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First published online 1 March 2006
doi: 10.1242/dev.02297

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Cooperative action of Sox9, Snail2 and PKA signaling in early neural crest development

¹ Center for Translational and Advanced Animal Research on Human Diseases, Division of Developmental Neuroscience, Graduate School of Medicine, Tohoku University, Sendai, Miyagi 980-8575, Japan.
² Department of Developmental Neurobiology, Graduate School of Medicine, Tohoku University, Sendai, Miyagi 980-8575, Japan.

View larger version (119K): [in a new window]   Fig. 1. Comparative analysis of the expression of transcription factors, such as Sox9, Msx1, Msx2, Foxd3, Sox10 and Snail2, from a dorsal view. Stage 6-9 embryos were sagittally bisected, and the right halves were hybridized with Snail2 probe, while the left halves were hybridized with other probes for close comparison. Arrowheads indicate expression in the head neural folds. Asterisks indicate expression of Sox9 and Foxd3 in the midline tissues.

View larger version (97K): [in a new window]   Fig. 2. Sox9 is required for Snail2 induction downstream of BMP signal. (A) BMP4 treatment (lower panels) of the neural plate explants effectively induces Sox9 expression and cell dispersal. (B) Neural plate explants were co-transfected with Snail2 and GFP expression vectors, and stained with anti-Sox9 antibody (upper panels) or phalloidin (lower panels). Snail2 misexpression fails to induce either Sox9 protein expression or epithelial-mesenchymal transition (EMT). (C) Sox9 transfection strongly induces Snail2 expression in neural plate explants and subsequent EMT indicated by an extensive cell dispersal, in the absence of BMP4. Insets indicate higher magnification, showing typical morphology of Sox9-transfected cells. (D) Electroporation of GFP and Sox9-En-nuc expression constructs was performed on quail embryos at stage 5, and the embryos were cultured for 7 hours. Misexpression of Sox9-En-nuc expression vector reduces the Snail2 mRNA expression (arrowheads), compared with the untransfected side, or a control embryo transfected with GFP alone. (E) Sox9 is required for Snail2 induction by BMP4. Neural plate explants, transfected with double-stranded RNA corresponding to Sox9 sequence (Sox9 dsRNA), show little expression of Snail2 or cell dispersal, in the presence of BMP4. Transfection of mutated dsRNA (Sox9mut dsRNA) has no effect (Sox9+Sox9 dsRNA) and the effect of Sox9 dsRNA is cancelled by a co-transfection of Sox9 (Sox10+Sox9 dsRNA), while co-transfection of Sox10 fails to cancel the effect of Sox9 dsRNA.

View larger version (45K): [in a new window]   Fig. 3. Snail2 activates its own promoter as a transcriptional activator. (A) A dose-dependent activation of D0.1-Luciferase reporter gene by Snail2 in NIH3T3 cells. Different amounts of Snail2 expression vector were co-transfected with the Snail2 promoter-Luciferase. (B) Snail2 protein binds to the E-Box2 sequence. Isotope-labeled probe containing the E-Box2 was incubated with Snail2 protein, and subjected to EMSA. The shifted band is diminished by a preincubation of the Snail2 protein with cold wild-type competitor, but persists by a preincubation with E-Box2-mutated competitor. (C) E-box2 is required for the Snail2 promoter activation by Snail2. When E-box2 in the D0.1 reporter is mutated (Em2), activation level of the promoter by Snail2 (+) is significantly decreased, compared with the wild type. (D) Snail2 acts as a transcriptional activator on the Snail2 promoter. An expression vector of VP16 activation domain and Snail2 zinc-finger motifs (VP) strongly activates the D0.1 promoter, while an expression vector of Engrailed2 repression domain and Snail2 zinc-finger fusion (En) has no significant effect on the Snail2 promoter activity. Wt, wild type Snail2. (E) Semi-quantitative RT-PCR analysis of endogenous Snail2 expression in neural plate explants, transfected with VP16-Snail2 and cultured without BMP4. Expression level is normalized with the value of amplified GAPDH. Result obtained from explants cultured for 18 hours with BMP4 are also indicated. (F) Transfection of En-Snail2 and VP16-Snail2 into ectoderm inhibits the expression of endogenous Snail2 protein in cultured embryos (arrowheads). GFP-fluorescence indicates the transfected area. Compare with the un-transfected, left neural folds. As the anti-Snail2 antibody recognizes the N-terminal sequence, it detects only endogenous Snail2 protein. (G) Transfection of VP16-Snail2 inhibits the induction of endogenous Snail2 expression in neural plate explants treated with BMP4.

View larger version (32K): [in a new window]   Fig. 4. Sox9 directly activates Snail2 promoter. (A) Activation of Snail2 promoter by Sox9 in neural plate explants. D1.2-Luciferase reporter gene and other `crest-specific' transcription factors were electroporated into neural plates, and cultured for 20 hours without BMP4. Only Sox9 can activate the reporter. Results are shown as fold-induction compared with the result obtained from explants, transfected with D1.2-Luciferase and empty pyDF30 plasmid. (B) Sox9 binds to the flanking sequence of E-box2. EMSA was performed to determine the binding site of Sox9 on Snail2 promoter. The sequence and the position of the probe and competitors are shown below. E-box2 is indicated by an open box. The Sox9 binding to the E-box probe is interfered by the addition of competitor 1, 3 and 5, but not by competitor 2 or 4. (C) The flanking sequence of E-box2 is required for the Snail2 promoter activation by Sox9. When the C-rich sequence next to the E-box2 in the D0.1 reporter is mutated (Em3), activation level of the promoter by Sox9 (+) is significantly decreased, compared with the wild-type D0.1. (D) Sox9 and Snail2 synergistically activate D0.1-Luciferase promoter. D0.1-Luciferase was co-transfected with Sox9 or Snail2 (or both) into NIH3T3 cells. Results are shown as a fold-induction compared with the result obtained from cells transfected with D0.1-Luciferase and an empty pyDF30.

View larger version (47K): [in a new window]   Fig. 5. Sox9 and Snail2 form a protein complex. (A) Schematic diagram of wild type (wt) and deletion mutants (C and N) of HA-tagged Sox9 and GST-tagged Snail2. (B) Interaction of GST-Snail2 deletions and full-length HA-Sox9. Extract of HA-Sox9-transfected COS7 cells were incubated with full-length (wt) and deletions of GST-Snail2 (N and C), and immunoprecipitated with anti-HA beads. Full-length and N-Snail2 is detected in precipitated fractions by anti-GST. (C) Interaction of full-length GST-Snail2 and deletions of HA-Sox9. Extracts of COS7 cells transfected with HA-tagged full-length (wt) Sox9 or deletions (N and C) were incubated with GST-Snail2 (N) and immunoprecipitated with anti-HA beads. With anti-GST, precipitation of Snail2 is detected when incubated with extracts containing either full-length Sox9 (wt) or C-Sox9.

View larger version (61K): [in a new window]   Fig. 6. PKA signal is required for BMP4 and Sox9 to induce neural crest cells in neural plate explants. (A) Effects of H89, a PKA inhibitor, on BMP4-mediated induction of neural crest. Neural plate explants were cultured in the presence of BMP4, and F-actin (phalloidin) and Snail2 were examined. Whereas explants treated with a vehicle (DMSO) show extensive EMT and Snail2 expression, treatment with H89 at low (1 µM) and high (10 µM) concentrations produces partial and nearly complete inhibition of EMT, respectively. Under both conditions, Snail2 induction by BMP4 is blocked. (B) H89 inhibits Sox9-mediated induction of EMT. Although Sox9 transfection strongly induces EMT in neural plate explants, indicated by an extensive cell dispersal in the presence of DMSO, H89 completely blocked EMT. (C) Transfection of Sox9m1m2, a PKA-mediated phosphorylation-deficient mutant, induces Snail2 expression, but fails to promote EMT.

View larger version (75K): [in a new window]   Fig. 7. Tissue distribution of phospho-CREB in stage 7 embryos. (A) A whole-mount preparation of anti-phospho-CREB stained embryo. Although anti-phospho-CREB immunoreactivity is broadly observed, stronger and weaker staining can be seen in the neural folds and midline tissue, respectively. The inset indicates a magnified view of boxed area and the neural fold is indicated by arrowheads. (B) Anti-phospho-CREB staining on section. Higher levels of CREB phosphorylation are observed in the prospective neural crest cells in the neural fold (arrow). DAPI staining shows cell nuclei.

View larger version (98K): [in a new window]   Fig. 8. PKA signal is important for Snail2 expression in the neural crest. Transfection of CA-PKA in the ectoderm of cultured embryo induces ectopic Snail2 expression in neural plate and non-neural ectoderm areas (arrowheads). By contrast, misexpression of PKIß expression vector reduces the Snail2 mRNA expression, compared with the untransfected side. Electroporation of GFP and CA-PKA or PKIß expression constructs into the ectoderm was performed on quail embryos at stage 5, and the embryos were cultured for 7 hours.

View larger version (28K): [in a new window]   Fig. 9. Enhancement of Snail2-mediated activation of the Snail2 promoter by PKA signal. Sox9, Sox9m1m2, Snail2 or Sox9+Snail2 were transfected into NIH3T3 cells along with D0.1-Luciferase reporter. To activate PKA signal, either 1 mM of 8-bromo-cAMP (+cAMP) was added in culture or constitutively active PKA (CA-PKA) was co-transfected. Results are shown as a fold-induction compared with the result obtained from cells transfected with D0.1-Luciferase and an empty pyDF30 vector.

View larger version (21K): [in a new window]   Fig. 10. Regulatory relationship of signals and transcription factors in avian cranial neural crest formation and EMT. Thick arrows indicate direct activation. Asterisks indicate direct activation of Snail2 transcription reported in our previous paper (Sakai et al., 2005).