Supplementary Figure 1. Etv4/5 exhibit dynamic expression in the developing chick limb bud. Both Etv4 and Etv5 are expressed in the limb mesenchyme from the earliest stages of limb development and become progressively restricted to the distal mesenchyme during limb outgrowth. The expression of these transcripts is highly conserved across species, when compared to their orthologous genes in mouse (see ).
Supplementary Figure 2. Small molecule mediated inhibition of FGF receptor-like kinase activities supports a role for Fgf signaling in the regulation of Etv4 and Etv5 expression. (A) Limb buds were dissected from Stage20/21 chick embryos and cultured in the presence of 30μM SU5402 dissolved in DMSO. Control limb buds were cultured in the presence of DMSO alone; In addition to the distal Etv4/5 expression characteristic of this developmental stage, some diffuse proximal expression was observed, an artifact of culturing conditions. Following 24h treatment, complete abolition of Etv4 and Etv5 expression (both proximal and distal) was observed in SU5402-treated limbs but not in control limbs, consistent with the requirement for an FGF receptor-like tyrosine kinase activity in Etv4/5 regulation. The maintenance of Tbx2 expression following SU5402 treatment suggests the loss of Etv4 and Etv5 is not a consequence of global loss of gene expression.
Supplementary Figure 3 (A) Schematic representation of the EtvEnR Rosa26 targeted allele, R26-EtvEnR. A cDNA fragment encoding the Etv4EnR fusion protein with a IRES-nuclear GFP tag was targeted into the ubiquitously expressed Rosa26 locus (R26) 3’ to a LoxP-flanked polyadenylation stop sequence cassette. (B) Constitutive repression of Etv4/5 does not perturb limb prepatterning. Whole mount in situ hybridization analysis of Gli3 and Hand2 in E9.25 wild-type and Prx1-Cre;R26-EtvEnR mouse embryos reveals no alterations in their expression domains.
Supplementary Figure 4. Shh expression in the zebrafish pectoral fin is dependent on a FGF receptor-like activity. Embryos were cultured from 12 hours post-fertilization in the presence of 30μM SU5402 dissolved in DMSO. Control embryos were cultured in the presence of DMSO alone. A complete abolition of erm, pea3 and etv5 expression was observed in SU5402-treated fin buds 12 hours after treatment commenced while Shh was robustly expressed in control embryos (fin buds marked with arrow), suggesting their transcription depends on Fgf signaling.
Supplementary Figure 5. Morpholino knockdown of erm, pea3 and etv5 in zebrafish. For each gene, the primary transcript is schematized; morpholino target sites are marked above with a red bar and the position of primers used to determine the resulting splicing pattern is indicated below. The DNA binding domain of erm, pea3 and etv5 is located in the 3’ region of each transcript and is spread across 3 exons as indicated in green. The predicted protein following MO injection is presented below: a non-specific amino acid fusion is indicated in red. erm morpholino was directed against the splice acceptor site upstream of exon 11 and results in a 79bp insertion of the entire intron 10. A stop codon is observed 3 amino acids into the predicted fusion protein, terminating the protein prior to critical elements required for DNA binding based on homology with other Ets-DNA binding transcription factors. pea3 morpholino was directed against the splice acceptor site upstream of exon 10 and results in a 173bp deletion, removing exon 9. This deletion puts the resultant protein out of frame creating an in-frame stop codon 15 amino acids into the predicted fusion protein. etv5 morpholino was directed against the splice acceptor site upstream of exon 7 and results in a 94bp insertion of the intron 6 resulting in translational termination at a stop codon 35 amino acids into the predicted fusion transcript.
Supplementary Figure 6. erm;pea3;etv5 morpholino knockdown results in expansion of Shh expression within the fin bud. (A) Wild-type series of Shh expression in the zebrafish embryo, from 29 hours post fertilization (hpf) to 48 hpf. A higher magnification image of the fin is shown below; scale bar represents 0.02mm. (B) Embryos were injected with triple MO mix or mismatch MO mix at the 1-2 cell stage and Shh expression assessed 48 hours post-fertilization. Shh expression in mismatch MO control embryos (n=40) appeared identical to wild-type embryos (n=20). In 20/52 experimental MO injected embryos (pea3;erm;etv5 MO_a), an increase in the intensity and domain of Shh expression was observed in the distal fin bud. For embryos where fin bud development was significantly delayed (pea3;erm;etv5 MO_b; n=20/52), an expanded domain of Shh was still observed relative to age-matched controls, the ectopic anterior Shh expression domain is arrowed. No change in Shh expression was observed in 9/52 embryos, while a decrease/loss of Shh was recorded in 3/52 embryos that also exhibited severe developmental defects.
Supplementary Figure 7. Limb development in Shh-Cre;R26-EtvEnR embryos. (A) Shh expression in the limb buds of Shh-Cre;R26-EtvEnR and R26-EtvEnR embryos at E10.5. (B) Skeletal analysis of forelimbs and hindlimbs development in R26-EtvEnR and Shh-Cre;R26-EtvEnR embryos at E16.5.
Supplementary Figure 8. Expression of EtvEnR in the chick limb bud results in anterior expansion of Shh and altered skeletal pattern. (A) EtvEnR was expressed throughout the chick limb bud using the RCAS system of gene delivery. Embryos were injected at Stage 10 into the lateral plate mesoderm and assayed for expression of Shh (blue) and Fgf8 (brown) at various stages of development. Anterior expansion of Shh was observed in EtvEnR infected limbs when compared to uninfected age-matched control embryos at early (Stage 21) and later (Stage25) of development, reflecting the pattern of ectopic Shh observed following expression of EtvEnR in mouse limb bud (compare with and ). A slight reduction in the robustness of ectopic Shh expression in some embryos likely reflects technical limitations of infection. (B) DAPI-stained cross-section through a developing chick embryo, infected with RCAS-EtvEnR at Stage 10 in the right lateral plate mesoderm. The degree of limb truncation can be appreciated when comparing infected limb (white arrow) with the contralateral control. (C) RCAS-EtvEnR infection in chick results in a range of limb abnormalities shown here by alcian blue staining of limb cartilage at Day 5 or 6 of development. Distal limb truncation was observed in a small number of embryos. More commonly, a reduction of the zeugopod with an expanded autopod with or without ectopic digits was observed.
Supplemental experimental procedures
Generation of mouse strains.
To generate the R26-EtvEnR
allele, a cDNA encoding the Etv4EnR fusion protein (Shepherd et al., 2001
) was cloned together a C-terminal IRES-nuclear GFP tag into the BigT vector (Srinivas et al., 2001). The resulting plasmid was digested to release the floxed Etv4EnR-IRES-GFP cassette, which was then inserted into pROSA26PAS (Mao et al., 2005). This targeting vector was linearized and electroporated into 3-1 embryonic stem cells (Mao et al., 2005). After G418 selection, recombinants at the ROSA26
locus were identified by visualization of loss of YFP expression, and the chimeras were then generated by blastocyst injection.
Prx1-Cre;Shhn/c;R26-EtvEnR mutant mice were obtained from the crosses of Prx1-Cre;Shhn/+ males and Shhc/+;R26-EtvEnR females, while Prx1-Cre;Fgfr1n/+;R26-EtvEnR mice were generated from the cross of Prx1-Cre;Fgfr1n/+ males and R26-EtvEnR females. To generate CAGGS-CreER; R26-EtvEnR; R26R embryos, CAGGS-CreER;R26R males were crossed to R26-EtvEnR females. Tamoxifen (Sigma) was administered at a single dose of 6mg/40g body weight by intraperitoneal injection into the pregnant dam at E8.5 and E9.5 and embryos collected at E10.5 and E11.5, respectively.
Mao, J., Barrow, J., McMahon, J., Vaughan, J., and McMahon, A. P. (2005). An ES cell system for rapid, spatial and temporal analysis of gene function in vitro and in vivo. Nucleic Acids Res 33, e155.
Shepherd, T. G., Kockeritz, L., Szrajber, M. R., Muller, W. J., and Hassell, J. A. (2001). The pea3 subfamily ets genes are required for HER2/Neu-mediated mammary oncogenesis. Curr Biol 11, 1739-1748.
Srinivas, S., Watanabe, T., Lin, C. S., William, C. M., Tanabe, Y., Jessell, T. M., and Costantini, F. (2001). Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4.
50 word summary for DC-D-08-00516
The FGF and Hedgehog pathways interlink to drive outgrowth and patterning of the vertebrate limb. We present evidence that an FGF/Etv pathway confines Sonic hedgehog expression to a posterior zone of polarizing activity. Our studies provide new insights into the signaling and transcriptional networks that govern vertebrate limb development.