January 1, 2014;
In vertebrates, the neural plate border
) is established by a group of transcription factors including Dlx3
. The crosstalk between these NPB
specifiers governs the separation of the NPB
region into placode and neural crest
) 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
is likely crucial for proper NPB
formation and neural crest
development in Xenopus.
[+] show captions
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.