(A–J’) WISH analysis of gene expression in embryos at 90% epiboly. Columns from left to right show WT embryos, WT embryos injected with 100 pg of gdf3 RNA, MZgdf3 mutants and MZgdf3 injected with gdf3 RNA. Each panel is a representative image of at least 15 embryos examined. (A–L) Midline and margin expression of Nodal signaling pathway genes ndr2 (A–D) and Lefty family members lft1 (E–H) and lft2 (I–L) were absent in MZgdf3 mutants and restored by gdf3 mRNA injection. (M–X) Analysis of early mesoderm transcription factor genes. (M–T). Expression domains of gsc, ta (ntl) and tbx16 (spt) were absent from the midline of MZgdf3 mutants, but restored by gdf3 RNA injection. (Q–X) Lateral and ventral mesendoderm expression domains of tbx16 (Q–T), and eve1 (U–X), which were reduced in width in MZgdf3, were restored to wild-type levels by gdf3 RNA. (Y–F’) Endoderm expression domains of transcription factors sox17 (Y–B’) and foxa2 (C’–F’) were absent in MZgdf3, and restored by gdf3 RNA, as was expression of foxa2 in midline neural tissues. (G’–J’). otx2 expression in the anterior neural plate is reduced in MZgdf3 but rescued to its normal extent by gdf3 RNA. (Second column from left) Strikingly, although injection of gdf3 RNA was capable of rescuing mesoderm, endoderm and neural tissues in MZgdf3 mutants it had no effect on gene expression in WT embryos.