Figure 5.

Zebrafish Dusp4 Is a Direct Nodal Target, Expressed in the First Two Cell Tiers, that Dephosphorylates Erk

(A) Sagittal and lateral sections of 50% epiboly embryo WISH-stained for dusp4.

(B) WISH at 50% epiboly for dusp4 and dusp6 after Nodal signaling inhibition (SB-505124) or Fgf signaling inhibition (SU-5402).

(C) qPCR using dissociated embryonic cells treated with increasing NODAL concentrations. Means ± SEM, n = 5.

(D) Chromatin immunoprecipitation for Smad2 on putative enhancers of the dusp4 gene (r1 and r2) and the dusp4 transcription start site (TSS) in 50% epiboly embryos treated with DMSO or the Nodal receptor inhibitor SB-505124. The mixl1 TSS and sox10 TSS are positive and negative controls respectively. Means ± SEM, n = 4, ^∗p < 0.05, t test.

(E and F) 50% epiboly zebrafish embryos injected with control MO (con MO) or mixl1 MO, stained for dusp4 (E) or lft1 (F) by WISH.

(G) Animal view of germ ring-stage embryo containing Ndr1-expressing clone, marked with dextran-fluorescein (dex) and stained for dusp4.

(H) Western blot showing attenuation of Fgf8a-induced P-Erk by Dusp4 at 50% epiboly.

(I) Quantification of embryos stained by FISH for dusp4 at 4.3 (dome), 4.7, and 5.0 hpf. For each time point n = 3, means are shown by the lines and the shading indicates the SD.

(J) A confocal Z-reconstruction of a 50% epiboly embryo stained for dusp4 and sox32. Nuclei are marked by DAPI. Arrow indicates a cell in the blastoderm labeled with both dusp4 and sox32. Dashed white line indicates the border of the YSL. Scale bar, 25 μm.

Scale bars, 100 μm unless otherwise stated. See also Figure S5.