Linker C et al. (2000), Relationship between gene expression domains of...

XB-ART-10566

Dev Biol 2000 Aug 15;2242:215-25. doi: 10.1006/dbio.2000.9723.

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Relationship between gene expression domains of Xsnail, Xslug, and Xtwist and cell movement in the prospective neural crest of Xenopus.

Linker C , Bronner-Fraser M , Mayor R .

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The markers Xslug, Xsnail, and Xtwist all are expressed in the presumptive neural folds and are thought to delineate the presumptive neural crest. However, their interrelationship and relative spatiotemporal distributions are not well understood. Here, we present a detailed in situ hybridization analysis of the relative patterns of expression of these transcription factors from gastrulation through neurulation and post-neural crest migration. The three genes mark the prospective neural crest and roof plate, coming on sequentially, with Xsnail preceding Xslug preceding Xtwist. By combining gene expression analysis with a fate map of the same region using DiI labeling, we determined the correspondence between early and late domains of gene expression. At the beginning of gastrulation, Xsnail is present in a unique domain of expression in a lateral region of the embryo in both superficial and deep layers of the ectoderm, as are Xslug and Xtwist. During gastrulation and neurulation, the superficial layer moves faster toward the dorsal midline than the deep layer, producing a relative shift in these cell populations. By early neurula stage, the Xsnail domain is split into a medial domain in the superficial ectoderm (fated to become the roof plate) and a lateral domain in the deep layer of the ectoderm (fated to become neural crest). Xsnail is down-regulated in the most anterior neural plate and up-regulated in the posterior neural plate. Our results show that changes in the expression of Xsnail, Xslug, and Xtwist are a consequence of active cell movement in some regions coupled with dynamic changes in gene expression in other regions.

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Species referenced: Xenopus
Genes referenced: snai1 snai2 twist1

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	FIG. 1. Expression of Xsnail, Xslug, and Xtwist. Late gastrula (A, F, and K), early neurula (B, G, and L), late neurula (C, H, and M), tailbud (D, I, and N), and tadpole stage (E, J, and O). Filled arrowheads show the border of the neural plate at early stages, the prospective neural crest at neurula stages, and the migrating neural crest at older stages. Open arrowheads in C and H show the medial expression domain of Xsnail and Xslug. Arrows show the mesodermal expression of the genes from late gastrula to late neurula stages. Note the complementary patterns of expression of Xsnail and Xslug in the mesoderm. Asterisks show the premigratory neural crest population at tailbud stages (D, I, and N; e, eyes). Neural crest migrating into the dorsal fin is revealed by Xslug expression (P) at tailbud stages. There was a marked anteroposterior wave of migration, with the most anterior cells farther away from their original position in the dorsal neural tube (a, anterior; p, posterior). Neural crest cells can be seen migrating as individual cells in the dorsal fin in Q (enlargement of the anterior part of P). At tailbud stages, migrating neural crest streams can be seen in the truncal region of the embryo (R).
	FIG. 2. In situ hybridization in serial sections for Xsnail, Xslug, and Xtwist. Late gastrula stage shows staining in the prospective neural fold population for Xsnail and Xslug (A and B), but only mesoderm expression for Xtwist (C). At late neurula stages, two bands of gene expression are observed, one more medial staining the superficial layer of cells (sf) and the other lateral comprising deeper layers of cells (nf). These two populations express all three genes (Dâ€“F) and are thought to represent premigratory neural crest cells. Mesodermal staining can be seen in the ventral part of the segmental plate and in the lateral plate. In cranial sections at the tailbud stage, migrating neural crest expressing all three markers can be observed surrounding the eyes and in the cranial mesenchyme (Gâ€“I). At the same stages at anterior truncal levels, neural crest cells lie in the dorsal neural tube as well as migrating toward the ventral part of the embryo (Jâ€“L). At tadpole stage in anterior truncal levels, premigratory neural crest cells are observed within the dorsal neural tube and migrating cells appear to move toward the dorsal fin (Mâ€“O). Expression in the mesoderm can be seen in the ventral part of the somites and in the lateral plate in truncal sections of tailbud and tadpole stages. Those sections lie between adjacent somites and therefore lack obvious Xsnail in this region. nf, neural folds; m, mesoderm; sf, superficial neural folds; n, notochord; c, neural crest cells; e, eyes; nt, neural tube; cg, cement gland; s, somites; df, dorsal fin; mi, myotome; sc, sclerotome; lp, lateral plate.
	FIG. 3. Fate map of Xsnail domains. The blue outline represents a model of a representative embryo at late gastrula stage (stage 11.5, A) and at neural stage (stage 16, F) made using a computational two-dimensional analysis program. Expression domains of Xsnail in 50 embryos were outlined (stage 11.5, B, or 16, G) and superimposed to generate a map showing the variation in the area of expression within the population; light yellow areas indicate where Xsnail was expressed by 20% of the embryos, whereas red areas indicate where Xsnail was expressed by 100% of the population (stage 11.5, C, or 16, H). Examples of five DiI injections made at stage 11.5 (D) and followed to stage 16 (I) are illustrated. 42 different injections were made and grouped as dorsal (red), medial (green), or ventral (blue) at stage 11.5 (E) and followed to stage 16 (J).
	FIG. 4. DiI label in the superficial cell layer was detected in thin sections. (A) Whole-mount view of an embryo after injection of DiI at stage 11.5; bar represents 100 mm. Inset shows a section through a similar embryo demonstrating a single domain of DiI labeling. (B) A thin transverse section of the same embryo at stage 16 in which the midline is to the right. DiI-stained regions are separated by more than 100 mm, suggesting that the superficial cell layer moves farther medially than the deep cell layer.
	FIG. 5. Time-lapse videomicroscopy of DiI-labeled embryos. Two DiI injections were made in the dorsal and ventral regions of the Xsnail expression area and followed for 6 h. Frames taken at 1-h intervals (AâF) show the movements of cells situated in different regions of the Xsnail expression domain (darker spots in A show the dorsal midline which was stained with Nile blue before the DiI injection to ensure accuracy in positioning the embryo. An arrow map with information from six movies shows a circular movement of cells which centers on the middle of Xsnail expression (G). Note that the cells are in the ventralmost part of the Xsnail expression. The data in G and H are deduced from the injections showed in Fig. 3 and from the six time-lapse recordings.
	FIG. 6. Origin of the cells expressing Xslug in the neural plate. (A) An embryo at the late neurula stage showing expression of Xslug in the neural crest population (arrows) and in individual cells in the neural plate surrounded by nonexpressing cells (arrowhead). (B) These cells could be Xslug-expressing cells derived from the neural plate that migrate into the neural fold (a); alternatively, they could originate from the neural folds and migrate into the neural plate (b). Embryos were injected at stage 15 in the neural plate (CâF) or in the neural folds (G, J) and in situ hybridization was performed immediately after staining (D, H) or after stage 20 (F, J). C, E, G, and I correspond to the fluorescent images of the sections showing the in situ hybridization for the Xslug gene in D, F, H, and J. Arrows, Xslug expression in the neural folds or neural crest cells; arrowhead, expression of Xslug in the neural plate. Notice that it is possible to detect Xslug-expressing cells in the neural plate only after the neural folds were labeled (GâJ), while no neural crest cells express Xslug when the neural plate is labeled (CâF).
	FIG. 7. Schematic diagram summarizing the dynamic pattern of neural crest marker expression. Embryos are shown from anterior, with dorsal being up. The superficial and deep layers of the ectoderm are illustrated, with gene expression shown in red (superficial) and blue (deep). (A) At midgastrula stage, gene expression is seen as a continuous band that surrounds the prospective neural plate, in the deep (blue) and superficial (red) layers of the ectoderm. Both layers move to the midline, but the superficial moves faster than the deep layer (arrows). At the same time, the gene expression domains extend posteriorly as a consequence of cell movement and up-regulation of genes in that region. Gene expression is down-regulated in the deep layer of the anterior neural fold by a signal likely coming from the anterior mesoderm. (B) At early neurula stage, gene expression in the anterior neural fold is observed only in the superficial ectoderm (red) and disappears from the anterior deep ectodermal layer (note that the blue band does not extend to the anterior end of the embryo). The nonequivalent movements of the deep and superficial ectoderm toward the dorsal midline produce a gap in gene expression between the deep (blue) and the superficial (red) regions. (C) At the late neurula stage, the neural tube/neural plate (dotted area) has already closed except in the superficial layer at the anteriormost region of the embryo. The split of the blue bar in the anterior region of the embryo marks the superficial lateral margins of the neural plate, while the red band marks the roof plate or deep layer of the neural plate, which has already closed along the anteriorâposterior axis of the embryo. As a consequence of neural tube closure in the posterior end, the deep layer of the ectoderm (blue) comes to lie on the top of the superficial layer (red). The blue cells are the prospective neural crest cells, and in the anterior neural plate, it is possible to visualize individual neural crest cells (blue dots) migrating (arrows) toward the dorsal midline in the region of the hindbrain.