Pax9 and Jagged1 act downstream of Gli3 in vertebrate limb development

Abstract

From early in limb development the transcription factor Gli3 acts to define boundaries of gene expression along the anterior-posterior (AP) axis, establishing asymmetric patterns required to provide positional information. As limb development proceeds, posterior mesenchyme expression of Sonic hedgehog (Shh) regulates Gli3 transcription and post-translational processing to specify digit number and identity. The molecular cascades dependent on Gli3 at later stages of limb development, which link early patterning events with final digit morphogenesis, remain poorly characterised. By analysing the transcriptional consequences of loss of Gli3 in the anterior margin of the E11.5 and E12.5 limb bud in the polydactylous mouse mutant extra-toes (Gli3(Xt/Xt)), we have identified a number of known and novel transcripts dependent on Gli3 in the limb. In particular, we demonstrated that the genes encoding the paired box transcription factor Pax9, the Notch ligand Jagged1 and the cell surface receptor Cdo are dependent on Gli3 for correct expression in the anterior limb mesenchyme. Analysis of expression in compound Shh;Gli3 mutant mouse embryos and in both in vitro and in vivo Shh signaling assays, further defined the importance of Shh regulated processing of Gli3 in controlling gene expression. In particular Pax9 regulation by Shh and Gli3 was shown to be context dependent, with major differences between the limb and somite revealed by Shh bead implantation experiments in the chick. Jagged1 was shown to be induced by Shh in the chick limb and in a C3H10T1/2 cell based signaling assay, with Shh;Gli3 mutant analysis indicating that expression is dependent on Gli3 derepression. Our data have also revealed that perturbation of early patterning events within the Gli3(Xt/Xt) limb culminates in a specific delay of anterior chondrogenesis which is subsequently realised as extra digits.

Expression analysis of limb element markers during mouse embryonic development

“…Gdf5 is expressed across developing joint sites, including interphalangeal joints, from E14.5 (Chen et al, 2016; Houweling et al, 2001; Huang et al, 2016; Sohaskey et al, 2009). Our findings for Scleraxis expression are in agreement with previously published analyses of Scleraxis , indicating expression in the autopod and in developing digit rays and cartilage condensations at E12.5, as well as defined expression in developing tendons at E13.5 (Huang et al, 2015; McGlinn et al, 2005; Schweitzer et al, 2001) and along developing digits, with strongest expression across sites of developing interzones at E14.5 (Murchison et al, 2007; Schweitzer et al, 2001). MyoD1 is expressed in muscle progenitors in the zeugopod, not the autopod, at E12.5.…”

Sonic Hedgehog regulates Hes1 through a novel mechanism that is independent of canonical Notch pathway signalling

“…We demonstrate that the regulation of Hes1 by Shh does not involve g-secretase-mediated Notch cleavage, and likely involves transcription factors other than RBP-Jk. This appears to be a separate effect to the recent discovery of Jagged upregulation via Shh/Gli3 (McGlinn et al, 2005). Therefore, in mammals the Hedgehog pathway influences the Notch pathway at multiple points.…”

A Novel Interaction Between hedgehog and Notch Promotes Proliferation at the Anterior–Posterior Organizer of the Drosophila Wing

“…Xt mutant (McGlinn et al 2005); reduced expression of Jagged1 and Notch2 in the cerebella of mice with reduced SHH signaling (Dakubo et al 2006); regulation of the Deltarelated ligand, DNER, by SHH in Purkinje neurons and fetal prostate (Dahmane and Ruiz i Altaba 1999;Wallace 1999;Tohgo et al 2006;Yu et al 2009); activation of N signaling in neuroblastomas in Ptch 1/À mice with elevated SHH signaling (Dakubo et al 2006); and Notch2 overexpression in mice carrying an activated allele of smo (Hallahan et al 2004). These studies establish a positive effect of SHH signaling on the N pathway, consistent with our data.…”