Bonstein L et al. (1998), Paraxial-fated mesoderm is required for neural ...

XB-ART-15359

Dev Biol 1998 Jan 15;1932:156-68. doi: 10.1006/dbio.1997.8795.

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Paraxial-fated mesoderm is required for neural crest induction in Xenopus embryos.

Bonstein L , Elias S , Frank D .

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Neural crest induction is thought to occur by a two-step process. Axially fated mesoderm induces neural plate, which is then recruited to neural crest by nonneural epidermal ectoderm at the neural plate border. This model suggests a rather indirect role for mesoderm in inducing neural crest. We extensively examined the role of mesoderm in neural crest induction by determining which types of mesoderm induce neural crest cells in Xenopus embryos. We found that noggin-dorsalized ventral marginal zone (VMZ) explants differentiate as melanocytes in the absence of axial mesoderm. Dorsalized VMZ is also a potent inducer of melanocytes when juxtaposed to animal cap ectoderm in recombinant explants. Dorsalized VMZ is analogous to the dorsal-lateral marginal zone (DLMZ) region of the embryo. Neural crest-inducing activities of gastrula stage DLMZ and dorsal marginal zone (DMZ) were also compared in recombinant explants. DLMZ was a stronger inducer of neural crest than was DMZ; DLMZ induced high levels of XSlug expression and melanocyte formation in recombinants, whereas DMZ weakly induced neural crest. In whole embryos lacking DLMZ, XSlug expression and melanocyte formation were significantly reduced; in contrast, no significant reduction of XSlug expression or melanocyte formation was seen in embryos lacking a DMZ. These results suggest that paraxial-fated mesoderm plays a central role in neural crest formation by inducing a novel type of lateral neural plate. This lateral neural plate is then recruited to neural crest by adjacent nonneural epidermal ectoderm.

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Species referenced: Xenopus
Genes referenced: acss2.2 acta4 actl6a ag1 b3gat1l eef1a2 hoxb9 myod1 ncam1 nog nrp1 snai2

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	FIG. 1. Melanocytes and neural markers are induced in noggin-dorsalized VMZ explants. In a representative experiment, 36 VMZ explants were removed from stage 10/ to 10.25 embryos. Eighteen explants were noggin-treated until stage 12.5–13 and the 18 remaining explants served as controls. All explants were grown to stage 41 and scored for melanocytes. Approximately 75% of the noggin-treated VMZs made melanocytes in comparison to 5% of the controls. Embryos were fixed in MEMFA according to Hemmati-Brivanlou et al. (1990a) and cleared in 2:1 benzyl benzoate:benzyl alcohol before photography. (A) Six representative noggin-dorsalized VMZ explants. (B) Six representative control VMZ explants. (C) Induction of neural markers in dorsalized VMZ explants. Eighteen VMZ explants were removed from embryos at stage 10/ to 10.25. Nine explants were noggin-treated until stage 12.5–13, and 9 untreated explants served as controls. Explants were grown to several stages (20, 26, 36, and 41), and total RNA was isolated for Northern analysis. Total RNA was also isolated from pools of five whole embryos. All 9 explants and one embryo equivalent of RNA were loaded per well. Filters were sequentially hybridized with Xenopus cDNA probes for muscle-specific actin (m-actin), XMyoD, NCAM, nrp-1, XIF-3, n-tubulin, Hoxb9, XAG-1, E13 (epidermal cytokeratin), and EF1a. The results from stage 26 explants are shown; similar results (not shown) were observed at stages 20, 36, and 41. All data were quantitated as described under Materials and Methods.
	FIG. 2. Dorsalized VMZ induces melanocytes in naive animal cap ectoderm. (A) Experimental strategy: VMZs were taken from homozygous albino embryos and recombined with AC ectoderm from heterozygous albino embryos (see Materials and Methods) or AC ectoderm taken from lightly pigmented embryos. This ensures that melanocytes detected in the recombinant explant AC are exclusively a result of induction. The use of heterozygous albino or lightly pigmented ACs enables easy visualization of black melanocytes on a light background. In a routine experiment 18 recombinant explants were compared between each group. In none of the experiments did ACs ever spontaneously differentiate as melanocytes. (B) VMZs were noggin-treated, washed, and recombined with ACs as described in the text. Seven representative recombinant explants (stages 39–41) are shown. (C) Control VMZs that underwent a parallel treatment in the absence of noggin were recombined with ACs as described in the text. Seven representative recombinant explants (stages 39–41) are shown. (D) Homozygous albino embryos were injected with 0.2–0.4 ng of BMP-4 DNR RNA. VMZs were removed at stage 10.25 and recombined with ACs taken from lightly pigmented embryos. Explants were grown to stages 39–41. Melanocytes are observed in trunkshaped explants (top) as well as cylindrical shaped explants (bottom). (E) Control VMZs were removed at stage 10.25 and recombined with ACs derived from lightly pigmented embryos. Explants were grown to stages 39–41. These explants elongated less than those shown in Fig. 4D and did not differentiate melanocytes or make muscle (not shown).
	FIG. 3. Paraxial-fated mesoderm is a strong inducer of neural crest cells. Stage 10.25–10.5 DLMZs and DMZs were removed from homozygous albino embryos and recombined with blastula stage ACs. DLMZs, DMZs, and ACs were also removed in parallel as controls. In a routine experiment, 18 explants from each group were scored for melanocytes at stage 41, and 9 explants from each group were grown until stage 17 for Northern analysis. (A) Three representative DLMZ–AC recombinant explants are shown. (B) Three representative DMZ– AC recombinant explants are shown. (C) Northern analysis of recombinant DLMZ and DMZ explants. Nine explant equivalents and one embryo equivalent (from a pool of five control embryos) of RNA were loaded per well. Filters were sequentially hybridized with Xenopus cDNA probes for XSlug, XAG-1, nrp-1, muscle-specific actin (m-actin), and EF1a. On all filters, the muscle-specific actin is the lower band; the upper band is cross-hybridization to cytoskeletal actin. (D) Whole-mount in situ hybridization with XSlug was performed on mid-neurula-stage embryos and explants (see Materials and Methods). Top: XSlug expression in a mid-neurula-stage embryo injected with b-gal, dorsal view. Bottom: DLMZ–AC recombinant explants in which AC cells were injected with b-gal; the arrows show the purple alkaline phosphatase staining on the red b-gal background.
	FIG. 4. Melanocyte formation is inhibited in embryos lacking paraxial mesoderm. (A) Embryos were dissected at stage 10.25–10.5. Either a 607 piece of the DMZ or both 607 pieces of the DLMZ were removed from nine embryos. Embryos were scored for melanocyte formation at stage 39–41. (B) A lateral view of a control embryo. (C) Embryos lacking a DLMZ region make heads and shortened trunks but lack melanocytes. (D) Embryos lacking a DMZ region make reduced amounts of anterior structures (eyes and cement glands) but contain large patches of trunk melanocytes (arrows mark patches of melanocytes).
	FIG. 5. Expression of XSlug in embryos lacking paraxial or axial mesoderm. (A) Northern analysis of RNA isolated from stage 17 embryos. Total RNA was isolated from 4 control, -DMZ, or -DLMZ embryos and from 10 control or LiCl-hyperdorsalized embryos. Two embryo equivalents of RNA were loaded per well for Northern analysis. Filters were sequentially hybridized with Xenopus cDNA probes for XSlug, nrp-1, muscle-specific actin (m-actin), and EF1a. (B) Whole-mount in situ hybridization with XSlug was performed on mid-neurula stage embryos (see Materials and Methods), dorsal view. (C) XSlug expression in mid-neurula-stage embryos (-DMZ), dorsal view. (D) XSlug expression in mid-neurula-stage embryos (-DLMZ), dorsal view.
	FIG. 6. Model of Xenopus neural crest induction. (A) The underlying axial mesoderm (AX) induces neural plate in ectoderm. Lateral neural plate interacts with the nonneural ectoderm to form neural crest cells in the neural folds region. (B) The underlying paraxial mesoderm (PX) induces/modifies lateral neural plate. Modified lateral neural plate interacts with the nonneural ectoderm to form neural crest cells in the neural folds region. (C) The underlying PX directly induces neural crest in the overlying cells of the neural folds region.