Regulation of Craniofacial Development by ALX Transcription Factors

摘要 额鼻发育不良（FND），也称为正中裂面综合征，是颅面发育不良的一个主要类别 严重影响面部形状和功能的出生缺陷。 FND患者需要多次矫正 手术并经常遭受终生损伤。虽然大多数 FND 病例都是零星发生，且不明原因 病因学方面，三个 ALX 家族基因 ALX1、ALX3 和 ALX4 中每一个的功能丧失突变已被证实。 被确定为常染色体隐性 FND 的遗传原因，ALX1 的破坏与严重的相关 FND3 患者出现面部裂和极度小眼症，而 ALX3 和 ALX4 突变导致 较轻但临床上独特的额鼻畸形。人们对ALX转录因子如何发挥作用知之甚少 调节颅面发育，以及控制额鼻发育的整体分子机制 人们对此知之甚少。在初步研究中，我们利用 CRISPR 介导生成了 Alx1 突变小鼠 基因组编辑，发现它们重现了 FND3 表型，包括额鼻骨减少 以及软骨、腭裂和小眼症。我们发现Alx1-/-胚胎表现出异位神经胶质细胞 额鼻突处外间充质基因表达的分化和减少。而且， Alx1-/-Alx4-/-双突变小鼠胚胎表现出异位颅神经节增多且严重得多 与 Alx1-/- 突变体相比，额鼻缺乏。先前对多种动物模型系统的研究表明 Twist1转录因子，其表达在脑神经嵴细胞中被激活 迁移对于外源间充质的规范至关重要。值得注意的是，虽然分子机制起作用 Twist1下游在促进外间充质命运中的作用仍不清楚，Twist1-/-小鼠胚胎未能 激活颅神经嵴细胞中所有三个 Alx 基因的表达。我们发现异位神经胶质细胞 Alx1-/- 和 Alx1-/-Alx4-/- 胚胎额鼻区的分化表明 Twist1 和 ALX 转录因子在同一分子网络中作用来调节颅神经嵴命运决定 外间充质谱系和神经胶质细胞谱系之间。该研究项目的具体目标是 确定介导额鼻ALX转录因子功能的细胞和分子机制 开发并解开和重建由 Twist1 和 ALX 组成的基因调控网络 调节颅神经嵴分化的转录因子。这些研究的结果将填补 颅面发育生物学中长期存在的关键差距，并导致分子生物学的新进展 大量颅面疾病的诊断和治疗/护理。

Abstract Frontonasal dysplasia (FND), also known as median cleft face syndrome, is a major class of craniofacial birth defects that profoundly impact the form and function of the face. FND patients require multiple corrective surgeries and often suffer life-long impairment. Whilst most FND cases occur sporadically with unknown etiology, loss-of-function mutations in each of the three ALX family genes, ALX1, ALX3, and ALX4, have been identified as the genetic causes for autosomal recessive FND, with disruption of ALX1 associated with severe facial clefting and extreme microphthalmia in FND3 patients while mutations in ALX3 and ALX4 resulted in milder but clinically distinctive frontonasal malformations. Little is known about how ALX transcription factors regulate craniofacial development, and the overall molecular mechanism controlling frontonasal development is poorly understood. In preliminary studies, we have generated Alx1 mutant mice using CRISPR-mediated genome editing and found that they recapitulated the FND3 phenotypes, including reduced frontonasal bones and cartilages, cleft palate, and microphthalmia. We found that Alx1-/- embryos exhibited ectopic neuroglial differentiation and reduction in ectomesenchymal gene expression in the frontonasal prominence. Moreover, Alx1-/-Alx4-/- double mutant mouse embryos exhibited increased ectopic cranial ganglia and much severer frontonasal deficiency than Alx1-/- mutants. Previous studies in multiple animal model systems revealed that the Twist1 transcription factor, whose expression is activated in cranial neural crest cells at the onset of migration, is critical for ectomesenchyme specification. Remarkably, while the molecular mechanism acting downstream of Twist1 in promoting ectomesenchymal fate is still unclear, Twist1-/- mouse embryos failed to activate the expression of all three Alx genes in cranial neural crest cells. Our finding of ectopic neuroglial differentiation in the frontonasal regions of Alx1-/- and Alx1-/-Alx4-/- embryos suggests that Twist1 and the ALX transcription factors act in the same molecular network to regulate cranial neural crest fate determination between the ectomesenchymal and neuroglial lineages. The specific aims of this research project are to determine the cellular and molecular mechanisms mediating ALX transcription factor function in frontonasal development and to unravel and reconstruct the gene regulatory network consisting of Twist1 and ALX transcription factors regulating cranial neural crest differentiation. Results from these studies will fill a longstanding critical gap in craniofacial developmental biology and lead to new improvements in molecular diagnosis and treatment/care of a large number of craniofacial disorders.