Inactivation of IL11 signaling causes craniosynostosis, delayed tooth eruption, and supernumerary teeth.

Craniosynostosis and supernumerary teeth most often occur as isolated developmental anomalies, but they are also separately manifested in several malformation syndromes. Here, we describe a human syndrome featuring craniosynostosis, maxillary hypoplasia, delayed tooth eruption, and supernumerary teeth. We performed homozygosity mapping in three unrelated consanguineous Pakistani families and localized the syndrome to a region in chromosome 9. Mutational analysis of candidate genes in the region revealed that all affected children harbored homozygous missense mutations (c.662C>G [p.Pro221Arg], c.734C>G [p.Ser245Cys], or c.886C>T [p.Arg296Trp]) in IL11RA (encoding interleukin 11 receptor, alpha) on chromosome 9p13.3. In addition, a homozygous nonsense mutation, c.475C>T (p.Gln159X), and a homozygous duplication, c.916_924dup (p.Thr306_Ser308dup), were observed in two north European families. In cell-transfection experiments, the p.Arg296Trp mutation rendered the receptor unable to mediate the IL11 signal, indicating that the mutation causes loss of IL11RA function. We also observed disturbed cranial growth and suture activity in the Il11ra null mutant mice, in which reduced size and remodeling of limb bones has been previously described. We conclude that IL11 signaling is essential for the normal development of craniofacial bones and teeth and that its function is to restrict suture fusion and tooth number. The results open up the possibility of modulation of IL11 signaling for the treatment of craniosynostosis.

Prevention of premature fusion of calvarial suture in GLI-Kruppel family member 3 (Gli3)-deficient mice by removing one allele of Runt-related transcription factor 2 (Runx2).

Mutations in the gene encoding the zinc finger transcription factor GLI3 (GLI-Kruppel family member 3) have been identified in patients with Grieg cephalopolysyndactyly syndrome in which premature fusion of calvarial suture (craniosynostosis) is an infrequent but important feature. Here, we show that Gli3 acts as a repressor in the developing murine calvaria and that Dlx5, Runx2 type II isoform (Runx2-II), and Bmp2 are expressed ectopically in the calvarial mesenchyme, which results in aberrant osteoblastic differentiation in Gli3-deficient mouse (Gli3(Xt-J/Xt-J)) and resulted in craniosynostosis. At the same time, enhanced activation of phospho-Smad1/5/8 (pSmad1/5/8), which is a downstream mediator of canonical Bmp signaling, was observed in Gli3(Xt-J/Xt-J) embryonic calvaria. Therefore, we generated Gli3;Runx2 compound mutant mice to study the effects of decreasing Runx2 dosage in a Gli3(Xt-J/Xt-J) background. Gli3(Xt-J/Xt-J) Runx2(+/-) mice have neither craniosynostosis nor additional ossification centers in interfrontal suture and displayed a normalization of Dlx5, Runx2-II, and pSmad1/5/8 expression as well as sutural mesenchymal cell proliferation. These findings suggest a novel role for Gli3 in regulating calvarial suture development by controlling canonical Bmp-Smad signaling, which integrates a Dlx5/Runx2-II cascade. We propose that targeting Runx2 might provide an attractive way of preventing craniosynostosis in patients.

Noggin null allele mice exhibit a microform of holoprosencephaly.

Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin(-/-) mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin(-/-) mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin(-/-) mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HPE.

Gli3Xt-J/Xt-J mice exhibit lambdoid suture craniosynostosis which results from altered osteoprogenitor proliferation and differentiation.

Gli3 is a zinc-finger transcription factor whose activity is dependent on the level of hedgehog (Hh) ligand. Hh signaling has key roles during endochondral ossification; however, its role in intramembranous ossification is still unclear. In this study, we show that Gli3 performs a dual role in regulating both osteoprogenitor proliferation and osteoblast differentiation during intramembranous ossification. We discovered that Gli3Xt-J/Xt-J mice, which represent a Gli3-null allele, exhibit craniosynostosis of the lambdoid sutures and that this is accompanied by increased osteoprogenitor proliferation and differentiation. These cellular changes are preceded by ectopic expression of the Hh receptor Patched1 and reduced expression of the transcription factor Twist1 in the sutural mesenchyme. Twist1 is known to delay osteogenesis by binding to and inhibiting the transcription factor Runx2. We found that Runx2 expression in the lambdoid suture was altered in a pattern complimentary to that of Twist1. We therefore propose that loss of Gli3 results in a Twist1-, Runx2-dependent expansion of the sutural osteoprogenitor population as well as enhanced osteoblastic differentiation which results in a bony bridge forming between the parietal and interparietal bones. We show that FGF2 will induce Twist1, normalize osteoprogenitor proliferation and differentiation and rescue the lambdoid suture synostosis in Gli3Xt-J/Xt-J mice. Taken together, we define a novel role for Gli3 in osteoblast development; we describe the first mouse model of lambdoid suture craniosynostosis and show how craniosynostosis can be rescued in this model.

Convergent signalling through Fgfr2 regulates divergent craniofacial morphogenesis.

Fibroblast growth factor receptor 2 (Fgfr2) has two splice variants IIIb and IIIc, which are unique in function and localization. Signalling through Fgfr2IIIb controls epithelial-mesenchymal interactions, which regulate morphogenesis during the development of several organs including the palate and tooth. In this study, we confirm that molar tooth development in Fgfr2IIIb(-/-) mice is arrested early in development and that the molar teeth of Fgf10(-/-) mice develop through all the normal stages of morphogenesis. We show that the molar phenotype of Fgfr2IIIb(-/-) mice is, in part, owing to reduced cell proliferation in both epithelial and mesenchymal compartments. We also show that the developing molar teeth of Fgf10(-/-) mice exhibit reduced cell proliferation. However, this reduction is not sufficient to arrest molar development. Recent evidence has indicated that Fgfr2IIIb/Fgf10 signalling is active in the calvaria in some pathological situations as heterozygous deletion of Fgfr2 exon IIIc in mice leads to ectopic expression of Fgfr2IIIb in the calvarial bones and causes craniosynostosis. Here, we investigate the mRNA expression of Fgfr2IIIb and Fgfr2IIIc as well as their ligands Fgf3, -7 and -10 in the developing murine tooth, palate and calvaria. We show that Fgf7 is expressed in the calvarial mesenchyme adjacent to the developing frontal bone and Fgf10 is expressed by osteoprogenitors in the developing frontal bone condensation. Taken together, we highlight the overlapping roles of Fgfr2IIIb/Fgf10 signalling in controlling epithelial-mesenchymal interactions during normal palate and tooth morphogenesis and how elevated signalling through Fgfr2IIIb/Fgf10 solely within the mesenchyme can result in abnormal calvarial morphogenesis.

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

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.

Loss-of-Function of Gli3 in Mice Causes Abnormal Frontal Bone Morphology and Premature Synostosis of the Interfrontal Suture.

Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder with polydactyly and syndactyly of the limbs and a broad spectrum of craniofacial abnormalities. Craniosynostosis of the metopic suture (interfrontal suture in mice) is an important but rare feature associated with GCPS. GCPS is caused by mutations in the transcription factor GLI3, which regulates Hedgehog signaling. The Gli3 loss-of-function (Gli3(Xt-J/Xt-J)) mouse largely phenocopies the human syndrome with the mice exhibiting polydactyly and several craniofacial abnormalities. Here we show that Gli3(Xt-J/Xt-J) mice exhibit ectopic ossification in the interfrontal suture and in the most severe cases the suture fuses already prior to birth. We show that abnormalities in frontal bones occur early in calvarial development, before the establishment of the interfrontal suture. It provides a model for the metopic suture pathology that can occur in GCPS.