Direct functional consequences of ZRS enhancer mutation combine with secondary long range SHH signalling effects to cause preaxial polydactyly

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.

Postaxial limb hypoplasia (PALH): the classification, clinical features, and related developmental biology

“…Point mutations in the ZRS gene can induce anterior, ectopic Shh expression and can cause preaxial polydactyly, triphalangeal thumb, Werner mesomelic syndrome, or tibial hypoplasia. 59,[61][62][63][64][65][66] Duplications and even triplications of the ZRS have been implicated in polysyndactyly disease, including triphalangeal thumb-polysyndactyly syndrome and Haas-type polysyndactyly syndrome. 65,67,68 Second, the activation and posterior restriction of Shh expression in the ZPA are maintained by mutual antagonism between the transcription factors HAND2 and GLI3.…”

“…59,[61][62][63][64][65][66] Duplications and even triplications of the ZRS have been implicated in polysyndactyly disease, including triphalangeal thumb-polysyndactyly syndrome and Haas-type polysyndactyly syndrome. 65,67,68 Second, the activation and posterior restriction of Shh expression in the ZPA are maintained by mutual antagonism between the transcription factors HAND2 and GLI3. 40 Initially, HAND2 and GLI3 are expressed throughout the presumptive limb field, and, during the onset of limb bud outgrowth, direct cross-regulation results in posterior restriction of HAND2 and anterior restriction of GLI3.…”

Novel <b><i>KIF7</i></b> Mutation in a Tunisian Boy with Acrocallosal Syndrome: Case Report and Review of the Literature

“…Polydactyly, cardiac defects and malformations of cerebral midline structures including agenesis of the corpus callosum or holoprosencephaly are also commonly observed in patients with Smith-Lemli-Opitz syndrome, an autosomal recessive metabolic disorder resulting from 7-dehydrocholesterol deficiency preventing the last step of cholesterol biosynthesis and its binding to SHH [Nowaczyk and Irons, 2012]. Moreover, gain-of-function mutations within an SHH regulatory element 1 Mb upstream of SHH , the zone of polarizing activity regulatory sequence, also cause a characteristic spectrum of limb malformations including polydactyly [Johnson et al, 2014].…”

Upstream regulation for initiation of restricted Shh expression in the chick limb bud

“…Shh gene expression is restricted posteriorly throughout limb development (Riddle et al, ; Echelard et al, ; Lewis et al, ; Endo et al, ), and Shh protein makes a density gradient from the posterior to the anterior side, specifying digit identities (Lewis et al, ; Harfe et al, ; Scherz et al, ; Towers et al, ). Alteration of Shh protein gradient results in changes in the number and identity of digits (Lewis et al, ; Li et al, ; Scherz et al, ; Johnson et al, ). Moreover, ectopic expression of Shh in the anterior limb bud gives rise to mirror‐image duplication of digits in a concentration‐dependent manner (Masuya et al, ; Masuya et al, ; reviewed by Hill et al, ).…”

“…Regulation of the restricted Shh expression can be separated into the phases of initiation, maintenance, and termination. In many studies, misregulation of Shh expression was performed at the maintenance phase after the initiation of Shh expression (Masuya et al, ; Yang et al, ; Fernandez‐Teran et al, ; Johnson et al, ; reviewed by Al‐Qattan, ). Alteration of the initial Shh expression pattern also has effects, including polydactylous phenotype, on limb morphogenesis (Zákány et al, ; Mao et al, ; Xu et al, ), indicating that initiation of the restricted Shh ‐expressing region is strictly regulated and is important for limb morphogenesis.…”