Fig. 4

Temporally regulated Gli3 repression of the Hh target gene fkd4. (A, B, D) Control MO-injected wild-type embryos with normal expression of fkd4 (A, B) and axial (D) in the ventral brain, medial floor plate, and lateral floor plate. (E, F) gli3MO-injected wild-type embryos had normal expression of fkd4 and gli1 (inset) at 19 h (E) but had dorsally expanded fkd4 (F, U) and gli1 (F inset) expression at 24 h, revealing a temporally regulated repressor role for zebrafish Gli3. (C, G, T, V) smu(smo) mutant embryos had reduced fkd4 expression (C, T, arrowheads), and gli3MO injections into smu(smo) mutant embryos did not lead to dorsal expansion of fkd4 (G, V, arrowheads) indicating that active Hh signaling is necessary for this expansion. As in smu(smo) mutants (Karlstrom et al., 2003), cyclopamine treatment starting at shield stage led to an extreme reduction of gli1 expression at 20 h (C inset). gli3MO injection resulted in a dramatic increase in gli1 expression in cyclopamine-treated embryos (G inset). (D, H) No expansion of axial was detected in gli3MO-injected embryos (H), indicating target specificity for the Gli3 repressor. (I, J) gli2MO injection into wild-type embryos did not lead to expanded fkd4 expression either at 19 h (I) or at 24 h (J). (K, L) In contrast, a few cells ectopically expressed fkd4 in smu(smo) mutants (K) and axial in wild-type embryos (L) (see insets) after gli2MO injection, revealing a weak, Hh-independent, repressor function for Gli2. (M, N) Coinjection of gli3MOs and gli2MOs resulted in dorsal expansion of fkd4 in wild-type embryos at 24 h (N) but not 19 h (M), similar to single gli3MO injections. (O) Double gli2MO + gli3MO injections also led to ectopic fkd4 expression in a few dorsal neural cells in smu(smo) mutants at 24 h and expanded axial expression (P), similar to gli2MO injections alone. (Q, R) shh RNA-injected wild-type embryos had dorsally expanded fkd4 expression at both 19 h (Q) and 24 h (R) compared to lacZ mRNA-injected embryos (inset), showing that Shh signaling is capable of inducing fkd4 expression at both of these ages. (S) Cross-section shows fkd4 expression in the medial (arrow) and lateral (arrowhead) floor plate cells. (T) In smu(smo) mutants, fkd4 is expressed only in the medial floor plate (arrow) and is lost in lateral floor plate (arrowhead). (U) gli3MO injection into wild-type embryos led to dorsally expanded fkd4 expression. (V) gli3MO injection into smu(smo) mutant embryos did not rescue of fkd4 expression in lateral floor plate cells or expand fkd4 expression dorsally. (W) Similarly, injection of gli3MOs and gli2MOs into smu(smo) mutants did not rescue fkd4 expression in lateral floor plate or expand fkd4 expression dorsally. (A–R) Side views of zebrafish embryos, yolks and eyes removed, anterior to the left. (S–W) Cross-sections of the trunk region, anterior up.