TFAP2A is required for both induction and specification of neural crest cells. (A) Whole-mount in situ hybridization for TFAP2A in chick embryos at six developmental stages (HH5–HH10). TFAP2A is enriched in both premigratory and migratory neural crest. Previous studies have shown regulation by TFAP2A of neural plate border genes MSX1, PAX7, and ZIC1 and for neural crest genes FOXD3, ETS1, and SOX10. (B) Transverse sections displaying TFAP2A expression in the neural plate border and the neural crest. (C) Whole-embryo quantitative RT-PCR for TFAP2A at six developmental stages (HH4–HH9) depicted as normalized mRNA expression relative to the housekeeping gene, HPRT1. (D,E) Electroporation scheme for stage-specific knockdown of TFAP2A. (D) Embryos were bilaterally electroporated with control and TFAP2A DsiRNA reagents at HH4 and screened for an induction phenotype at HH8−. Quantitative RT-PCR for induction genes MSX1, PAX7, ZIC1, and GBX2 in control versus TFAP2A DsiRNA-treated sides of bilaterally electroporated embryos, represented as fold change compared with control. Embryos were surveyed at HH8− (n = 11). (E) Embryos electroporated at HH7 were screened for a specification phenotype at HH9. Quantitative RT-PCR for specification genes, FOXD3, ETS1, SOX9, and SOX10 in control versus TFAP2A DsiRNA-treated sides of bilaterally electroporated embryos, represented as fold change compared with control. Embryos were surveyed at HH9 (n = 10). The neural marker SUFU was included as a control to ensure defects were neural crest specific. (F) Immunohistochemistry (whole-mount and transverse sections) for TFAP2A (green), as well as the neural plate border PAX7 (red), upon bilateral TFAP2A DsiRNA treatment. Orientation of knockdown sections has been flipped for comparison. (G) Immunohistochemistry (whole-mount and transverse sections) for TFAP2A (green) and the neural crest marker SOX9 (red) upon bilateral TFAP2A DsiRNA treatment. (HH) Hamburger Hamilton; (NP) neural plate; (NPB) neural plate border; (NNE) nonneural ectoderm; (NC) neural crest; (kd) knockdown; (n.s.) not significant. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001 (for number of embryos analyzed, see also Supplemental Table S1).
