Regulation of Craniofacial Development by ALX Transcription Factors

摘要 额鼻发育不良(FND)，又称正中裂面裂综合征，是颅面部的一个主要类型 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病例是零星发生的，但不明原因 三个ALX家族基因ALX1、ALX3和ALX4的病因学、功能丧失突变已被证实 被确认为常染色体隐性遗传性FND的遗传原因，ALX1的中断与严重 FND3患者的面部裂伤和极小眼炎，而ALX3和ALX4的突变导致 额鼻畸形较轻，但临床上有独特之处。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 人们对此知之甚少。在初步研究中，我们使用CRISPR介导的方法培育了ALX1突变小鼠 基因组编辑发现，他们重述了FND3的表型，包括减少的额鼻骨 还有软骨、腭裂和小眼炎。我们发现ALX1-/-胚胎表现为异位神经胶质细胞 额鼻突外胚间充质基因表达的分化和减少。此外， ALX1-/-ALX4-/-双突变小鼠胚胎表现出异位颅神经节增加和更严重 额鼻缺乏症的发生率高于ALX1-/-突变体。之前在多种动物模型系统中的研究表明， Twist1转录因子，其表达在脑神经脊细胞发病时被激活 迁移，是外生组织规范的关键。值得注意的是，当分子机制作用于 Twist1下游在促进外胚间充质分化中的作用尚不清楚，Twist1-/-小鼠胚胎未能 激活三种Alx基因在脑神经脊细胞中的表达。异位神经胶质细胞的发现 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，调节脑神经脊命运的决定 外胚间充质系和神经胶质系之间。这项研究项目的具体目的是 确定额鼻区ALX转录因子功能的细胞和分子机制 Twist1和ALX基因调控网络的构建与构建 转录因子调节脑神经隆起分化。这些研究的结果将填补 颅面发育生物学长期存在的关键差距并导致分子生物学的新进展 大量头面部疾病的诊断和治疗/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.