☆ 4.7 Article

Crucial and Overlapping Roles of Six1 and Six2 in Craniofacial Development

JOURNAL OF DENTAL RESEARCH (2019)

Journal

JOURNAL OF DENTAL RESEARCH

Volume 98, Issue 5, Pages 572-579

Publisher

SAGE PUBLICATIONS INC

DOI: 10.1177/0022034519835204

Keywords

BMP4; frontonasal dysplasia; median facial cleft; neural crest; transcription factor; Alx1

Funding

National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR) [DE018401, DE027046]
China Scholarship Council scholarship

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Abstract

SIX1 and SIX2 encode closely related transcription factors of which disruptions have been associated with distinct craniofacial syndromes, with mutations in SIX1 associated with branchiootic syndrome 3 (BOS3) and heterozygous deletions of SIX2 associated with frontonasal dysplasia defects. Whereas mice deficient in Six1 recapitulated most of the developmental defects associated with BOS3, mice lacking Six2 function had no obvious frontonasal defects. We show that Six1 and Six2 exhibit partly overlapping patterns of expression in the developing mouse embryonic frontonasal, maxillary, and mandibular processes. We found that Six1(-/-)Six2(-/-) double-mutant mice were born with severe craniofacial deformity not seen in the Six1(-/-) or Six2(-/-) single mutants, including skull bone agenesis, midline facial cleft, and syngnathia. Moreover, whereas Six1(-/-) mice exhibited partial transformation of maxillary zygomatic bone into a mandibular condyle-like structure, Six1(-/-)Six2(+/-) mice exhibit significantly increased penetrance of the maxillary malformation. In addition to ectopic Dlx5 expression at the maxillary-mandibular junction as recently reported in E10.5 Six1(-/-) embryos, the E10.5 Six1(-/-)Six2(+/-) embryos showed ectopic expression of Bmp4, Msx1, and Msx2 messenger RNAs in the maxillary-mandibular junction. Genetically inactivating 1 allele of either Ednra or Bmp4 significantly reduced the penetrance of maxillary malformation in both Six1(-/-) and Six1(-/-)Six2(+/-) embryos, indicating that Six1 and Six2 regulate both endothelin and bone morphogenetic protein-4 signaling pathways to pattern the facial structures. Furthermore, we show that neural crest-specific inactivation of Six1 in Six2(-/-) embryos resulted in midline facial cleft and frontal bone agenesis. We show that Six1(-/-)Six2(-/-) embryos exhibit significantly reduced expression of key frontonasal development genes Alx1 and Alx3 as well as increased apoptosis in the developing frontonasal mesenchyme. Together, these results indicate that Six1 and Six2 function partly redundantly to control multiple craniofacial developmental processes and play a crucial neural crest cell-autonomous role in frontonasal morphogenesis.

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

Paul P. R. Iyyanar, Zhaoming Wu, Yu Lan, Yueh-Chiang Hu, Rulang Jiang

Summary: The loss of ALX1 function leads to frontonasal dysplasia syndrome FND3, which is characterized by severe facial clefting and microphthalmia. This study generated an Alx1-deletion mouse model using CRISPR/Cas9-mediated genome editing and found that Alx1 is strongly expressed in frontonasal neural crest cells, playing a role in periocular and frontonasal mesenchyme development. Alx1(del/del) embryos showed increased apoptosis in periocular mesenchyme, decreased expression of ocular developmental regulators Pitx2 and Lmxb1, and disrupted frontonasal mesenchyme identity.

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