Xenbase Image ID: 144696

Figure 2. CHD7 and its ATP-ase function are required for neural crest migration in vivo. (A) CHD7 mRNA expression during Xenopus embryogenesis. CHD7 expression was visualized by RNA in situ hybridization at indicated stages of development, showing diffuse pattern at the gastrula stage, but expression localized to the neural (arrow 1), neural crest (arrow 2) and preplacodal ectodermal (arrow 3) tissues at the late neurula stage. At the tailbud stages, CHD7 is expressed in the pharyngeal arches (arrow 4) and optic placode (arrow 5), as well as alongside the neural tube (arrow 6). (B) Morpholino mediated knockdown of CHD7 protein levels in Xenopus laevis embryos. Embryos were injected on both sides at the two-cell stage with morpholino oligonucleotide (MO) targeting either CHD7 or BRD7 at 3.3 uM concentration. Nuclei were extracted from these as well as uninjected control embryos at neurula stage. Whole nuclear lysates were normalized for protein concentrations and analyzed by immunoblotting with α-CHD7xl and α-RNA pol II antibodies. (C) Neural crest migration defect in CHD7 MO and hCHD7 ATPK998R injected embryos. Two-cell stage embryos were injected with mRNA encoding a photo-activatable protein Kaede alone (a and b), Kaede and CHD7 MO (3.3 uM) (c), Kaede, CHD7 MO, and hCHD7 wt mRNA (1 ng) (d), or Kaede and hCHD7 ATPK998R mRNA (9 ng) (e). At the neurula stage embryonic structures corresponding to a subset of the anterior neural and neural crest tissues were UV-irradiated to photo-convert Kaede protein from green to red fluorescence (schematics in Supplementary Figure 8A). Cell migration to the pharyngeal arches (PA) was assayed at the tailbud stage (orange arrows). (D) Effect of CHD7 knockdown on expression of transcription factors involved in neural crest formation. Two cell-stage embryos were injected with CHD7 MO at 3.3 uM asymmetrically into a single blastomere and analyzed by whole mount RNA in situ hybridization at neurula stage to visualize expression patterns transcription factors controlling: neural plate border territory specification (Msx1, Zic1, Pax3), maintenance of the competent border (MycII) and early neural crest formation (Sox9, Twist, Slug). Sox9 is detected in two major expression domains: the neural crest (blue arrow) and prospective otic placode (red arrow). (E) hCHD7 mRNA rescues defects in Sox9 and Twist expression. Two cell-stage embryos were co-injected with CHD7 MO (3.3 uM) and wild type full-length hCHD7 mRNA (1 ng) asymmetrically into a single blastomere. At late neurula stages the embryos were analyzed by whole mount RNA in situ hybridization. Representative examples of full and partial rescue are shown. Image published in: Bajpai R et al. (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Macmillan Publishers Ltd. Larger Image Printer Friendly View

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