Dataset Information

Six2 regulates Pax9 expression, palatogenesis and craniofacial bone formation.

ABSTRACT: In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2^-/- embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5' upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.

SUBMITTER: Sweat YY

PROVIDER: S-EPMC7568837 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Six2 regulates Pax9 expression, palatogenesis and craniofacial bone formation.

Sweat Yan Yan YY Sweat Mason M Mansaray Maurisa M Cao Huojun H Eliason Steven S Adeyemo Waisu L WL Gowans Lord J J LJJ Eshete Mekonen A MA Anand Deepti D Chalkley Camille C Saadi Irfan I Lachke Salil A SA Butali Azeez A Amendt Brad A BA

Developmental biology 20191123 2

In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulati ...[more]

PMID: 31765609

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Project description:Human CLCN7 encodes voltage-gated chloride channel 7 (ClC-7); mutations of CLCN7 lead to osteopetrosis which is characterized by increased bone mass and impaired osteoclast function. In our previous clinical practice, we noticed that osteopetrosis patients with CLCN7 mutations had some special deformities in craniofacial morphology and tooth dysplasia. It is unclear whether these phenotypes are the typical features of CLCN7 involved osteopetrosis and whether ClC-7 could regulate the development of craniofacial bone and tooth in some signaling pathways. Methods: First, we collected 80 osteopetrosis cases from the literature and compared their craniofacial and dental phenotypes. Second, four osteopetrosis pedigrees with CLCN7 mutations were recruited from our clinic for gene testing and clinical analysis of their craniofacial and dental phenotypes. Third, we used a zebrafish model with clcn7 morpholino treatment to detect the effects of ClC-7 deficiency on the development of craniofacial and dental phenotypes. General observation, whole mount alcian blue and alizarin red staining, whole mount in situ hybridization, scanning electron microscope observation, lysoSensor staining, Q-PCR and western blotting were performed to observe the in vivo characteristics of craniofacial bone and tooth changes. Fourth, mouse marrow stromal cells were further primarily cultured to detect ClC-7 related mRNA and protein changes using siRNA, Q-PCR and western blotting. Results: Over 84% of osteopetrosis patients in the literature had some typical craniofacial and tooth phenotypes, including macrocephaly, frontal bossing, and changes in shape and proportions of facial skeleton, and these unique features are more severe and frequent in autosomal recessive osteopetrosis than in autosomal dominant osteopetrosis patients. Our four pedigrees with CLCN7 mutations confirmed the aforementioned clinical features. clcn7 knockdown in zebrafish reproduced the craniofacial cartilage defects and various dental malformations combined the decreased levels of col10a1, sp7, dlx2b, eve1, and cx43. Loss of clcn7 function resulted in lysosomal storage in the brain and jaw as well as downregulated cathepsin K (CTSK). The craniofacial phenotype severity also presented a dose-dependent relationship with the levels of ClC-7 and CTSK. ClC-7/CTSK further altered the balance of TGF-β/BMP signaling pathway, causing elevated TGF-β-like Smad2 signals and reduced BMP-like Smad1/5/8 signals in clcn7 morphants. SB431542 inhibitor of TGF-β pathway partially rescued the aforementioned craniofacial bone and tooth defects of clcn7 morphants. The ClC-7 involved CTSK/BMP and SMAD changes were also confirmed in mouse bone marrow stromal cells. Conclusion: These findings highlighted the vital role of clcn7 in zebrafish craniofacial bone and tooth development and mineralization, revealing novel insights for the causation of osteopetrosis with CLCN7 mutations. The mechanism chain of ClC-7/CTSK/ TGF-β/BMP/SMAD might explain the typical craniofacial bone and tooth changes in osteopetrosis as well as pycnodysostosis patients.

Dataset Information

Six2 regulates Pax9 expression, palatogenesis and craniofacial bone formation.

Publications

Six2 regulates Pax9 expression, palatogenesis and craniofacial bone formation.

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