(A–C) gdf3 mutants were generated using TALEN-mediated mutagenesis. (A) Gdf3, a TGFβ family member, comprises a signal peptide, pro-domain and mature TGFβ domain. TALENs were designed to target genomic sequences located near the amino end of the pro-domain. (B) Mutants were identified by high-resolution melt analysis (HRMA). (C) Three mutant alleles, gdf3^zy51, gdf3^zy52, gdf3^z53, had 8, 1 and 6 bp deletions respectively. (D–G) Morphological phenotypes of gdf3 mutants at 24 hpf. (D) Wild-type (WT) and (E) zygotic (Z) gdf3 mutants were phenotypically indistinguishable. (F) Maternal-zygotic (MZ) gdf3 mutants lacked notochord, pharyngeal endoderm and had reduced anterior neural tissues. (G) MZgdf3 mutants were completely rescued by injection of 100 pg gdf3 RNA at the one-cell stage. (H–W) WISH analysis of gene expression in WT (columns H-T and J-V) and MZgdf3 (columns I-U and K-W) mutants at 24 hpf. (H, I) ta (ntl) expression in the notochord was absent from MZgdf3 although tailbud expression was maintained. (J, K) tbx16 (spt) expression in spinal cord neurons was absent in MZgdf3 while tailbud expression is unaffected. (L, M) Trunk and tail somites expressing myod1 were reduced in number and altered in shape in MZgdf3. (N, O) Expression of hand2 in the heart, pharyngeal arch mesoderm and pectoral fin buds is absent in MZgdf3. (P–S) Ventral neural tissues and pharyngeal endoderm expressing foxa2 (axial) were absent in MZgdf3. Patterns of expression of otx2 in the forebrain and midbrain (T, U), and erg2b (krox20) in hindbrain rhobomeres 3 and 5 (V, W) were altered in MZgdf3 mutants. All embryos (D–W) are shown in lateral view with rostral to the left except N, O, R, S which are dorsal views in the same orientation. Each panel is a representative image from at least 15 embryos.

(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.

(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.

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.