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Fig. 1
Zebrafish fscn1a is a direct TGF-β/Nodal target gene.
(a) The spatiotemporal expression pattern of fscn1a during zebrafish embryogenesis was examined by in situ hybridization at the indicated stages. Stages and directionality are as follows: From the 1-cell to 1k-cell stages, lateral views with animal pole to the top; from 30% epiboly to mid-gastrulation stages, lateral views with dorsal to the right; bud stage, dorsal view with anterior to the top; 24 h.p.f., lateral views with anterior to the left. epi, epiboly. (b) Temporal expression profile of fscn1a was determined by semi-quantitative RT–PCR (upper panels) and western blot (lower panels). Total RNA isolates and proteins were prepared from embryos at the indicated stages. (c) fscn1a expression was assessed by in situ hybridization in wild-type (WT) and MZoep mutant embryos. Embryos injected with 1 pg sqt mRNA were also collected at the 30% epiboly and shield stages and subjected to in situ hybridization. The ratios of affected embryos are indicated. (d) 0.05 pg sqt mRNA was injected into a single cell located in the animal pole at the 64-cell stage. 20 pg egfp mRNA was co-injected as a cell lineage tracer. Left panel, EGFP-labelled cells were observed at the shield stage by fluorescence microscopy; middle and right panels, in situ hybridization analysis of fscn1a expression in shield stage embryos following single-cell injection. (e) fscn1a expression in sqt mRNA injected embryos after CHX treatment. Embryos were injected with 1 pg sqt mRNA at the one-cell stage and incubated with 50 µg ml-l CHX from the 128-cell to shield stages and then collected for in situ hybridization. Animal pole views with dorsal to the right. Scale bar, 200 µm.