Woda JM et al. (2003), Dlx proteins position the neural plate border a...

XB-ART-6081

Development 2003 Jan 01;1302:331-42. doi: 10.1242/dev.00212.

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Dlx proteins position the neural plate border and determine adjacent cell fates.

Woda JM , Pastagia J , Mercola M , Artinger KB .

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The lateral border of the neural plate is a major source of signals that induce primary neurons, neural crest cells and cranial placodes as well as provide patterning cues to mesodermal structures such as somites and heart. Whereas secreted BMP, FGF and Wnt proteins influence the differentiation of neural and non-neural ectoderm, we show here that members of the Dlx family of transcription factors position the border between neural and non-neural ectoderm and are required for the specification of adjacent cell fates. Inhibition of endogenous Dlx activity in Xenopus embryos with an EnR-Dlx homeodomain fusion protein expands the neural plate into non-neural ectoderm tissue whereas ectopic activation of Dlx target genes inhibits neural plate differentiation. Importantly, the stereotypic pattern of border cell fates in the adjacent ectoderm is re-established only under conditions where the expanded neural plate abuts Dlx-positive non-neural ectoderm. Experiments in which presumptive neural plate was grafted to ventral ectoderm reiterate induction of neural crest and placodal lineages and also demonstrate that Dlx activity is required in non-neural ectoderm for the production of signals needed for induction of these cells. We propose that Dlx proteins regulate intercellular signaling across the interface between neural and non-neural ectoderm that is critical for inducing and patterning adjacent cell fates.

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Species referenced: Xenopus laevis
Genes referenced: dlx3 dlx5 gal.2 hes4 krt12.4 msx1 ncam1 six1 snai1 snai2 sox2 tubb2b

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	Fig. 1. Dlx gene expression becomes restricted to the ventral ectoderm. (A) At blastula stage (stage 9), Xdlx3 is expressed broadly throughout the ectoderm. Animal pole is oriented up. (B) By early gastrula stage (stage 10), Xdlx3 expression is restricted to the more ventral ectoderm (black arrowhead). Lateral view with dorsal oriented to the left. Dorsal lip is on the left (green arrowhead). (C) By the beginning of neurulation (stage 13), Xdlx3 expression is completely absent from the neural plate and is expressed throughout the non-neural ectoderm (dorsal view with anterior to the top). (D) Schematic of Dlx homeodomain constructs. The activating Dlx construct was made by ligating regions encoding the Dlx3 homeodomain (blue) to the VP16 activation domain (yellow). A conditional version was generated by fusion to the ligand-binding domain of the human glucocorticoid receptor (GR; green). Inhibitory constructs were made similarly using the Engrailed repressor domain (EnR; red). Identical constructs were made with the Dlx5 homeodomain. The homeodomains are highly conserved among Dlx family members; thus the fusion proteins are envisaged to regulate target genes of all family members comparably. See Materials and Methods for details of construct preparation.
	Fig. 2. Dlx activity restricts neural plate expansion but does not induce epidermal differentiation. β-galactosidase and either EnR-Dlx3hd, EnR-Dlx5hd or VP16-Dlx3hd mRNAs were injected into one dorsal animal blastomere of 4-cell stage embryos. The embryos were then stained for the β-galactosidase (as a lineage label; magenta stain) and assayed for Xsox2 (stage 13), NCAM (stage 17-18) or keratin (stage 17-18) expression by whole-mount in situ hybridization (blue stain), or by EpA immunostaining (stage 17-18) to reveal neural plate or epidermal differentiation. All views are dorsal with anterior to the top except M, which is lateral with anterior to the right and H and I, which are transverse histological sections through the neural tube. Dashed lines indicate the dorsal midline. (A) Xsox2 expression is the same on the injected and uninjected sides of control embryos injected with β -galactosidase mRNA alone. (B) Injection of VP16-Dlx3hd mRNA reduces the Xsox2 domain on the injected side. (C,D) In contrast, embryos injected with EnR-Dlx3hd (C) or EnR-Dlx5hd (D) mRNA shows expanded Xsox2 expression on the injected side. (E-G) The NCAM domain was similarly reduced by or expanded by VP16-Dlx3hd and EnR-Dlx3hd, respectively. (H,I) Transverse sections through stage 25 embryos (H) the control embryo has a symmetrical, closed neural tube. (I) An embryo expressing EnR-Dlx3hd illustrates that the neural tube closed properly but was expanded on the injected side where β-gal-positive cells populate the neural tube. Dorsal is to the top. (J-M) keratin expression; a marker expressed throughout the non-neural ectoderm. (J) Normal expression of epidermal keratin in a control embryo injected with β-galactosidase mRNA alone. (K) Injection of VP16-Dlx3hd mRNA did not expand epidermal keratin expression. (L) Injection of EnR-Dlx3hd mRNA inhibited epidermal keratin expression. White arrows mark the loss of keratin expression in the injected region. (M) A lateral view illustrates the loss of keratin (blue) in the injected region (magenta β-galactosidase stain). (N-P) VP16-Dlx3hd did not affect the epidermal epitope EpA (O), whereas EnR-Dlx3hd (P) prevented normal expression (N).
	Fig. 4. Alterations in cell lineages that border the neural plate in embryos with localized expression of VP16-Dlx3hd or EnR-Dlx3hd. Embryos were injected unilaterally with EnR-Dlx3hd or VP16-Dlx3hd at the 8-16 cell stage, cultured until the appropriate developmental stage and then examined by in situ hybridization (blue stain). β-galactosidase stain (magenta) indicates progeny of injected blastomeres. All embryos are shown as dorsal views with anterior to the top except for J and K which are lateral views, anterior to the right, and V-Y, anterior views, dorsal to the top. (A-F) Stage 13 embryos showing that markers of cells at the border of the neural plate (Xhairy2A and Xmsx-1) are ablated by VP16-Dlx3hd and shifted laterally by EnR-Dlx3hd. Arrows mark displacement caused by EnR-Dlx3hd relative to uninjected side of the embryo. (G-P) Stage 13 embryos showing identical effects on markers of neural crest precursors (Xsnail and Xslug). Lateral views of Xsnail expression illustrate the extent of the shift (J,K) seen in dorsal view (I, arrows). Transverse sections (O,P) showing Xslug expression (blue) and β-galactosidase (magenta) illustrate that the size of the Xslug expression domain is unaltered but occurs at the lateral margin of the cells expressing the injected mRNA. (Q-U) Primary neurons (marked by N-tubulin) are also ablated by VP16-Dlx3hd and displaced laterally by EnR-Dlx3hd in stage 14 Xenopus (Q-S) and 2-somite stage zebrafish (T,U). (V-Y) Stage 18 embryos showing cranial placode precursors (marked by Xsix1) shifted medially or laterally by localized expression of VP16-Dlx3hd and EnR-Dlx3hd, respectively (W,X). Note that the anterior domain of Xsix1 is unaffected, even where widespread expression of EnR-Dlx3hd ablates Xsix1 more laterally (Y).

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Adamska, Inner ear and lateral line expression of a zebrafish Nkx5-1 gene and its downregulation in the ears of FGF8 mutant, ace. 2000, Pubmed