Ossipova O and Sokol SY (2011), Neural crest specification by noncanonical Wnt ...

XB-ART-44514

Development 2011 Dec 01;13824:5441-50. doi: 10.1242/dev.067280.

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Neural crest specification by noncanonical Wnt signaling and PAR-1.

Ossipova O , Sokol SY .

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Neural crest (NC) cells are multipotent progenitors that form at the neural plate border, undergo epithelial-mesenchymal transition and migrate to diverse locations in vertebrate embryos to give rise to many cell types. Multiple signaling factors, including Wnt proteins, operate during early embryonic development to induce the NC cell fate. Whereas the requirement for the Wnt/β-catenin pathway in NC specification has been well established, a similar role for Wnt proteins that do not stabilize β-catenin has remained unclear. Our gain- and loss-of-function experiments implicate Wnt11-like proteins in NC specification in Xenopus embryos. In support of this conclusion, modulation of β-catenin-independent signaling through Dishevelled and Ror2 causes predictable changes in premigratory NC. Morpholino-mediated depletion experiments suggest that Wnt11R, a Wnt protein that is expressed in neuroectoderm adjacent to the NC territory, is required for NC formation. Wnt11-like signals might specify NC by altering the localization and activity of the serine/threonine polarity kinase PAR-1 (also known as microtubule-associated regulatory kinase or MARK), which itself plays an essential role in NC formation. Consistent with this model, PAR-1 RNA rescues NC markers in embryos in which noncanonical Wnt signaling has been blocked. These experiments identify novel roles for Wnt11R and PAR-1 in NC specification and reveal an unexpected connection between morphogenesis and cell fate.

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Species referenced: Xenopus laevis
Genes referenced: ctnnb1 dvl2 foxd3 hes4 krt70 mark2 mark3 myc myod1 ocln pax3 ror2 snai2 sox2 sox3 sox8 tfap2a twist1 wnt11
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	Fig. 1. Modulation of neural crest markers by noncanonical Wnt signaling. (A-H)Albino Xenopus embryos were injected animally with 0.5-1 ng Wnt11 (A-E) or dnWnt5/11 (F-H) mRNA at the four-cell stage, cultured until stage 14/16 and the expression of FoxD3 (A,F), Snail2 (B), Sox8 (C), Sox2 (D,H), Pax3 (E) and MyoD (G) analyzed by in situ hybridization. b-galactosidase is a lineage tracer (light blue) marking the injected side. Arrows point to altered marker expression. Dorso-anterior view is shown. (I) RT-PCR analysis of gene expression in neuralized animal caps. Animal caps were isolated from embryos at stage 9, injected with RNA as indicated, and cultured until stage 16/20 for RT-PCR analysis. Snail2, Twist, FoxD3, Sox8 and Pax3 are induced by Wnt signaling in the absence of significant changes in the pan-neural marker Sox3. no Wnt or Frizzled stimulation; uni, uninjected animal caps; WE, whole embryos; T, no reverse transcriptase.
	Fig. 3. Neural crest markers in embryos with modulated Dvl and Ror2 function. (A-H)Xenopus embryos were injected with the indicated Dvl (Dsh construct) RNAs (1 ng each) and processed for in situ hybridization as described in Fig. 1. Schematic representations of the Dvl mutant constructs are shown at the top. Dsh-delta-N activates Sox8 (A) and FoxD3 (B) but not Sox2 (C). Dsh-DEP+ slightly expands Sox2 (D) but inhibits Sox8 (E), FoxD3 (F), Pax3 (G) and AP2 (H). (I-L)The effects of Ror2 interference on NC marker expression. Ror2-delta-C and Ror2MO, but not wild-type Ror2, inhibit FoxD3 (I,K,L). MyoD is not affected by Ror2-delta-C (J). Arrows point to altered marker expression. Dorso-anterior view is shown.
	Fig. 4. Wnt11R is essential for neural crest specification. (A-F)Xenopus embryos were injected with the indicated MOs or RNAs and processed for in situ hybridization as described in Fig. 1. Wnt11R MO1 (A-C) and MO2 (D-F) inhibit FoxD3 (A,D) and Sox8 (B,E) but do not significantly affect Sox2 (C,F). (G)Control MO (CO MO) does not alter FoxD3. (H,I)Wnt11 and delta-N-Dsh RNAs partially suppress the inhibitory effect of Wnt11R MO1 on Sox8 (H) and FoxD3 (I).
	Fig. 5. Wnt5 and Wnt 11 influence PAR-1 localization and activity. (A)Wnt5a or Wnt11 RNAs (1 ng each) trigger the relocalization of Myc- PAR-1 (co-injected as 0.3 ng of RNA) from the basolateral cortex to the cytoplasm in embryonic ectoderm (arrow, anti-Myc staining). The asterisk indicates cytoplasmic staining. By contrast, PAR-1 distribution is not significantly affected by dnWnt11, E-cadherin depletion or b- catenin overexpression (lower panels). The location of the superficial ectoderm cells used for this analysis is illustrated beneath. (B)Western analysis reveals comparable Myc-PAR-1 levels in the presence of Wnt or control GFP RNAs in stage 10.5-11 embryonic lysates. alpha-tubulin is a loading control. (C)Wnt signaling increases Flag-PAR-1 protein kinase activity as assessed by hTau phosphorylation at Ser262/356 detected by a phospho-specific antibody. Recombinant hTau protein was added at 1ug per sample. Amounts of precipitated PAR-1 were assessed by anti-Flag antibody.
	Fig. 6. PAR-1 plays an essential role in neural crest specification. Xenopus embryos were injected with RNAs or MO and in situ hybridization analysis was carried out as described in Fig. 1. (A-D)PAR-1 RNA (0.3 ng) upregulates Sox8 (A), FoxD3 (B) and Snail2 (C) but does not affect Sox2 (D). (E-I)Effects of PAR-1 depletion. (E)ï€ Control MO (COMO) does not change Sox8. (J)PAR-1B MO2, which has a different sequence, has a similar effect to PAR-1B MO (G). Arrows indicate a change in marker gene expression. Dorso-anterior view is shown.
	Fig. 7. PAR-1 functions in the Wnt5/11 pathway upstream of Pax3. (A)ï€ PAR-1 RNA promotes Pax3 expression (stage 17). (B)ï€ PAR-1 is required for Pax3 expression (stage 14/15). (C,D)ï€ PAR-1 rescues the FoxD3 defect in embryos that were injected with dnWnt5/11 RNA. Dorso-anterior view, except A (dorsal view). Arrows indicate altered gene expression. (E)ï€ Model for Wnt11R signaling during NC specification. Dvl and the associated PAR-1 protein kinase are essential regulators of NC-specific transcription in response to Wnt11R/Ror2 signaling. Dashed lines represent indirect effects via unknown intermediates
	Fig. S1. Wnt11 signaling is involved in neural crest formation. Embryos were injected with Wnt11 (1 ng), dnWnt11 (2 ng) mRNAs or Wnt11R MO1 (40 ng) and were analyzed by in situ hybridization at stage 14/15 as described in Fig. 1. Wnt11 RNA expands the neural plate border marked by Hairy2a (A, horizontal lines), upregulates AP2 (B) and reduces XK70 keratin expression (C). dnWnt11 inhibits FoxD3 (D) but does not have an effect on Sox2 (E). Wnt11R MO1 reduces AP2 expression (F). In A-C, dorsal view is shown, anterior is to the bottom; in D-F, dorso-anterior view is shown. Arrows indicate changes in marker expression.
	Fig. S2. Interference with Ror2 function prevents NC specification. Embryos were injected with mRNAs and analyzed by in situ hybridization at stage 14/15 as described in Fig. 1. Ror2Î´C inhibits Sox8 (A) but not Sox2 (C). Wild-type Ror2 has no effect on Sox8 (B). Arrows indicate changes in marker expression. Dorso-anterior view is shown.
	Fig. S3. Efficiency of Wnt11R depletion. (A) The efficiency of Wnt11R depletion was assayed in vivo by co-injecting 500 pg wnt11r:GFP RNA containing MO target sequence or control GFP RNA with 20 ng Wnt11R MO1. Wnt11R MO1 does not significantly affect control GFP RNA translation (top panels). By contrast, wnt11r:GFP RNA-injected sample fluorescence is strongly reduced by Wnt11R MO1 (middle panels). An uninjected embryo is shown at the bottom as a negative control. (B) Western analysis of GFP levels from embryos shown in A; Î±-tubulin is a loading control. Wnt11R MO1 efficiently blocks protein translation from wnt11r:GFP RNA but not from control GFP RNA.
	Fig. S4. Wnt11R depletion reduces the Pax3 domain. Embryos were injected and processed as described in Fig. 1. (A) Pax3 is not changed by control MO (CO MO) injection (20 ng). (B,C) Wnt11R MO1 (20 ng; B) or Wnt11R MO2 (40 ng; C) partially suppresses Pax3 in stage 14/15 embryos. Arrows indicate changes in marker expression. Dorso-anterior view is shown.
	Fig. S5. Redistribution of PAR-1 in response to Wnt11. (A) The basolateral localization of GFP-PAR-1 is disrupted by Wnt11 in ectodermal cells. Methods are as in Fig. 3B, except that GFP-PAR-1 was used instead of Myc-PAR-1. (B) Wnt11 causes the redistribution of Myc-PAR-1 from the basolateral cortex to the cytoplasm, but does not alter the basolateral distribution of occludin. Arrows indicate basolateral Myc-PAR-1 or occludin. Asterisk indicates cytoplasmic Myc-PAR-1 in the presence of Wnt11.
	Fig. S5. Redistribution of PAR-1 in response to Wnt11. (A) The basolateral localization of GFP-PAR-1 is disrupted by Wnt11 in ectodermal cells. Methods are as in Fig. 3B, except that GFP-PAR-1 was used instead of Myc-PAR-1. (B) Wnt11 causes the redistribution of Myc-PAR-1 from the basolateral cortex to the cytoplasm, but does not alter the basolateral distribution of occludin. Arrows indicate basolateral Myc-PAR-1 or occludin. Asterisk indicates cytoplasmic Myc-PAR-1 in the presence of Wnt11.

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Acloque, Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. 2009, Pubmed