Ectopic Activation of Shh and Shh Targets Results from an Inhibition of Etv-Dependent Regulation

(A) Shh expression at different stages of limb development in R26-EtvEnR and Prx1-Cre;R26-EtvEnR embryos.

(B) Expression of Gli1, Hand2, Alx4, and Gremlin transcripts in the limb buds of R26-EtvEnR and Prx1-Cre;R26-EtvEnR embryos at E10.5.

(C) Dorsal view of Shh expression in the zebrafish pectoral fin bud 48 hr postfertilization (upper); a higher-magnification view (lower). The scale bars represent 0.02 mm.

(D) Quantitative real-time PCR analysis of Gli1 mRNA levels after culturing of anterior limb bud cells for 24 hr in the presence or absence of Fgf8.

Shh Expression in the Zebrafish Pectoral Fin Is Dependent on a FGF Receptorlike 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.

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.

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.