Zhang Z et al. (2014), Xenopus Nkx6.3 is a neural plate border specifi...

XB-ART-50416

PLoS One 2014 Dec 15;912:e115165. doi: 10.1371/journal.pone.0115165.

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Xenopus Nkx6.3 is a neural plate border specifier required for neural crest development.

Zhang Z , Shi Y , Zhao S , Li J , Li C , Mao B .

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In vertebrates, the neural plate border (NPB) is established by a group of transcription factors including Dlx3, Msx1 and Zic1. The crosstalk between these NPB specifiers governs the separation of the NPB region into placode and neural crest (NC) territories and also their further differentiation. Understanding the mechanisms of NPB formation and NC development is critical for our knowledge of related human diseases. Here we identified Nkx6.3, a transcription factor of the Nkx family, as a new NPB specifier required for neural crest development in Xenopus embryos. XNkx6.3 is expressed in the ectoderm of the neural plate border region at neurula stages, covering the epidermis, placode and neural crest territories, but not the neural plate. Inhibition of Nkx6.3 by dominant negative construct or specific morpholino leads to neural crest defects, while overexpression of Nkx6.3 induces ectopic neural crest in the anterior neural fold. In animal caps, Nkx6.3 alone is able to initiate the whole neural crest regulatory network and induces neural crest fate robustly. We showed that overexpression of Nkx6.3 affects multiple signaling pathways, creating a high-Wnt, low-BMP environment required for neural crest development. Gain- and loss-of-function of Nkx6.3 have compound effects on the expression of known NPB genes, which is largely opposite to that of Dlx3. Overexpression of Dlx3 blocks the NC inducing activity of Nkx6.3. The crosstalk between Nkx6.3, Dlx3 and Msx1 is likely crucial for proper NPB formation and neural crest development in Xenopus.

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Species referenced: Xenopus
Genes referenced: dlx3 dlx5 eya1 fgf8 foxd3 fzd3 hdc lgals4.2 msx1 nkx6-3 npb pax3 six1 snai2 sox2 wnt8a zic1
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	Figure 2. Nkx6.3 is required for neural crest development in Xenopus.Injection of a inducible dominant negative form of Nkx6.3, Nkx6.3-HDC, induced severe defects in pigmentation at tadpole stages (A) when dexamethasone was added, which was rescued by co-expression of the wild type Nkx6.3-GR construct (B). The pigmentation patterns of the trunk region of an Nkx6.3-HDC injected embryo and a control embryo were highlighted in (C) and (D) respectively. Injection of Nkx6.3HDC-GR reduced the expression of Slug (E), which can be restored by co-injected Nkx6.3GR mRNA (F). (G) and (H) Injection of specific morpholino against Nkx6.3 but not a control morpholino impaired the expression of the neural crest marker Slug. The injected sides in (E)–H) are on the left, labeled by the red staining of the co-injected tracing lacZ. In (E)–H), the numbers of embryos showing similar changes of gene expression and total injected embryos in each group are indicated.
	Figure 3. Nkx6.3 is able to induce neural crest dependent on Wnt signaling.(A) Nkx6.3GR mRNA was injected into one blastomere at 2-cell stage and DEX was added at stage 11, the expression of neural crest marker FoxD3 was inhibited on the injected side (arrow). (B, C) Local injection of Nkx6.3 mRNA at 32-cell stage induced neural crest (arrowheads) in the anterior neural folds. (D) In animal caps, overexpression of Nkx6.3 induced the expression of neural crest markers and that of Wnt8 and FGF8 (lanes 1, 2). The VpHDC construct, in which the eh1 repressor domain was replaced by the Vp16 activation domain, repressed the neural crest inducing effect of Nkx6.3 (compare lanes 2, 3). Inhibiting Wnt signaling by GSK3β blocked most of the effects of Nkx6.3 on marker gene expression (lane 4). (E) The effect of different Nkx6.3 constructs on the expression of luciferase reporter genes of Wnt (Top-Flash, middle panel) and BMP (ID-Luc, lower panel) signaling in Xenopus embryos. The domain structures of Nkx6.3 and the HDC, NKHD constructs were shown in the upper panel. eh1, eh1 repressor domain; HD, homeodomain; C, C-terminal domain. (F) Effects of the fusion constructs of GAL4 and various Nkx6.3 domains on the expression of a GAL4 luciferase reporter gene. Wild type Nkx6.3 and that lacking the C terminal domain worked as repressors (bars 1, 2) while the EHL region with or without the eh1 domain both activated the transcription of the reporter (bars 3, 6). EHL, the linker region between the eh1 repressor domain and the HD domain. , p<0.05; *, p<0.01. The expression of the different constructs were confirmed by Western blot (data not shown).
	Figure 4. Msx1 is an immediate target gene in response to Nkx6.3.(A) 1 ng Nkx6.3GR mRNA was injected into each cell at 2-cell stage. Animal caps were dissected at Stage 9, treated with cycloheximide for 30 minutes to inhibit protein synthesis, and then cultured in media containing dexamethasone for 2 hours before processed for RT-qPCR to monitor the expression of the marker genes indicated. Among the tested genes, Msx1 was the only one up-regulated clearly under such condition. (B) Dynamic induction of Msx1 by Nkx6.3. The induction of Msx1 was monitored at different time points after induction as described in (A). The expression level of Msx1 was up-regulated in one hour, but was then down-regulated at later stages.
	Figure 5. Nkx6.3 is a neural plate border specifier.The effects of Nkx6.3 overexpression (A–J) or knockdown (A′–J′) on the expression of indicated neural and non-neural ectodermal markers and that of Wnt8. The injected sides were all on the left marked by red β-galactosidase staining. In (E) and (E′), the arrowed lines indicate the width of the neural plates. The numbers of embryos showing similar changes of gene expression and total injected embryos in each group are indicated.
	Figure 6. Dlx3 blocks the neural crest induction activity of Nkx6.3.RT-PCR analysis in animal cap assay showing the effect of Dlx3 on the expression of the indicated neural crest genes induced by Nkx6.3. Note that the expression of endogenous Dlx3 and Dlx5 were all reduced upon Nkx6.3 over-expression. -RT, negative control with reverse-transcriptase omitted in the RT reaction; embryo, RNA template from whole embryos was used as a positive control.
	Figure 1. XNkx6.3 is expressed in the neural plate border ectoderm.(A) Experimental strategy to verify the expression domain of Nkx6.3 by qPCR. A transverse slice of tissue of the potential neural plate border region of single stage 17 embryos was dissected out and separated sequentially into 7 pieces, which were expected to represent epidermis, placode, neural crest and neural plate identities respectively. The explants were then processed to real-time RT-PCR analysis. E, epidermis; P, placode; NC, neural crest; NP, neural plate. (B) Nkx6.3 is expressed predominantly in the ectoderm. A piece of tissue corresponding to region 3 in (A) was dissected out and separated into surface ectodermal and deep mesodermal parts. The expression of Nkx6.3 and known ectodermal (Keratin, Dlx3 and Msx1) and mesodermal (MyoD) genes were monitored by qPCR. (C) The expression of Nkx6.3 and known neural plate border markers in a representative series of dissected epidermis, placode, neural crest, and neural plate explants from a single embryo at stage 17. The series of explants from each embryo were checked first for the expression of Dlx3, Six1, FoxD3 and Sox2, and only those with relative clean separation of the epidermis, placode, neural crest and neural plate were further analyzed for the expression of Nkx6.3 and additional markers. RE, relative expression.

References [+] :

Ahrens, Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis. 2005, Pubmed, Xenbase