Abstract
Sonic hedgehog (SHH) plays a central role in patterning numerous embryonic tissues including, classically, the developing limb bud where it controls digit number and identity. This study utilises the polydactylous Silkie (Slk) chicken breed, which carries a mutation in the long range limb-specific regulatory element of SHH, the ZRS. Using allele specific SHH expression analysis combined with quantitative protein analysis, we measure allele specific changes in SHH mRNA and concentration of SHH protein over time. This confirms that the Slk ZRS enhancer mutation causes increased SHH expression in the posterior leg mesenchyme. Secondary consequences of this increased SHH signalling include increased FGF pathway signalling and growth as predicted by the SHH/GREM1/FGF feedback loop and the Growth/Morphogen models. Manipulation of Hedgehog, FGF signalling and growth demonstrate that anterior-ectopic expression of SHH and induction of preaxial polydactyly is induced secondary to increased SHH signalling and Hedgehog-dependent growth directed from the posterior limb. We predict that increased long range SHH signalling acts in combination with changes in activation of SHH transcription from the Slk ZRS allele. Through analysis of the temporal dynamics of anterior SHH induction we predict a gene regulatory network which may contribute to activation of anterior SHH expression from the Slk ZRS.
Highlights
The zone of polarising activity (ZPA) is a transient area of posterior limb bud mesenchyme with the ability to induce and pattern extra digits when grafted to the anterior border of a host wing (Saunders and Gasseling, 1968)
Demonstrate that induction of Slk polydactyly is genotype specific, as ectopic Sonic hedgehog (SHH) is not induced in anterior Wt tissue recombined with Slk posterior leg mesenchyme (Dunn et al, 2011). Based on these observations we have previously proposed a model, based on the Growth/Morphogen model (Towers et al, 2008) which suggests that extra SHH signalling observed in the posterior Slk leg may cause growth and long-range patterning effects which leads to preaxial polydactyly (Dunn et al, 2011)
The anterior Slk leg develops an ectopic area of SHH expression at late stage 25HH, which leads to preaxial polydactyly (Arisawa et al, 2006; Dunn et al, 2011)
Summary
The zone of polarising activity (ZPA) is a transient area of posterior limb bud mesenchyme with the ability to induce and pattern extra digits when grafted to the anterior border of a host wing (Saunders and Gasseling, 1968). SHH co-ordinates limb growth and digit patterning simultaneously by maintaining Hedgehogdependent growth from the posterior limb during the early digit patterning phase, resulting in digit pattern that is regulated by concentration and length of exposure to SHH directly and by the expansion of the limb field at later stages of limb development (the Growth/Morphogen model; (Harfe et al, 2004; Towers et al, 2008; Zhu et al, 2008)) This is mediated through a positive feedback loop with Fibroblast Growth Factors (FGFs) expressed in the overlying ectoderm, mediated by mesenchymal BMPantagonist Gremlin, promoting outgrowth of the limb (The SHH/ GREM1/FGF feedback loop; Laufer et al, 1994; Niswander et al, 1994; Lewandoski et al, 2000; Michos et al, 2004; Bénazet et al, 2009; Galli et al, 2010; Zeller, 2010). Both SHH expression and limb out-growth is terminated when high levels of FGF signalling inhibits GREM1 expression which disrupts the SHH/GREM1/FGF feedback loop (Verheyden and Sun, 2008)
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