The site of ectopic Nodal/Gdf3 signaling influences the efficacy of MZgdf3 mutant rescue and the severity of overexpression phenotypes in WT embryos. (A) Experimental Approach: 4–8 cell WT and MZgdf3 embryos were injected with 5 pg ndr2 RNA +25 pg eGFP RNA or with 50 pg gdf3 RNA +25 pg eGFP RNA. At 50% epiboly embryos expressing eGFP in 25% or less of the blastoderm were selected. These embryos were grown until 24 hpf and photographed with transmitted light and fluorescent illumination or were grown until shield stage and processed by WISH with a gsc probe then by IHC with anti-GFP. (B, C) 24 hpf WT embryos injected with gdf3/eGFP RNA had normal phenotypes, regardless of whether (B) midline or (C) non-midline lineages were targeted. (D) MZgdf3 embryos expressing Gdf3/eGFP in dorsally-derived midline lineages including the notochord and polster showed complete morphological rescue of notochord, somites, brain and eyes. (E) MZgdf3 embryos with expression of Gdf3/eGFP in non-midline lineages including the skin and somites showed rescue of somites and notochord but lacked normal development of anterior neural tissues and eyes. (F) Expression of Ndr2/eGFP in dorsal midline lineages in WT embryos resulted in embryos that were predominantly normal but some exhibited slightly narrower head and trunk and kinked notochords. This is strikingly distinct from (G) expression of Ndr2/eGFP outside midline lineages, which strongly dorsalized the embryos, or from ubiquitous expression in WT embryos injected with ndr2 RNA at the 1–2 cell stage (Figure 3S–U). (H, I) MZgdf3 embryos injected with ndr2/eGFP RNA showed no rescue of the MZgdf3 mutant phenotype regardless of what lineages were targeted. (J–Y) Shield stage embryos that were injected at the 4–8 cell stage with the indicated RNAs were processed by WISH for gsc and by IHC for GFP. Purple cells express gsc RNA; brown cells express GFP from RNA injection. Panels show embryos representative of each phenotypic class. (J–M) WT embryos at shield stage that were injected with gdf3/eGFP RNA showed no alteration in, or ectopic expression of gsc regardless of the location of expressing cells. (N–Q) MZgdf3 embryos injected with gdf3/eGFP RNA showed rescue of gsc expression when the expressing cells were located at (O), or adjacent to the presumptive dorsal shield (P). (R–U) WT embryos injected with ndr2/eGFP RNA showed ectopic gsc expression associated with the clone of expressing cells, regardless of the location of these cells within the embryo. (V–Y) MZgdf3 embryos injected with ndr2/eGFP RNA were unresponsive to this nodal ligand and showed no gsc expression. Note: Due to the lack of gsc expression, the location of the GFP-expressing clone of cells relative to the dorsal axis could not be reliably assigned in these embryos.