Xenbase Image ID: 139643

Fig. 4. In animal cap explants barhl2, otx2 and irx3 co-expression enables induction of shh expression by Shh. (A) Experimental design: AAC were prepared from embryos injected with RNA as indicated. AACs were sandwiched to yield ßGal/GFP conjugates and cultured for 48 h. In experiments shown in (D) the N-Shh-expressing part of the explant was replaced by beads. AACs were analyzed by ISH for expression of Xenopus (X) shh. The % of explants showing the phenotype is indicated. The scale bar stands for 0.5 mm. Representative sandwiched explants are shown. (B) Explants of anterior, or p2-like, neuroepithelial identity cultured in the presence of N-Shh are not competent to express shh. AACs expressing noggin (a) or barhl2 and otx2 (b) were sandwiched with AACs expressing N-Shh. Neither AACs (100%, n=72), nor p2-like explants (barhl2+otx2) (100%, n=24) expressed endogenous Xshh when exposed to N-Shh. (C, D) In the presence of N-Shh pre-ZLI’s cells are competent to express Xshh. Pre-ZLI (barhl2, otx2, irx3) explants expressing or not ßGal (red) as indicated were sandwiched with: (C) (a) AACs (100%, n=72), or (b) AACs expressing N-Shh (b) (55%, n=96), (c) (72%, n=25). Enlarged image are shown in (b). Clusters of cells expressing Xshh appear along the border between the (barhl2, otx2, irx3)- and the Shh-expressing parts of the explants (white arrow). (D) beads (a) without N-Shh (88%, n=25) or (b) with N-Shh (76%, n=25). (c) Enlarged image of (b): clusters of cells expressing Xshh developed in close contact with the N-Shh-impregnated beads. The gray circle indicates the bead’s location. (E) Induction of Xshh occurs within barhl2, otx2, irx3 expressing cells and when any of the transcription factors barhl2 or otx2 or irx3 is absent, Xshh expression is not induced in explants. (a) Pre-ZLI (barhl2, otx2, irx3) explants expressing ßGal (red) were sandwiched with AACs expressing N-Shh (72%, n=25). Representative explants and vibratome sections (30 µm) are shown. The Xshh expressing-cells exhibit ßGal activity. Representative sandwiched AACs are shown for: (b) AACs (barhl2+irx3) with AACs N-Shh (100%, n=24), (c) AACs (otx2+irx3) with AACs N-Shh (92%, n=24). The scale bar stands for 0.5 mm. Image published in: Juraver-Geslin HA et al. (2014) Copyright © 2014. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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