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，是一种主要类型的颅面畸形， 严重影响面部形态和功能的先天缺陷。FND患者需要多次矫正 手术，并经常遭受终身损伤。虽然大多数FND病例零星发生， 病因学，三个ALX家族基因ALX 1，ALX 3和ALX 4中的每一个的功能缺失突变，已经被 被确定为常染色体隐性FND的遗传原因，ALX 1的破坏与严重的 FND 3患者的面部裂开和极端小眼球，而ALX 3和ALX 4的突变导致 轻度但临床上独特的额鼻畸形。关于ALX转录因子是如何 调节颅面发育，以及控制额鼻发育的整体分子机制 是很难理解的。在初步研究中，我们已经使用CRISPR介导的基因工程技术产生了Alx 1突变小鼠。 基因组编辑，并发现它们重现了FND 3表型，包括减少额鼻骨 软骨，腭裂，小眼症我们发现Alx 1-/-胚胎表现出异位神经胶质细胞， 分化和减少外胚间充质基因表达的额鼻突。此外，委员会认为， Alx 1-/-Alx 4-/-双突变小鼠胚胎显示异位颅神经节增加， 与Alx 1-/-突变体相比，先前在多种动物模型系统中的研究表明， Twist 1转录因子，其表达在脑神经嵴细胞中激活， 迁移对于外生间充质的特化至关重要。值得注意的是，虽然分子机制作用于 Twist 1下游在促进外胚间充质命运方面的作用尚不清楚，Twist 1-/-小鼠胚胎未能 激活颅神经嵴细胞中所有三种Alx基因的表达。我们发现异位神经胶质细胞 Alx 1-/-和Alx 1-/-Alx 4-/-胚胎额鼻区的分化表明，Twist 1和ALX 转录因子在相同的分子网络中起作用以调节颅神经嵴命运决定 外胚间充质和神经胶质细胞之间的区别本研究项目的具体目标是 确定在额鼻粘膜中介导ALX转录因子功能的细胞和分子机制， 发展，并解开和重建由Twist 1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 在颅面发育生物学的长期关键差距，并导致新的改进，分子 诊断和治疗/护理大量颅面疾病。

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.