Shh and Twhh have both redundant and unique function during MDT differentiation. Wild-type embryos or syu mutant embryos were injected with twhh-MO (0.5 mM), twhh-mRNA or both shh- and twhh-mRNAs. In wild type, ptc1 is expressed in hypothalamus, ZLI and basal plate (A). The width of the expression of ptc1 anterior and posterior to the ZLI is around 16 cells (bracket). twhh morphants show an overall reduction of ptc1 expression and the width of ptc1 domain is reduced to 14 cell diameters (B, bracket). In the syu mutants, ptc1 is down-regulated (C), and its domain at the ZLI shrinks to 5 cell diameters (bracket). In syu mutants additionally knocked-down for twhh, ptc1 expression is strongly reduced at the ZLI (D). (E) dlx2a expression in wild-type embryo at 28 hours. twhh morphants show a reduction of dlx2a in the prethalamus (F). Similarly, dlx2a is reduced in syu mutant embryos and the anterior ventral domain is not detectable (G, arrowheads). dlx2a expression is absent in the shh/twhh mutant/knockdown embryos (H), as in smu mutant embryos (I). Mis-expression of twhh-mRNA leads increased dlx2a expression in the prethalamus (J, bracket) and can be further enhanced by synchronous mis-expression of shh-mRNA (150 pg) (K, bracket). twhh morphants show similar thalamic expression of dbx1a compared with wild-type siblings (L,M). By contrast, syu mutant embryos show no detectable dbx1a in the thalamus (N), whereas expression in the tegmentum (asterisks) and ZLI (black arrow) seem unchanged. In shh/twhh mutant/knockdown embryos dbx1a expression is absent from the thalamus (O). smu mutants phenocopy the shh/twhh mutant/knockdowns (P). Mis-expression of twhh leads to weak expansion of thalamic dbx1a expression (Q), whereas combined shh/twhh mis-expression leads to a strong increase in the anteroposterior direction (R).
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