Sonic hedgehog signaling in craniofacial development.

Mutations in SHH and several other genes encoding components of the Hedgehog signaling pathway have been associated with holoprosencephaly syndromes, with craniofacial anomalies ranging in severity from cyclopia to facial cleft to midfacial and mandibular hypoplasia. Studies in animal models have revealed that SHH signaling plays crucial roles at multiple stages of craniofacial morphogenesis, from cranial neural crest cell survival to growth and patterning of the facial primordia to organogenesis of the palate, mandible, tongue, tooth, and taste bud formation and homeostasis. This article provides a summary of the major findings in studies of the roles of SHH signaling in craniofacial development, with emphasis on recent advances in the understanding of the molecular and cellular mechanisms regulating the SHH signaling pathway activity and those involving SHH signaling in the formation and patterning of craniofacial structures.

Developmental origin of the mammalian premaxilla.

The evolution of jaws has played a major role in the success of vertebrate expansion into a wide variety of ecological niches. A fundamental, yet unresolved, question in craniofacial biology is about the origin of the premaxilla, the most distal bone present in the upper jaw of all amniotes. Recent reports have suggested that the mammalian premaxilla is derived from embryonic maxillary prominences rather than the frontonasal ectomesenchyme as previously shown in studies of chicken embryos. However, whether mammalian embryonic frontonasal ectomesenchyme contributes to the premaxillary bone has not been investigated and a tool to trace the contributions of the frontonasal ectomesenchyme to facial structures in mammals is lacking. The expression of the Alx3 gene is activated highly specifically in the frontonasal ectomesenchyme, but not in the maxillary mesenchyme, from the beginning of facial morphogenesis in mice. Here, we report the generation and characterization of a novel Alx3CreERT2 knock-in mouse line that express tamoxifen-inducible Cre DNA recombinase from the Alx3 locus. Tamoxifen treatment of Alx3CreERT2/+;Rosa26mTmG/+ embryos at E7.5, E8.5, E9.5, and E10.5, each induced specific labeling of the embryonic medial nasal and lateral nasal mesenchyme but not the maxillary mesenchyme. Lineage tracing of Alx3CreERT2-labeled frontonasal mesenchyme from E9.5 to E16.5 clearly showed that the frontonasal mesenchyme cells give rise to the osteoblasts generating the premaxillary bone. Furthermore, we characterize a Dlx1-Cre BAC transgenic mouse line that expresses Cre activity in the embryonic maxillary but not the frontonasal mesenchyme and show that the Dlx1-Cre labeled embryonic maxillary mesenchyme cells contribute to the maxillary bone as well as the soft tissues lateral to both the premaxillary and maxillary bones but not to the premaxillary bone. These results clearly demonstrate the developmental origin of the premaxillary bone from embryonic frontonasal ectomesenchyme cells in mice and confirm the evolutionary homology of the premaxilla across amniotes.

Alx1 Deficient Mice Recapitulate Craniofacial Phenotype and Reveal Developmental Basis of ALX1-Related Frontonasal Dysplasia.

Loss of ALX1 function causes the frontonasal dysplasia syndrome FND3, characterized by severe facial clefting and microphthalmia. Whereas the laboratory mouse has been the preeminent animal model for studying developmental mechanisms of human craniofacial birth defects, the roles of ALX1 in mouse frontonasal development have not been well characterized because the only previously reported Alx1 mutant mouse line exhibited acrania due to a genetic background-dependent failure of cranial neural tube closure. Using CRISPR/Cas9-mediated genome editing, we have generated an Alx1-deletion mouse model that recapitulates the FND craniofacial malformations, including median orofacial clefting and disruption of development of the eyes and alae nasi. In situ hybridization analysis showed that Alx1 is strongly expressed in frontonasal neural crest cells that give rise to periocular and frontonasal mesenchyme. Alx1 del/del embryos exhibited increased apoptosis of periocular mesenchyme and decreased expression of ocular developmental regulators Pitx2 and Lmxb1 in the periocular mesenchyme, followed by defective optic stalk morphogenesis. Moreover, Alx1 del/del embryos exhibited disruption of frontonasal mesenchyme identity, with loss of expression of Pax7 and concomitant ectopic expression of the jaw mesenchyme regulators Lhx6 and Lhx8 in the developing lateral nasal processes. The function of ALX1 in patterning the frontonasal mesenchyme is partly complemented by ALX4, a paralogous ALX family transcription factor whose loss-of-function causes a milder and distinctive FND. Together, these data uncover previously unknown roles of ALX1 in periocular mesenchyme development and frontonasal mesenchyme patterning, providing novel insights into the pathogenic mechanisms of ALX1-related FND.

Htra1 is a Novel Transcriptional Target of RUNX2 That Promotes Osteogenic Differentiation.

BACKGROUND/AIMS: Runt-related transcription factor 2 (Runx2) is a master regulator of osteogenic differentiation, but most of the direct downstream targets of RUNX2 during osteogenesis are unknown. Likewise, High-temperature requirement factor A1 (HTRA1) is a serine protease expressed in bone, yet the role of Htra1 during osteoblast differentiation remains elusive. We investigated the role of Htra1 in osteogenic differentiation and the transcriptional regulation of Htra1 by RUNX2 in primary mouse mesenchymal progenitor cells. METHODS: Overexpression of Htra1 was carried out in primary mouse mesenchymal progenitor cells to evaluate the extent of osteoblast differentiation. Streptavidin agarose pulldown assay, chromatin immunoprecipitation assay, and dual luciferase assay were carried out to investigate the interaction of RUNX2 protein at the Htra1 promoter during osteoblast differentiation. RESULTS: Overexpression of Htra1 increased the production of mineralized bone matrix, upregulating several osteoblast genes, such as Sp7 transcription factor (Sp7) and Alkaline phosphatase, liver/bone/kidney (Alpl). In addition, Htra1 upregulated osteogenesis-related signalling genes, such as Fibroblast growth factor 9 (Fgf9) and Vascular endothelial growth factor A (Vegfa). A series of experiments confirmed Htra1 as a direct RUNX2 transcriptional target. Overexpression of Runx2 resulted in the upregulation of Htra1 mRNA and protein. Chromatin immunoprecipitation and streptavidin agarose pull-down assays showed that RUNX2 binds a proximal -400 bp region of the Htra1 promoter during osteogenic differentiation. Dual luciferase assays confirmed that RUNX2 activates the proximal Htra1 promoter during osteogenic differentiation. Mutation of putative RUNX2 binding sites revealed that RUNX2 interacts with the Htra1 promoter at -252 bp and -84 bp to induce Htra1 expression. CONCLUSION: We demonstrate that Htra1 is a positive regulator of osteogenic differentiation, showing for the first time that Htra1 is a direct downstream target of RUNX2.

Six2 Plays an Intrinsic Role in Regulating Proliferation of Mesenchymal Cells in the Developing Palate.

Cleft palate is a common congenital abnormality that results from defective secondary palate (SP) formation. The Sine oculis-related homeobox 2 (Six2) gene has been linked to abnormalities of craniofacial and kidney development. Our current study examined, for the first time, the specific role of Six2 in embryonic mouse SP development. Six2 mRNA and protein expression were identified in the palatal shelves from embryonic days (E)12.5 to E15.5, with peak levels during early stages of palatal shelf outgrowth. Immunohistochemical staining (IHC) showed that Six2 protein is abundant throughout the mesenchyme in the oral half of each palatal shelf, whereas there is a pronounced decline in Six2 expression by mesenchyme cells in the nasal half of the palatal shelf by stages E14.5-15.5. An opposite pattern was observed in the surface epithelium of the palatal shelf. Six2 expression was prominent at all stages in the epithelial cell layer located on the nasal side of each palatal shelf but absent from the epithelium located on the oral side of the palatal shelf. Six2 is a putative downstream target of transcription factor Hoxa2 and we previously demonstrated that Hoxa2 plays an intrinsic role in embryonic palate formation. We therefore investigated whether Six2 expression was altered in the developing SP of Hoxa2 null mice. Reverse transcriptase PCR and Western blot analyses revealed that Six2 mRNA and protein levels were upregulated in Hoxa2-/- palatal shelves at stages E12.5-14.5. Moreover, the domain of Six2 protein expression in the palatal mesenchyme of Hoxa2-/- embryos was expanded to include the entire nasal half of the palatal shelf in addition to the oral half. The palatal shelves of Hoxa2-/- embryos displayed a higher density of proliferating, Ki-67 positive palatal mesenchyme cells, as well as a higher density of Six2/Ki-67 double-positive cells. Furthermore, Hoxa2-/- palatal mesenchyme cells in culture displayed both increased proliferation and elevated Cyclin D1 expression relative to wild-type cultures. Conversely, siRNA-mediated Six2 knockdown restored proliferation and Cyclin D1 expression in Hoxa2-/- palatal mesenchyme cultures to near wild-type levels. Our findings demonstrate that Six2 functions downstream of Hoxa2 as a positive regulator of mesenchymal cell proliferation during SP development.

Hoxa2 Inhibits Bone Morphogenetic Protein Signaling during Osteogenic Differentiation of the Palatal Mesenchyme.

Cleft palate is one of the most common congenital birth defects worldwide. The homeobox (Hox) family of genes are key regulators of embryogenesis, with Hoxa2 having a direct role in secondary palate development. Hoxa2-/- mice exhibit cleft palate; however, the cellular and molecular mechanisms leading to cleft palate in Hoxa2-/- mice is largely unknown. Addressing this issue, we found that Hoxa2 regulates spatial and temporal programs of osteogenic differentiation in the developing palate by inhibiting bone morphogenetic protein (BMP) signaling dependent osteoblast markers. Expression of osteoblast markers, including Runx2, Sp7, and AlpI were increased in Hoxa2-/- palatal shelves at embryonic day (E) 13.5 and E15.5. Hoxa2-/- mouse embryonic palatal mesenchyme (MEPM) cells exhibited increased bone matrix deposition and mineralization in vitro. Moreover, loss of Hoxa2 resulted in increased osteoprogenitor cell proliferation and osteogenic commitment during early stages of palate development at E13.5. Consistent with upregulation of osteoblast markers, Hoxa2-/- palatal shelves displayed higher expression of canonical BMP signaling in vivo. Blocking BMP signaling in Hoxa2-/- primary MEPM cells using dorsomorphin restored cell proliferation and osteogenic differentiation to wild-type levels. Collectively, these data demonstrate for the first time that Hoxa2 may regulate palate development by inhibiting osteogenic differentiation of palatal mesenchyme via modulating BMP signaling.