Fgf-dependent Etv4/5 activity is required for posterior restriction of Sonic Hedgehog and promoting outgrowth of the vertebrate limb - PubMed (original) (raw)

Fgf-dependent Etv4/5 activity is required for posterior restriction of Sonic Hedgehog and promoting outgrowth of the vertebrate limb

Junhao Mao et al. Dev Cell. 2009 Apr.

Abstract

Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. Here, we show that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development.

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Figures

Figure 1

Figure 1. Etv4/5 and Fgf8 exhibit dynamic expression in the developing mouse limb bud

Whole mount in situ hybridization with antisense probes for Etv4, Etv5 and Fgf8 at different stages of wild type limb bud development. Correlated expression in the limb ectoderm (Fgf8) and adjacent mesenchyme (Etv4/5) was detected at as early as E8.75 and maintained until E12.5.

Figure 2

Figure 2. Repressing Etv target genes results in an FgfR1 dosage sensitive shortening of limb elements and Shh dependent polydactyly

(A) Skeletal analysis of wild type R26-EtvEnR and mutant Prx1-Cre;R26-EtvEnR limbs at postnatal day 1 (P1). (B) Skeletal analysis of P1 forelimbs of R26-EtvEnR, Prx1-Cre;R26-EtvEnR, Prx1-Cre;Shhn/c, and Prx1-Cre;R26-EtvEnR;Shhn/c mice. (C) Skeletal analysis of P15 forelimbs of Prx1-Cre;Fgfr1 n/+, Prx1-Cre;R26-EtvEnR and Prx1-Cre;R26-EtvEnR;Fgfr1 n/+ mice. Arrow head (black) points to the enhanced zeugopod phenotype in the Prx1-Cre;R26-EtvEnR;Fgfr1 n/+ forelimb.

Figure 3

Figure 3. 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 the zebrafish pectoral fin bud 48 hours post fertilization (upper); a higher magnification view (lower), scale bar represents 0.02 mm. (D) Quantitative real time PCR analysis of Gli1 mRNA levels following culturing of anterior limb bud cells for 24h in the presence or absence of Fgf8.

Figure 4

Figure 4. Etv4/5 function in early distal mesenchyme to restrict Shh expression

(A) CAGGS-CreER;R26-EtvEnR;R26R embryos were treated with tamoxifen at E8.75 or E9.5 and collected at E10.5 or E11.5, respectively. Left panels showedβ-gal staining in contralateral limbs indicating recombination after tamoxifen administration. (B) Effect of AER removal and/or RCAS-EtvEnR infection on Shh expression (black arrow heads) in the chick limb. AER removal was verified by assaying Fgf8 expression (white arrow). (C) Whole mount in situ hybridization analysis of Shh expression in control chick limbs (left panels) following removal of the posterior quarter of the limb (including the ZPA) at stage 17/18 (right panels) with (lower) or without (upper) RCAS-EtvEnR infection.

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