Differential regulation of gene expression in the digit forming area of the mouse limb bud by SHH and gremlin 1/FGF-mediated epithelial-mesenchymal signalling

Abstract

Spatially and temporally coordinated changes in gene expression are crucial to orderly progression of embryogenesis. We combine mouse genetics with experimental manipulation of signalling to analyze the kinetics by which the SHH morphogen and the BMP antagonist gremlin 1 (GREM1) control gene expression in the digit-forming mesenchyme of mouse limb buds. Although most mesenchymal cells respond rapidly to SHH signalling, the transcriptional upregulation of specific SHH target signals in the mesenchyme occurs with differential temporal kinetics and in a spatially restricted fashion. In particular, the expression of the BMP antagonist Grem1 is always upregulated in mesenchymal cells located distal to the SHH source and acts upstream of FGF signalling by the apical ectodermal ridge. GREM1/FGF-mediated feedback signalling is, in turn, required to propagate SHH and establish the presumptive digit expression domains of the Notch ligand jagged 1 (Jag1) and 5ЈHoxd genes in the distal limb bud mesenchyme. Their establishment is significantly delayed in Grem1-deficient limb buds and cannot be rescued by specific restoration of SHH signalling in mutant limb buds. This shows that GREM1/FGF feedback signalling is required for regulation of the temporal kinetics of the mesenchymal response to SHH signalling. Finally, inhibition of SHH signal transduction at distinct time points reveals the differential temporal dependence of Grem1, Jag1 and 5ЈHoxd gene expression on SHH signalling. In particular, the expression of Hoxd13 depends on SHH signal transduction significantly longer than does Hoxd11 expression, revealing that the reverse co-linear establishment, but not maintenance of their presumptive digit expression domains, depends on SHH signalling.

A Gli silencer is required for robust repression of gremlin in the vertebrate limb bud

“…−/− embryos (Aoto et al, 2002;Litingtung et al, 2002;Panman et al, 2006;te Welscher et al, 2002;Zúñiga et al, 1999). Collectively, these studies illustrate the profound importance of Shh in counteracting Gli3-mediated repression of target genes.…”

“…To determine the time period during which GRE1 requires Gli activator for enhancer activity in the posterior limb, we used an established ex vivo limb bud culture assay, treating GRE1LacZ +/− limb buds with the Hh pathway inhibitor cyclopamine (Panman et al, 2006). We cultured one forelimb in media containing cyclopamine while the contralateral side was cultured in control media, providing an internal control for staging and embryo variability (Fig.…”

“…To circumvent this problem, we performed additional limb bud cultures on 32-and 38-somite embryos and measured lacZ expression by qRT-PCR. As a control to ensure that the experimental conditions resulted in robust inhibition of Shh signaling, we measured the expression of the obligate Shh target gene Gli1 (Panman et al, 2006). When forelimbs from 32-somite embryos were cultured, they had an 84% reduction in Gli1 gene expression and a 75% reduction in lacZ expression.…”

Shh pathway activation is present and required within the vertebrate limb bud apical ectodermal ridge for normal autopod patterning

“…The expression of the genes involved in the Shh/Grem1/Fgf feedback loop is dynamic and dependent on the timing and levels of other components in the regulatory network (16)(17)(18)(19). To investigate the effect of removal of Hh signaling from the AER on the components of the Shh/Grem1/Fgf feedback loop, double RNA in situ hybridizations for Shh and Fgf4 or Fgf8 were performed on normal and Msx2-Cre;Smo flox/flox embryos.…”

SHH propagates distal limb bud development by enhancing CYP26B1-mediated retinoic acid clearance via AER-FGF signalling

“…We have investigated the transcriptome alterations between Shh-deficient and wild-type forelimb buds around E10.5 (35-36 somites), which corresponds to the period during which limb bud development largely depends on SHH signalling Zhu et al, 2008;Panman et al, 2006). Unexpectedly, this analysis revealed that forelimb buds of Shh-deficient mouse embryos were molecularly proximalised as the expression of several proximal genes was upregulated and their domains extended distally.…”