Regulation of Calvarial Osteogenesis by Concomitant De-repression of GLI3 and Activation of IHH Targets

Abstract

Loss-of-function mutations in GLI3 and IHH cause craniosynostosis and reduced osteogenesis, respectively. In this study, we show that Ihh ligand, the receptor Ptch1 and Gli transcription factors are differentially expressed in embryonic mouse calvaria osteogenic condensations. We show that in both Ihh−/− and Gli3Xt−J/Xt−J embryonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone development. RUNX2 is a master regulatory transcription factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expanded and aberrant osteogenesis observed in Gli3Xt−J/Xt−J mice, and consistent with Runx2-I expression by relatively immature osteoprogenitors. Ihh−/− mice exhibited small calvarial bones and HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and Ihh, we generated Ihh−/−;Gli3Xt−J/Xt−J compound mutant mice. Even in the absence of Ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the craniosynostosis phenotype of Gli3Xt−J/Xt−J mice is not dependent on IHH ligand. Also, we found that Ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH, we performed RUNX2 siRNA knock down experiments in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embryonic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentiation. As GLI3 represses the expression of Runx2-II and Ihh, and also elevates the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regulatory feedback circuit to control calvarial osteogenesis and suture patency. Taken together, RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstream targets.

Structural Genome Variations Related to Craniosynostosis

“…Depending on which node of these craniosynostosis-related networks is affected, a distinct craniosynostosis syndrome or set of phenotypes arises. Recent findings have raised the image of digenic inheritance in some cases of craniosynostosis, which calls for genome-wide investigation involving array-CGH, WES or WGS [Timberlake et al, 2017;Veistinen et al, 2017]. A molecular diagnosis of familial, syndromic craniosynostosis cases can be achieved through targeted sequencing of the genes identified by linkage analyses (e.g., EFNB1 , FGFR1 , FGFR2 , FGFR3 , MSX2 , POR , RAB23 , and TWIST1 ) [Kutkowska-Kaźmierczak et al, 2018].…”

“…In cases with nonsyndromic midline craniosynostosis, a digenic mechanism, involving concurrent presence of risk alleles of BMP2 and SMAD6 , has also been discovered [Timberlake, et al, 2016]. Also concomitant de-repression of GLI3 and activation of IHH targets has been demonstrated [Veistinen et al, 2017]. Thus, molecular diagnosis of craniosynostosis beyond Saethre-Chotzen, Crouzon, Pfeiffer, Apert, and Jackson-Weiss syndromes based on mutations in the TWIST1 , FGFR1 and FGFR2 genes has been complemented with a widening spectrum of loci and genes ( Table 1 ) [Jabs et al, 1993[Jabs et al, , 1994Muenke et al, 1994;Reardon et al, 1994;Park et al, 1995;Rutland et al, 1995;Wilkie et al, 1995;Wieland et al, 2004;Roscioli et al, 2013;Twigg and Wilkie, 2015;Miller et al, 2017;Timberlake et al, 2017;Timberlake and Persing, 2018].…”

Identification of Gli1‐interacting proteins during simvastatin‐stimulated osteogenic differentiation of bone marrow mesenchymal stem cells

“…Ihh regulates osteogenic differentiation by modulating Runx2‐controlled osteoblastic cell fates . Gli1‐positive cells located beneath the growth plate contribute to both chondrocytes and osteoblasts during bone fracture healing .…”

The Right Gene, Expressed at the Wrong Time, or at the Wrong Place

“…At the epiphysis, the thyroid hormone fine tunes levels of IHH and thereby controls the transit of proliferating immature chondrocytes into mature hypertrophic chondrocytes to eventually become osteoblasts [Aghajanian et al, 2017]. While RUNX2 is the master regulatory transcription factor controlling osteogenesis, it is itself regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstream targets [Veistinen et al, 2017]. This suggests that IHH is a key element in a regulatory feedback circuit controlling skull osteogenesis and suture patency [Veistinen et al, 2017].…”

“…While RUNX2 is the master regulatory transcription factor controlling osteogenesis, it is itself regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstream targets [Veistinen et al, 2017]. This suggests that IHH is a key element in a regulatory feedback circuit controlling skull osteogenesis and suture patency [Veistinen et al, 2017]. Together with other In medical genetics, clinically relevant mutations are usually thought to damage a gene or to alter its copy number.…”