Movements of the Inducing and Induced Tissues

1.3.1. Early Events

Gastrulation nominally begins as the cuboidal prospective BC constrict their apices and elongate in the apical-basal direction, acquiring their definitive

Fig. 1. These diagrams show important aspects of the fates and movements of the prospective neural tissue and the neural-inducing or "Organized tissue during gastrulation and neuru-lation. The embryos are viewed from the right sides such that dorsal is to the left, the AP is at the top, and vegetal pole is at the bottom. The Organizer occupies the dorsal sector of the involuting marginal zone, spreading laterally about 30° on either side of the midline, but for simplicity, only the prospective tissues at the dorsal midline are shown, including prospective prechordal (PM) and prospective notochord (N), covered on the outside by the prospective endoderm (E) of the archenteron roof. Both the midline and the lateral aspects of the prospective neural plate, including the prospective fore and midbrain (F, M), rhombencephalon (RH), and spinal cord (SC) are shown. Movements are indicated by arrows. Also indicated are the BC, the blastocoel (BL), the archenteron (A), and the respread bottle cells (RBC).

Fig. 1. These diagrams show important aspects of the fates and movements of the prospective neural tissue and the neural-inducing or "Organized tissue during gastrulation and neuru-lation. The embryos are viewed from the right sides such that dorsal is to the left, the AP is at the top, and vegetal pole is at the bottom. The Organizer occupies the dorsal sector of the involuting marginal zone, spreading laterally about 30° on either side of the midline, but for simplicity, only the prospective tissues at the dorsal midline are shown, including prospective prechordal (PM) and prospective notochord (N), covered on the outside by the prospective endoderm (E) of the archenteron roof. Both the midline and the lateral aspects of the prospective neural plate, including the prospective fore and midbrain (F, M), rhombencephalon (RH), and spinal cord (SC) are shown. Movements are indicated by arrows. Also indicated are the BC, the blastocoel (BL), the archenteron (A), and the respread bottle cells (RBC).

shape (15,16) (Fig. 1, stage 10-, 10+). This process begins about 30 min before stage 10, as described by Nieuwkoop and Faber (17), and is progressive. The number of cells with constricted apices and the degree of constriction of individual cells increas, resulting in an increasing concentration of black pigment in the apices of these cells, transforming them from yellow to gray, and finally to black (see Fig. 2 of ref. 16). As a result, IMZ rotates, the outside being pulled vegetally by the constricting BC, and the deep prospective PM, formerly associated with the deep ends of the prospective BC, is displaced upward (16) (Fig. 1, stages 10-, 10+). At this time, radial intercalation of cells occurs in the dorsal region of the gastrula, including both the prospective nervous system and the Organizer, and these regions become thinner and extend vegetally (see 18,19) (see arrows in Fig. 1, stages 10-, 10+). This vegetal extension and the formation of BC act together to rotate the vegetal edge of the IMZ inward and upward, such that it comes to lie beneath and in contact with the overlying prospective neural ectoderm (Fig. 1, stage 10+). This process of early, precocious movement of the deep prospective mesoderm was described in detail by Nieuwkoop and Florshutz (20).

1.3.2. A Stage of Rapid Transition

Because this early rapid movement of PM beneath the prospective neural ectoderm is a critical event in neural induction (see Subheading 4.1.), we distinguish among and characterize several forms of this stage. Stage 10- is characterized by a small area of prospective BC initiating constriction, their apices darkening slightly, to gray (Fig. 2, stage 10-, top). In most 10- embryos, the PM has neither rotated completely nor reached the overlying prospective neural ectoderm. The dorsal blastocoel wall is connected to the blastocoel floor by a smooth concave surface (Fig. 2, stage 10-, bottom). In the transition from stage 10- to stage 10, the BC apices constrict and darken further to form the blastoporal pigment line. Initially, this line is straight and extends about one-fifth to one-quarter the diameter of the embryo (stage 10 of Nieuwkoop and Faber, ref. 17) (Fig. 2, stage 10, top). The interior of the embryo at this stage is variable (Fig. 2, stage 10, bottom). It may be identical to that of the previous or the following stages, or intermediate between the two. As more BC are recruited, and those that have already formed constrict more, the field of BC widens and a shallow groove forms (Fig. 2, stage 10+, top; cf Fig. 1, stage 10+). Inside, the involuting cells have rotated upward, contacting the inner surface of the prospective neural tissue (arrows, Fig. 2, stage 10+, bottom). What was previously the smooth concave surface at the margin of the blasto-coel has become a cleft along which the PM and the overlying neural tissue are in contact, a fissure called the "cleft of Brachet" (pointer, Fig. 2, stage 10+, bottom). Thus the involuted tissue may come into initial apposition with the

Stage 10- Stage 10 Stage 10+

Fig. 2. Video frames taken through the stereomicroscope during dissections to make explants show the external view of the progressive formation of BC (top panels) and the involution of the leading edge of the Organizer mesoderm beneath the prospective neural tissue, on the inside (bottom panels). At stage 10-, only a few of the prospective BC have begun to undergo apical constriction, and these appear in a broad area of gray cells (pointer, stage 10-, top). Internally, the dorsal blastocoel wall (DBW) is continuous with the blastocoel floor (BLF) across a smooth curvature at the lateral edge of the blastocoel (stage 10-, bottom). By stage 10, more BC have formed, and the ones that have formed are darker, indicating greater contraction of their apices (stage 10, top panel). Internally, the curvature of the lateral margin of the blastocoel decreases in radius as the blastocoel floor rises and the blastocoel wall is pulled vegetally (arrows, stage 10, bottom). By stage 10+, more BC have formed, the constriction of those already formed is greater, and a shallow invagination has developed (stage 10+, top). Internally, the leading edge of the Organizer has rotated upward and outward, contacting the overlying neural tissue across the cleft of Brachet (pointer, stage 10+, bottom).

overlying neural tissue in stage 10 embryos, and this has almost always occurred by stage 10+. Thereafter, the cells spread on, and begin to migrate anteriorly on, the inner surface of the prospective neural tissue.

The prospective fate of this early involuting tissue is heterogeneous. A tongue of smaller, grayish or brownish PM cells extends animally from the bottle cells along the inner surface of the neural ectoderm (stippled, Fig. 1, stage 10+). By stage 10+ to 10.25, this tongue of PM cells reaches the leading edge of the involuted material in some embryos. In others, the tongue falls short of the leading edge. Central to these PM cells, and anterior to them as well (in embryos in which they do not reach the leading edge of the involuted tissue), larger, light-colored cells with coarse yolk platelets are found. These larger cells are indistinguishable from the rest of the central endodermal cells extending upward from the vegetal pole. Because cells in this region map to the pharyngeal region of the embryo, or anterior to the pharynx, they are thus referred to as pharyngeal endoderm (20-22).

According to Nieuwkoop and Faber (17), these definitive mesodermal mantle (PM) cells are "delimited" from the central endoderm at approx stage 10.25. This appears to be the case in our experience, although the timing of their appearance and the ease with which they can be distinguished from the remaining central endodermal cells varies between spawnings and sometimes between embryos in a single spawning. Nakatsuji (23) distinguished these populations of cells on the basis of the size distribution of their yolk platelets, as seen in sections. Markers, such as goosecoid (24), noggin (4), and Otx2 (25), are expressed in this PM or "head" mesoderm, although on the basis of location, the "pharyngeal endoderm" may also express these markers (see also ref. 25a). Vodicka and Gerhart (26) correlated the early regions and movements of the organizer tissues with the expression of molecular markers, specifically, Xbra, noggin, goosecoid, and XNR3, in fate maps (also see the fate map in ref. 27). Whether the PM, the pharyngeal endoderm, or both, contribute to the inductive activity of the anterior organizer, and whether their contributions are the same or different from one another is a matter of speculation, since no assays of induction have clearly distinguished between them.

Since stage 10 involves rapid internal transitions, the external staging criteria by which it is defined is not a reliable predictor of internal events. Nieuwkoop and Faber (17) describe the dorsal gastrula wall of this stage as having one epithelial layer from two or three to five or six layers of deep cell. This range reflects the rapid radial intercalation (see 19) and vegetal extension (see ref. 18) in the dorsal gastrula wall from stage 10- through stage 10+, a process that appears not to be strictly correlated with the BC formation on which external staging is based. Thus, in cases where the presence or absence of this early contact with the neural tissue appears to be important, we directly determine the amount of involution that has occurred at a given external stage (see Subheading 4.2.3.), particularly during the transitional stage 10, which is less consistent internally, than stage 10- or 10+.

1.3.3. Convergent Extension: a Good Way to Make an Axis (and a Good Way to Fool the Investigator!)

The posterior regions of both the neural ectoderm and the dorsal mesoderm are originally very wide and very short, and they acquire their final form by extreme convergence (narrowing) and extension (lengthening) during gastru-lation and neurulation (10). One can be seriously misled in designing and interpreting neural induction experiments if these convergent extension movements and their contribution to embryonic development are misunderstood. The common impression of events following BC formation (Fig. 1, stage 10.5) is that the length of the dorsal axial structures (the nervous system, the notochord, and the archenteron) is generated as the involuted mesoderm crawls anteriorly a long distance, and finally comes to rest beneath the appropriate part of the prospective neural ectoderm. It is assumed that at that time, or perhaps before, neural induction occurs as signals are passed from the mesoderm to the neural ectoderm.

In fact, this is not what occurs. After BC formation, the mesoderm contacts the neural ectoderm at or slightly above the equator (Fig. 1, stage 10+), and it only migrates approximately to the animal pole (Fig. 1, stages 12-17). Thus the leading edge of the involuted mesoderm actually migrates only about a quarter of the circumference of the embryo. Although this migration is very important, it contributes little to the elongation of the dorsal aspect of the embryo. The length of the dorsal embryonic tissues is generated as the nervous system extends posteriorly, across the VE, and converges transversely (mediolaterally), squeezing the blastopore shut over the ventral aspect of the yolk plug (see arrows, Fig. 1; Fig. 3A). As this occurs, the prospective PM/ endodermal tissues of the IMZ involute and converge, and extend on the inside, more or less in concert with the overlying posterior neural tissue (see refs. 28 or 29). As the dorsal sector of the embryo elongates through neurulation, the ventral sector shortens. The prospective ventral epidermis diverges around both sides of the ventral midline and moves dorsally with the rise of the neural folds (see arrows, Fig. 1, stages 12-17) (21). These shortening movements on the ventral side contribute to the overall dominance of the sagittal profile at the end of neurulation by the dorsal, extending tissues (Fig. 1, stage 17). The powerful and continuing role of convergent extension in elongating the posterior axis means that in the early fate maps, the prospective RH and SC, the two neural regions undergoing most of the convergent extension, appear very broad and very short (Fig. 3B). They elongate and narrow (converge and extend) greatly in the course of gastrulation and neurulation (10).

In summary, we present a list of facts important for designing and interpreting experiments on neural induction:

The potential inducing tissues of the Organizer involute and make contact with the inner surface of the potential responding tissues earlier than previously thought, usually during stage 10, and nearly always by 10+.

Because the prospective neural tissues are very short in the animal-vegetal axis at the early gastrula stage, the first, early contact of the inducing tissues on the undersurface of the ectoderm is in the anterior neural region.

Inducing and responding tissues shear relatively little, since the first contact of the two is relatively anterior, and because their posterior regions extend more or less together. Thus, corresponding anterior-posterior regions spend more time together than previously thought.

stage 1IK5 stage 11 stage 12,5 stage 15 stage 17

Stage II Slage 15 Stage 17

Fig. 3. Video frames from a time-lapse recording of the vegetal view of an embryo during gastrulation and neurulation show the extreme extension of the neural tissue across the yolk plug of the embryo. This embryo was held in clay with its AC fixed. Dorsal is at the top of the frames. In (A), the Spemann Organizer involutes (curved arrows, stage 10.511), whereas the prospective neural plate converges and extends across the yolk plug (straight arrows, stage 10.5-12.5), to close over the ventral part of the VE (asterisk). After blastopore closure, the neural tissue continues to extend posteriorly, pushing the blasto-pore posteriorly (stages 12-17). The involuted PM converges and extends coordinately with the overlying neural plate. In (B), the fate map of the stage 15 neural plate, redrawn from Eagleson and Harris (91) and projected onto the video image of the stage 15 neural plate, was mapped backward to stage 11 and forward to stage 17 by tracing individual cells at the junctions of map coordinates. Shown are the diencephalon (DI), mesencephalon (MES), RH, SC. This is the same embryo shown in panel (A).

Stage II Slage 15 Stage 17

Fig. 3. Video frames from a time-lapse recording of the vegetal view of an embryo during gastrulation and neurulation show the extreme extension of the neural tissue across the yolk plug of the embryo. This embryo was held in clay with its AC fixed. Dorsal is at the top of the frames. In (A), the Spemann Organizer involutes (curved arrows, stage 10.511), whereas the prospective neural plate converges and extends across the yolk plug (straight arrows, stage 10.5-12.5), to close over the ventral part of the VE (asterisk). After blastopore closure, the neural tissue continues to extend posteriorly, pushing the blasto-pore posteriorly (stages 12-17). The involuted PM converges and extends coordinately with the overlying neural plate. In (B), the fate map of the stage 15 neural plate, redrawn from Eagleson and Harris (91) and projected onto the video image of the stage 15 neural plate, was mapped backward to stage 11 and forward to stage 17 by tracing individual cells at the junctions of map coordinates. Shown are the diencephalon (DI), mesencephalon (MES), RH, SC. This is the same embryo shown in panel (A).

Since the prospective nervous system is initially very short, most of it, particularly its posterior region, is very close to the inducing, Organizer tissue. Inducing signals passing through the plane of the tissue do not have to travel very far.

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