitors with only mesoderm descendants open red circles and squares, progenitors with paraxial mesoderm and axial mesoderm or endoderm descendants filled brown circles and squares, progenitors with only axial mesoderm descendants. The lower figures in each set show the lineal AP distribution of each clone on a schematic representation of the neural tube the regions of the neural tube and the corresponding `segments' in the paraxial mesoderm are shown on the left. The position of the node is indicated by an asterisk. The presence of one of more labelled cells in a `segment' is indicated by a coloured line,absence of labelled cells by a broken line connecting the clone to the node. Blue, ectoderm red, paraxial mesoderm (unless otherwise indicated) brown,axial mesoderm (notochordal plate) yellow, endoderm. The clones generated by individual progenitors can be identified as follows: for the node region,anterior precede posterior subregions and, within subregions, proximal precedes distal. Anterior towards the node, the anterior axial subregion precedes the paraxial subregion, and anterior or proximal precedes posterior or distal within each subregion. The subregional divisions are indicated by broken vertical lines on the clonal distribution scheme. Clones initiated in mesoderm follow the last epiblast subregion. Anterior clonal limits predicted by the hypothesis that Hox codes are fixed in the node region and carried further by lineage transmission are indicated by blue (neurectoderm) and red(paraxial mesoderm) arrowheads at the appropriate stages for Hoxb1and Hoxb8. i.m., intermediate mesoderm l.p., lateral plate mesodermMes, mesencephalon Pros, prosencephalon r, rhombomere Rh, rhombencephalonS, somite SC, spinal cord Sm, somitomere(Meier and Tam, 1982) +,additional to paraxial mesoderm.

If Hox codes are acquired at the node, specific predictions about neurectoderm and mesoderm separately are not necessarily dependant on the validity of the general prediction about relative dispersion of neurectoderm and mesoderm from the node region. The specific predictions for neurectoderm,based on later anterior expression boundaries of Hoxb1, Hoxb4 and Hoxb8, and the stage at which expression reaches the node(Table 1), are that the colonising population of rhombomeres 3 and 4 (r3/r4, for the anterior limit of Hoxb1) (Wilkinson et al.,1989) is generated at the node at LSEB/NP stages, that of r6/r7,at the level of the first somite (S1), (Hoxb4)(Gould et al., 1998) is generated at the node at the EHF stage, and that of neurectoderm at the level of S5/S6, in the anterior spinal cord, (Hoxb8)(Deschamps and Wijgerde, 1993Charité et al., 1998) is generated at the node at the HF stage. Analysis of neurectoderm descendants of epiblast clones generated in the node region (Fig. 7, upper set) and anterior to it (Fig. 7, lower set) showed that the most anterior limit of any neurectoderm clone derived from the node region at LSEB and NP stages was at the level of S1 and S2,respectively (median at S5), and not more anteriorly in r3,r4,r5, as expected from Hoxb1 expression. Contribution to r3,r4,r5 came from the most anterior descendants of clones generated in the node region at the earlier LS stage, and from a region ∼100 μm anterior to the node at the NP stage. Contribution to neurectoderm at the level of S1 by anterior descendants of epiblast near the node came not from LNP/EHF stage embryos as expected from Hoxb4 expression, but from the earlier LS stage and from ectoderm anterior to the node at the LSEB stage. At the HF stage, the most anterior contribution of epiblast at the node region to the neurectoderm was at the level of S7 (median at S9/S10), not at S5/S6 as predicted from the Hoxb8 expression pattern. Contribution to the S5 level by anterior clonal descendants came from the node region of younger LSEB (median at S4/S5)and NP stage (median at S5) embryos as well as from epiblast anterior to the node at NP and HF stages(Fig.7). Therefore cells that will eventually occupy the anterior boundary regions of Hoxb1 and Hoxb4 expression in the hindbrain or Hoxb8 in the spinal cord, are already in positions anterior to the node when the anterior expression boundaries reach the node region(Fig. 8).

Rostral spread of Hox expression in relation to the precursors of cells that will eventually occupy the rostral expression boundaries in the neurectoderm The anteroposterior (A,P) axis is shown at different stages. The node is represented by a star, the primitive streak as an open bar. The extent of Hoxb1, Hoxb4 and Hoxb8 expression is indicated by coloured lines below the axis. The position of precursors of rhombomere 4 (R4,definitive anterior boundary of Hoxb1) and different somite (S)levels (S5 level, definitive anterior boundary of Hoxb8) in the neural axis are shown above the axis.

Rostral spread of Hox expression in relation to the precursors of cells that will eventually occupy the rostral expression boundaries in the neurectoderm The anteroposterior (A,P) axis is shown at different stages. The node is represented by a star, the primitive streak as an open bar. The extent of Hoxb1, Hoxb4 and Hoxb8 expression is indicated by coloured lines below the axis. The position of precursors of rhombomere 4 (R4,definitive anterior boundary of Hoxb1) and different somite (S)levels (S5 level, definitive anterior boundary of Hoxb8) in the neural axis are shown above the axis.

The specific prediction with regard to mesoderm is that the precursors of the first somite, which is the anterior limit of Hoxb1 expression in the mesoderm (Murphy and Hill,1991) are present at the level of the node at the LSEB/NP stage,those of S5/6 (Hoxb4) (Gould et al., 1998) at the node at the EHF stage, and those of S10/11(Hoxb8) (Charité et al., 1998 Deschamps and Wijgerde, 1993)at the node at the HF stage. Although the anterior limits of mesoderm clones generated near the node became progressively more posterior with advancing initial stage, there was no general agreement of prediction with the levels at which anterior descendants were found. The anterior limit of two mesodermal clones was in S1 and one was just anterior, as predicted for Hoxb1expression after labelling at the LSEB stage, and two clones derived at the NP stage were in S2, but the anterior limits of the other four clones at LSEB and NP stages were more posterior (median of nine clones at S2). The eight mesoderm clones generated at the HF stage had anterior limits between S4 and S9 (median at S6/7), instead of more posteriorly at S10/11 as expected from the AP level of the definitive anterior expression boundaries of Hoxb8. Therefore, although lineage transmission of Hoxb1expression in paraxial mesoderm has not been excluded, mesoderm generated at the node does not behave as predicted if anterior Hox boundaries were being consistently established by the lineage transmission of specific Hox codes acquired sequentially at the node between LS and HF stages.

Hindbrain and anterior spinal cord elongate both by addition from the node and by internal growth

Sequential addition of material into the hindbrain and spinal cord from the node is indicated by the progressively more caudal position of the anterior limits of clones generated in the node region between LS and HF stages(Fig. 7, upper set). Of the clones contributing to neurectoderm, 45% (14/31) extended to the node, whereas no (0/9) clones generated in the axial region anterior to the node did(Fig. 7, lower set). This is supporting evidence of a self maintaining pool of precursors in the node region for the spinal cord (Mathis and Nicolas, 2000 Mathis et al.,2001) and also for part of the hindbrain.

Comparison of the anterior limits of neurectoderm clones shows a consistency in result in clones generated anterior to the node(Fig. 7, lower set) compared with those generated in the node region(Fig. 7, upper set): the anterior limit is well correlated with the distance from the node of anterior axial progenitors (those anterior to the node and within 55 μm either side of the midline) at all stages. The AP position of the anterior limits of the clones was compared with the position of their anterior axial progenitors by linear regression analysis in order to assess quantitatively whether the neural axis anterior to the node at LSEB to HF stages, representing prospective r3 to spinal cord at the level of S8, is stabilised or is continuing to grow within itself (Fig. 9). The value of the regression coefficient (b) was 2.769(s.e.=0.463, n=9, P<0.001). This value is also significantly greater than 1 (P<0.01), implying that the part of the axis representing precursors of hindbrain and spinal cord to the level of S8, anterior to the node at LSEB to HF stages, increases in length within 24 hours. The axis is therefore extending within itself anterior to the node, and not only at the node by the sequential insertion of new material. In addition,the posterior displacement of the anterior limits of paraxially generated clones relative to anterior axial ones (LSEB and NP, Fig. 7, lower set) suggests that convergence and extension occur in the more lateral ectoderm until at least the NP stage. These results could explain why labelling epiblast just anterior to the node at the HF stage gives clones with anterior limits in the neurectoderm that are more posterior than might have been expected from the shape of the embryo and the space apparently available for the first somites[compare Fig. 1F with Fig. 7 (HF)]. The results also underline the dynamic nature of the relative AP positions of cells that are leaving, and have recently left, the node during a period when Hox expression domains are traversing rostrally through the region.

AP extension of hindbrain and spinal cord. (A,B) Schematic representation of the curved AP axis at the time of injection (A) and after culture (B). The position of the node is indicated by a star. For regression analysis, the position of the injected cell (x) was measured from the anterior embryonic/extra-embryonic junction (thick curved line in A). The position of the most anterior descendant (y) in the resulting clone was measured along the midline (thick curved line in B) from the boundary between the first and second somites (S1/S2). Values of y anterior to S1/S2 are negative. (C)Regression of the AP position of the most anterior descendant (y1,y2. ) on progenitor position (x1, x2. ). Initial stages: Square, LSEB dots, NP triangles, HF. Only clones with anterior axial progenitors (see Fig. 7) were used, and were suitable for this approach because there is no significant increase in length or cell mixing along the midline of prospective forebrain, midbrain and anterior hindbrain between the LS and 4S stage (K.A.L., unpublished). The values in the regression equation Y=a + bX are 137.11=–867.60 + 2.7686 (362.89).

AP extension of hindbrain and spinal cord. (A,B) Schematic representation of the curved AP axis at the time of injection (A) and after culture (B). The position of the node is indicated by a star. For regression analysis, the position of the injected cell (x) was measured from the anterior embryonic/extra-embryonic junction (thick curved line in A). The position of the most anterior descendant (y) in the resulting clone was measured along the midline (thick curved line in B) from the boundary between the first and second somites (S1/S2). Values of y anterior to S1/S2 are negative. (C)Regression of the AP position of the most anterior descendant (y1,y2. ) on progenitor position (x1, x2. ). Initial stages: Square, LSEB dots, NP triangles, HF. Only clones with anterior axial progenitors (see Fig. 7) were used, and were suitable for this approach because there is no significant increase in length or cell mixing along the midline of prospective forebrain, midbrain and anterior hindbrain between the LS and 4S stage (K.A.L., unpublished). The values in the regression equation Y=a + bX are 137.11=–867.60 + 2.7686 (362.89).


MATERIALS AND METHODS

Whole Embryo Culture and Staging

Fertilized chicken eggs were incubated at 38°C to obtain embryos at stages ranging from X (Eyal-Giladi and Kochav, 1976 ) to 6 (Hamburger and Hamilton, 1951 ). Substages of HH Stage 3 were defined as described previously (Schoenwolf et al., 1992 ), with embryos from Stages 3a and 3b grouped together as Stage 3a/b, and those from Stages 3d and 4 grouped together as Stage 3d/4. Culture dishes and embryos were prepared as described by Darnell and Schoenwolf ( 2000 ) for modified New ( 1955 ) culture.

Scanning and Transmission Electron Microscopy

Chick embryos were fixed overnight with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer and then washed briefly with 0.1 M phosphate buffer. After this, they were postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer and dehydrated with an ascending graded ethanol series up to 100%. After this, embryos for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were separated. Those for SEM were passed through one 10-min change of 1:1 100% ethanol:hexamethyldisilazane (HMDS, Electron Microscopy Sciences, Fort Washington, PA), followed by two 10-min changes in HMDS, after which the HMDS was allowed to evaporate overnight, drying the samples. Dried samples were then mounted onto stubs, coated with gold/palladium, and examined with an Hitachi S2460N scanning electron microscope. Embryos for TEM were transferred to propylene oxide and embedded in Epon. Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with an Hitachi H-7100 transmission electron microscope.

Many of the embryos collected for SEM were dissected or cut into slices. Dissection involved the removal of the hypoblast or definitive endodermal layer using fine cactus needles while the embryos were in buffer after primary fixation. Careful observations of embryos during this process suggested that such dissection had little or no adverse effect on the underlying mesoderm, with the possible exception that occasional mesodermal cells may have become detached with the endoderm. Similarly, embryos were transversely sliced using razor blades, again in buffer after primary fixation.

Fate Mapping Experiments

Injections of the primitive streak.

Chick embryos at Stages 2, 3a/b, and 3c, cultured as described above, were selected for injections of the primitive streak. A mixture of 5-carboxytetramethylrhodamine, succinimidyl ester (CRSE Molecular Probes, Inc., Eugene, OR) and 1,1′-dioctadecyl-3, 3, 3′-tetramethylindocarbocyanine perchlorate (DiI Molecular Probes, Inc.) was injected as described by Darnell et al. ( 2000 ) into the primitive streak at three levels (i.e., rostral, middle, and caudal, designated as Sites 1, 2, and 3, respectively). Embryos were immediately examined with a fluorescence microscope, to ascertain the size and site of each injection, and then re-incubated for 4 hr. After fixation in 4% paraformaldehyde in PBS, they were processed for immunocytochemistry and subsequent paraffin histology as described below. Some embryos were fixed after the initial examination for Time 0 injections. All embryos were processed for immunocytochemistry and subsequent paraffin histology as described below. Control embryos were treated exactly as experimentals, except that they were not injected.

Injections of the parastreak epiblast.

For injections of the parastreak epiblast, chick embryos were selected at Stages 2–3c and cultured as described above. A mixture of CRSE and DiI was made into the epiblast at three levels, rostral, middle, and caudal, also designated (like primitive-streak injections) as Sites 1, 2, and 3, respectively. Embryos were immediately examined with a fluorescent microscope and then re-incubated for 5, 7, and 24 hr before fixation. All embryos were processed for immunocytochemistry and subsequent paraffin histology as described below. Control embryos were treated exactly as experimentals, except that they were not injected.

Immunocytochemistry

All embryos labeled with the fluorescent markers CRSE and DiI were processed for whole-mount immunocytochemistry (ICC) as described previously by Patel et al. ( 1989 ), except that the embryos were fixed in 4% paraformaldehyde in PBS and the peroxidase reaction product was intensified by the addition of 2% CoCl2 and 2% Ni (NH4)2 (SO4) per ml of DAB-PBT. To label the rhodamine groups of CRSE-DiI labeled embryos with a permanent reaction product detectable in paraffin section, we used anti-rhodamine (rabbit IgG polyclonal, primary Molecular Probes, Inc.) and horseradish peroxidase-conjugated goat anti-rabbit IgG (secondary antibody Boehringer Mannheim, Germany). After their examination and photography as whole-mounts, embryos were processed for paraffin histology as described below.

Additional embryos were processed as whole mounts for ICC at stages XII-4 using anti-fibronectin and anti-laminin antibodies (Developmental Studies Hybridoma Bank) (secondary antibody for both consisted of horseradish peroxidase-conjugated goat anti-mouse IgG Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Before each primary antibody treatment, either the hypoblast or endoderm was removed to ensure antibody penetration. Subsequently, embryos were processed for paraffin histology as described below.

Paraffin Histology

Histological analysis was carried out on embryos previously labeled by whole-mount ICC. Embryos were dehydrated in an ascending ethanol series up to 100%. Those labeled for ICC were taken through two 5-min changes of Histosol. After infiltration in Paraplast, they were finally embedded in Paraplast and sectioned at 10 μm.

Measurements of the Primitive Streak

Chick embryos at Stages 2, 3a/b, and 3c (five for each stage) were used for measuring the length and width of the primitive streak in living embryos, cultured as described above. Embryos were selected carefully to ensure that only blastoderms with margins completely attached to the vitelline membrane were used thus, we avoided measuring embryos with artifactually distorted primitive streaks. For each embryo, an eyepiece graticule mounted in the eyepiece of a dissecting microscope was used to measure the length and width of the primitive streak. The width of the primitive streak was measured at two levels: rostral and mid-streak. These initial measurements represented Time 0 values. Embryos were re-incubated further, and measurements were subsequently taken at 2-hr intervals up to 8 hours of re-incubation. The values obtained were analyzed by calculating the mean length and width of the primitive streak for each stage and time of re-incubation. The percentage increase in the length of the primitive streak, the percentage increase in the width of the rostral primitive streak, and the percentage decrease in the width of the mid-primitive streak was computed from the mean values obtained.


Embryonic Induction: Its Types, Experimental Evidence, Characteristics, and Mechanism

In amphibian embryos, the dorsal ectodermal cells in a mid-longitudinal region differentiate to form a neural plate, only when the chorda-mesoderm is below it. Chorda-mesoderm is the layer formed by invagination cells from the region of the dorsal blastoporal lip, which form the roof of archenteron. Mangold (1927) selected a small part of dorsal blastoporal lip from an early gastrula of Triturus cristatus and grafted it at a place near the lateral lip of the blastopore of the host gastrula of T. taeniatus.

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The graft cells grew in number and spread inside the host gastrula to form an additional chorda-mesoderm at this place. This chorda-mesoderm, subsequently induced the ectoderm of the host gastrula to form an additional neural tube.

The graft cells themselves formed an additional notochord. As the host gastrula developed further, it grew into a double embryo joined together. One of the embryos was the regular one, while the second was the induced one. The latter did not develop a complete head.

This experiment clearly showed that the dorsal blastoporal lip of the blastula had the ability to induce the formation of the neural plate in the ectoderm of the host. This phenomenon is called neural induction. Other parts of an embryo can similarly induce the formation of other structures. This influence of one structure in the formation of another structure is called embryonic induction.

In fact, the entire development of an organism is due to a series of inductions. The structure, which induces the formation of another structure, is called the inductor or organizer. The chemical substance that is emitted by an inductor is called an evacuator. The tissue on which an evacuator or inductor acts is called the responsive tissue.

Historical Background of Embryonic Induction:

For the discovery of neural induction, the German embryologist, Hans Spemann and his student, Hilde Mangold (1924) worked a lot and for his work Spemann received Nobel Prize in 1935.

These two scientists performed certain heteroblastic transplantations between two species of newt, i.e., Triturus cristatus and Triturus taeniatus and reported that the dorsal lip of their early gastrula has the capacity of induction and organization of presumptive neural ectoderm to form a neural tube and also the capacity of evocation and organization of ectoderm, mesoderm and endoderm to form a complete secondary embryo.

They called the dorsal lip of the blastopore the primary organizer since it was first in the sequence of inductions and as it had the capacity to organize the development of a second embryo. Later on, the primary organizer was reported to exist in many animals, e.g. in frogs (Daloq and Pasteels, 1937) in cyclostomes (Yamada, 1938) in bony fishes (Oppenheimer, 1936) in birds (Waddington, 1933) and in rabbit (Waddington, 1934).

Primary organizer and neural induction have been reported in certain pre-vertebrate chordates, such as ascidians and Amphioxus (Tung, Wu and Tung, 1932). In 1960 and 1963 Curtis investigated and reported that the organizer of gastrula of Xenopus laevis can be distinguished in the cortex of gray crescent of a fertilized egg.

Holtfreter (1945) gave an account of how an enormous variety of entirely unspecific substances-organic acids, steroids, kaolin, methylene blue, sulphhydryl compounds, which had nothing in common except the property of being toxic to sub-ectodermal cells-produced neurulation in explants. Barth and Barth (1968, 69) provided further information about the chemical nature of embryonic induction.

Types of embryonic induction:

Lovtrup (1974) classified different types of embryonic induction into two basic categories-endogenous and exogenous inductions.

1. Endogenous induction:

Certain embryonic cells gradually assume new diversification pattern through the inductors that are produced by them endogenously. Due to these inductors, these cells undergo either self-transformation or self-differentiation. Examples of such induction were reported in Mesenchymal cells of ventral pole of Echinoid and in small sized, yolk-laden cells of dorsal lip of amphibian blastopore.

2. Exogenous induction:

When some external agent or a cell or a tissue is introduced into an embryo, they exert their influence by a process of diversification pattern upon neighbouring cells through contact induction. This phenomenon is called exogenous induction. It may be homotypic or heterotypic depending on the fact that whether the inductor provokes the formation of same or different kind of tissues respectively (Grobstein, 1964).

In homotypic induction, a differentiated cell produces an inductor. The inductor not only serves to maintain the state of the cell proper, but also induces adjacent cells to differentiate according to it, after crossing the cell boundaries. Best example of the heterotypic exogenous induction is the formation of a secondary embryonic axis by an implanted presumptive notochord in amphibians.

Experimental evidences to induction:

Spemann and Mangold (1924) transplanted heteroplastically a piece of the dorsal lip of the blastopore of an early gastrula of pigmented newt, Triturus cristatus and grafted it near the ventral or lateral lip of the blastopore of the early gastrula of pigmented newt T. taeniatus. Most of the graft invaginated into the interior and developed into notochord and somite’s and induced the host ectoderm to form a neural tube, leaving a narrow strip of tissue on the surface.

With the development of host embryo, an additional whole system of organs was induced at the graft – placement area. Except for the anterior part of the head, almost a complete secondary embryo comprising of the additional organs was formed. Posterior part of the head was present as indicated by a pair of ear rudiments.

Since in this experiment the type of transplantation involved was heteroplastic, it was found that notochord of secondary embryo consisted exclusively of graft cells the somites consisted partly of graft and partly of host cells (Fig. 1).

Few cells, which did not invaginate during gastrulation, were left in the neural tube. The bulk of the neural tube, part of the somites, kidney tubules and the ear rudiments of the secondary embryo consisted of host cells.

The graft becomes self-differentiated and at the same time induces the adjoining host tissue to form spinal cord and other structures including somites and kidney tubules. Spemann (1938) described dorsal lip of the early gastrula as a “primary organizer” of the gastrulative process.

However, organization of the secondary embryo results from a series of both inductive interactions and self-differentiative changes in the host and donor tissues. Hence, now a days the term “embryonic induction” or “inductive interactions” is preferred. The part, which is the source of induction, is called “inductor”.

Characteristics of the organizer:

Organizer has the ability for self-differentiation and organization. It also has the power to induce changes within the cell and to organize surrounding cells, including the induction and early organization of neural tube. Primary organizer determines the main features of axiation and organization of the vertebrate embryo.

Induction is a tool-like process, utilized by this center of activity through which it affects changes in surrounding cells and as such influences organization and differentiation. These surrounding cells, changed by the process of induction, may in turn act as secondary inductor centers with abilities to organize specific sub-areas.

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

Regional specificity of the organizer:

Vital-staining experiments of Vogt with newt eggs have shown that the material successively forming the dorsal blastoporal lip moves forward as the archenteron roof. Transplants taken from this region are also able to induce a secondary embryo or the belly of a new host i.e. the archenteron roof acts as a primary inductor in essentially the same way as does the dorsal lip tissue proper. The inductions of neural inductor are found to be regionally specific and the regional specificity is imposed on the induced organ by the inductor.

Therefore, the inductive capacity of the blastoporal lip varies both regionally and temporally. Most of the dorsal and dorso-lateral blastoporal material is necessary for a graft to induce a more or less complete secondary embryo. Spemann (1931) demonstrated that during gastrulation anterior part of the archenteric roof invaginates over the dorsal lip of the blastopore earlier.

Dorsal blastopore lip of the early gastrula contains the archenteric and deuterocephalic organizer and the dorsal blastopore lip of the late gastrula contains the spinocaudal organizer. Inductions produced by the dorsal lip of the blastopore taken from the early and the late gastrula differ in accordance with exception the first tends to produce head organs and the second tends to produce trunk and tail organs (Fig. 2).

As invagination continues and the dorsal lip no longer consists of prospective head endo-mesoderm but progressively becomes prospective trunk mesoderm it acts as a trunk-tail inductor. The most caudal region of the archenteron roof, in fact, specifically induces tail somites and probably other mesodermal tissues. The archenteron roof induces entirely different class of tissues various neural and meso-ectodermal tissues by its anterior region and various mesodermal tissues by its most posterior region.

Therefore, differences in specific induction capacities exist between head and trunk level of archenteron roof and are related to the regional differentiation of the neural tissue into archencephalic (including fore-brain, eye, nasal pit), deuterencephalic (including hind-brain, ear vesicle) and spinocaudal components. Thus, archenteron roof consists of an anterior head inductor including an archencephalic inductor and a deuterencephalic inductor and a trunk or spinocaudal inductor.

Primary induction and gray crescent:

The dorsal lip region of the blastopore at the onset of gastrulation can be traced back to the gray-crescent of the undivided fertilized amphibian egg. It was conceived by some developmental biologists that the crescent material of egg cortex initiated gastrulation and has the capacity of neural induction. A.S.G. Curtis (1963) performed a series of experiments of transplanting parts of the cortex of the fertilized egg of the clawed toad, Xenopus laevis at the beginning of cleavage.

In one experiment, the gray-crescent cortex was excised from the fertilized egg and it was observed that the cell division though proceeded undisturbed, the gastrulation failed to take place (Fig. 3А). In another experiment, the gray crescent cortex of uncleaved fertilized egg was excised and transplanted into a ventral position of a second egg, so that the egg receiving the graft had two gray crescents on opposite sides.

As a result, egg cleaved to form a blastula, which underwent two separate gastrulation movements to produce two separate primary nervous systems, notochord and associated somites (Fig. 3D). Similar experiments conducted on the eight-cell stage showed that something had happened during the short – interval represented by the first three cleavages.

Gray crescent cortex of the eight-cell stage still retained its inductive capacity when grafted to younger stages (Fig. 3C). Removal of the gray crescent at this stage no longer inhibits subsequent gastrulation and normal development, the missing crescent properties being replaced from adjacent cortical regions (Fig. 3B).

According to Curtis, a change in cortical organization spreads across the surface of the egg during the second and third cleavages, starting from the gray crescent when this change is completed, interactions, probably of a biophysical nature, can take place among various parts of the cortex.

Mechanism of neural induction:

Development of the ectoderm overlying the roof of the archenteron into neural tissue suggests a direct action upon the ectodermal cells, either by surface interaction or by chemical mediation.

(1) One of the broad possibility is surface interaction of the cells at the inductive interface. The contact of the two cellular layers may provide a device whereby the structural pattern or geometry or behaviour of the ectodermal cell membranes is altered directly by the underlying chorda mesodermal cells.

Thus, the spatial configuration of the latter membranes might induce a change in the spatial configuration of the ectodermal cell membranes, this in turn producing in the interior of the cell changes that determine its development into neural plate. A morphological arrangement of this kind could account for quick and effective transmission of the inductive effect.

(2) Another broad possibility is a chemical mediation of the inductive effect. Therefore, a chemical substance or substances produced and released by inducing chorda mesoderm cells at the archenteron -ectoderm interface may act upon or enter the ectodermal cells to initiate cellular activities leading to neural development. A great deal of evidence favours the idea of an exchange of material between cells and also suggests that a diffusible substance may act as effective inductive stimulus.

Chemical basis of neural induction:

The results of numerous studies to elucidate the mechanism of induction and to identify the chemical substance or substances presumed to be involved have not yielded good results. It was found that many different tissues, embryonic or adult, from a great variety of different species, were capable of inducing nervous tissue in amphibian embryos. Moreover, some foreign tissues were found to be much more potent inductors after they had been killed by heat or alcohol treatment.

This fact remains against the concept of a universally present ‘masked organizer’, released in the primary inductor region. Few inorganic agents as iodine and kaolin, local injury, exposure to saline solutions of excessively high or low pH, cause neural differentiation in ectoderm. These findings establish the early grand concept of master-chemical embryonic organizer of Holtfreter’s sublethal cytolysis. It has the concept of reversible cell injury liberating neural inductor.

Different chemical substances of either gray crescent or dorsal lip or chordamesoderm are separated by different biochemical methods to find out the molecule which causes the neural induction and then the inductive capacity of each molecule was tested separately. Few experiments show that evocator or inducing substance is a protein.

Exhaustive attempts were made by different embryologists to understand the real mechanism of neural induction. Some theories have been put forward to understand the mechanism of neural induction, out of which the most important are as follows:

1. Protein denaturation theory of neural induction:

According to Ranzi (1963) neural induction and notochord formation are related to protein denaturation. Site of notochord formation is amphibian gray crescent, which is a center of high metabolic activity. Such centers of greater metabolic activity correspond to sites of protein denaturation.

2. Gradient theory of neural induction:

Toivonen (1968) and Yamada (1961) stated that two chemically distinct factors are involved in the action of the primary inductor. Out of these two factors, one is neuralizing agent and the other is mesodermalizing agent. These experiments were conducted with denatured bone marrow and liver as the inductors.

Regional specificity of the embryonic axis arises from the interaction between two gradients: neutralizing principle has its highest concentration in the dorsal side of the embryo and diminishes laterally, while the mesodermalizing principle is present as an antero-posterior gradient with its peak in the posterior region.

Anteriorly the neutralizing principle acts alone to induce forebrain structures, more posteriorly the mesodermalizing principle acts along with the neutralizing one to induce mid-brain and hind-brain structures, while even more posteriorly the high concentration level of the mesodermal gradient produces spino-caudal structures (Fig. 4).

3. One factor hypothesis of neural induction:

Nieuwkoop (1966) using living notochord as the inductor, postulated that only one factor which first evokes ectoderm to form neural tissue and later causes ectoderm to transform into more posterior and mesodermal structure (Fig. 5) is involved.

In one experiment, consisting of combining isolated gastrular ectoderm with a piece of notochord and then removing the notochord tissue after varying lengths of time, it was found that only 5 minutes exposure to inductor caused a part of the ectoderm to transform into brain and eye structures.

4. Ionic theory of neural induction:

According to Barth and Barth (1969), the actual process of induction may be initiated by release of ions from bound form, representing a change in the ratio between bound to free ions within the cell of the early gastrula. Induction of nerve and pigment cells in small aggregates of prospective epidermis of the frog gastrula were found to be dependent on the concentration of the sodium ions.

Normal induction of nerve and pigment cells by mesoderm in small explants from the dorsal lip and lateral marginal zones of the early gastrula is dependent on the external concentration of sodium. Thus, normal embryonic induction depends on an endogenous source of ions and that an intracellular release of such ions occurs during late gastrulation.

Genic basis of neural induction:

There are evidences that the component tissues of neural inductor become differentiated prior to ectodermal cells. During this process, the rate of transcription of mRNA and differential activation of genes becomes many fold, while the differentiation of ectodermal cells is set in only after mid-gastrulation.

According to experiments conducted by Tiedemann (1968), after 2 to 7 days of cultivation of dorsal blastopore lip of young Triturus gastrula with adjacent ectoderm in a medium containing sufficient quantities of Actinomycin-D to inhibit RNA synthesis, induction could not take place, but some differentiation of muscle and notochord occurred. It shows that mRNA by transcription from the DNA was required, which also requires the presence of Actinomycin-D. Therefore, no neural induction could be detected in this experiment.

Time of neural induction:

Neural induction occurs at the time when the material of chordamesoderm moves from the dorsal lip of blastopore inward and forward (Saxen and Toivonen 1962). The inductive stimuli exhibit a time gradient, which may be crucial with regard to action and reaction events.

Embryonic induction in different chordates:

Although neural induction was first discovered in urodele amphibians, it was found that the dorsal lip of the blastopore and the roof of the archenteron of other vertebrates have the same function. The chordamesoderm in all vertebrates induces the nervous system and sense organs. Neural inductor has been investigated in the following chordates:

(1) In Cyclostomes, especially in lampreys, the property of neural induction lies in the presumptive chorda mesodermal cells of dorsal lip of the blastopore.

Prior to cyclostomes, in Ascidians different blastomeres of eight cell stage have the following presumptive fates-(i) the two anterior animal pole blastomeres produce head epidermis, palps and the brain with its two pigmented sensory structures, (ii) two posterior animal pole blastomeres produce epidermis, (iii) two anterior vegetal blastomeres produce notochord, spinal cord and part of the intestine (iv) two posterior vegetal cells produce mesenchyme, muscles and part of the intestine.

From these experiments, Raverberi (1960) concluded that the formation and differentiation of brain by two anterior animal blastomeres is dependent on the induction of two anterior vegetal blastomeres, which act as neural inductors. It was further concluded that the two anterior vegetal blastomeres gave rise to diverse tissues, namely, endoderm, notochord and spinal cord.

(2) Wu and Tung (1962) proved the existence of the primary organizer and neural induction in Amphioxus. They transplanted pieces of tissues from the inner surface of the dorsal blastopore lip of an early gastrula of Amphioxus into the blastocoel of another embryo in the same stage (Fig. 6) and observed that secondary embryo developed in the ventral region of the host with a notochord and mesoderm produced by the graft and the neural tube from host tissue.

Thus, the chordal tissue of Amphioxus gastrula possesses the power of neural induction, while mesodermal and endodermal tissues have little such inductive power.

(3) In bony fishes, induction of secondary well developed embryos were produced by transplanting the posterior edge of the blastodisc which corresponds to the dorsal lip of the blastopore, into the blastocoel of another embryo (Fig. 7) or by transplanting the chordamesoderm and ectoderm. Neural inductions were also obtained by transplanting the dorsal lip of the blastopore in the sturgeon.

(4) In frogs, the induction of secondary embryo can be produced by the dorsal lip of the blastopore transplanted into the blastocoel of a young gastrula, in very much the same way as in newts and salamandars.

(5) In reptiles archenteron has the same inducing activity as in other vertebrates but there is no experimental proof of occurrence of neural inductor.

(6) In birds the existence of primary organizer was established by Waddington and co-workers. Anterior half of the primitive streak was the inducing part similar to the lips of the blastopore in amphibians. In the experiment whole blastoderms were removed from the egg in early gastrulation and cultivated in vitro on the blood plasma clot.

From another embryo, parts of the primitive streak were then inserted between epiblast and hypoblast, inductions of secondary embryos obtained. Primitive streak was found dependent on the underlying hypoblast for its formation (Fig. 8).

(7) A successful neural induction was performed in a rabbit embryo by cultivating the early blastodisc on a plasma clot and implanting the primitive streak of the chick as inductor. Tissues of the mammalian gastrula were found having competence for neural induction. Anterior end of a rabbit embryo, with two pairs of somites, induced a neural plate in a chick embryo when placed under a chick blastoderm.

Other types of embryonic inductions:

Along with gastrulation growth, various organ systems of the embryo begin to differentiate and acquire the power of inducing the differentiation of later formed structures or organs such as eyes, ears, limbs and lungs, etc. These organs develop organizing property and become the source of induction.

Therefore, this series of organizers can be called as secondary, tertiary and quaternary organizers. Progressive development of embryonic organs is dependent on sequential induction. One embryonic tissue interacts with the adjacent one and induces it to develop and this process continues in sequence.

Chorda mesoderm, the primary organizer induces the formation of fore-brain and optic area in the anterior part of the embryo. The optic area evaginates forming the optic vesicle. By invagination it changes into a double walled cup-like structure, the optic cup which acts as secondary organizer to induce the formation of tertiary organizer to form cornea.

The layer of mesenchyme left in front of the anterior chamber of eye combines with the overlying somatic ectoderm (epidermis) and forms cornea, choroid and sclera (Fig. 9). Thus the whole process of development seems to be a cause of induction and interaction only. Number of inductions are secondary or tertiary such as nasal-groove, optic vesicle, lens, cornea and so on involve ectodermal reactions.


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