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Fig. 7. When ectopically expressed in the developing neuroepithelium and continuously exposed to Shh, pre-ZLI-like cells recapitulate the characteristic features of their developmental program. (A) Experimental design: AAC were prepared from embryos injected with RNA as indicated. At st. 8/9 cells were dissociated and reaggregated to generate explants made of mixed cell types. Pieces of explants were grafted into sibling’s neural plate at st. 14. Embryos were let to develop until st. 35. (B, C) Grafted cells expressing barhl2, otx2, irx3 develop ectopic ZLI when continuously exposed to Shh. (B) ISH using Xshh as probe on st. 35 embryos grafted with AACs (otx2+N-Shh) (a), or AACs (barhl2, otx2, irx3+N-Shh) (b) mixed explants. The grafted sides of st. 35 representative neural tubes are shown, side view, dorsal up, anterior left. ZLI: zona limitans intrathalamica; fp: floor plate. (c) Enlarged view of grafted cells from (b). (C) Analysis of grafting experiments. In embryos grafted with (otx2+N-Shh) mixed explants no ectopic ZLI develop in ßGal-expressing cell clusters (shown in blue) (100%, n=18). In contrast in embryos grafted with (barhl2, otx2, irx3+N-Shh) mixed explants most embryos developed ectopic ZLI (89%, n=26). From 35 identified ectopic ZLI, 25 developed in contact with an endogenous source of Shh – floor or basal plates, the ZLI – (shown in orange) and 10 developed autonomously (shown in green). (D) Injected pre-ZLI cells develop ectopic ZLI when continuously exposed to Shh. ISH using Xshh as probe on st. 36 embryos injected into the same dorsal blastomere of 16-cell stage embryos with (a) ßgal alone, or together with barhl2, otx2, irx3 genes (b, c). The injected sides of st. 36 neural tubes are shown, side view, dorsal up, anterior left. When in contact with a source of secreted Shh, pre-ZLI-like cells (red) formed cellular clusters inside which Xshh expression is detected (stars). (c) Enlarged view of an “ectopic ZLI”. Image published in: Juraver-Geslin HA et al. (2014) Copyright © 2014. Image reproduced with permission of the Publisher, Elsevier B. V. Gene Synonyms Species Stage(s) Tissue shh.L hedgehog, vhh-1, X-SHH, Xhh, xshh X. laevis Throughout NF stage 35 and 36 zona limitans intrathalamica basal forebrain Image source: Published Larger Image Printer Friendly View

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