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Abstract：
Heterozygous deletion of Six2, which encodes a member of sine oculis homeobox family transcription factors, has recently been associated with the frontonasal dysplasia syndrome FND4. Previous studies showed that Six2 is expressed in multiple tissues during craniofacial development in mice, including embryonic head mesoderm, postmigratory frontonasal neural crest cells, and epithelial and mesenchymal cells of the developing palate and nasal structures. Whereas Six2 -/- mice exhibited cranial base defects but did not recapitulate frontonasal phenotypes of FND4 patients, Six1 -/- Six2 -/- double mutant mice showed severe craniofacial defects including midline facial clefting. The complex phenotypes of FND4 patients and of Six1 -/- Six2 -/- mutant mice indicate that Six2 plays crucial roles in distinct cell types at multiple stages of craniofacial morphogenesis. Here we report generation of mice carrying insertions of a pair of loxP sites flanking exon-1 of the Six2 gene (Six2 f allele) using CRISPR/Cas9-mediated genome editing. We show that the Six2 f allele functions normally and is effectively inactivated by Cre-mediated recombination in vivo. Furthermore, we show that Six2 f/f ;Wnt1-Cre mice recapitulated cranial base defects but not neonatal lethality of Six2 -/- mice. These results indicate that Six2 f/f mice enable systematic investigation of cell type- and stage-specific Six2 function in development and disease.
摘要：
Six2的杂合缺失，编码sineoculis同源异型盒家族转录因子的成员，最近与额鼻发育不良综合征FND4有关。先前的研究表明，Six2在小鼠颅面发育过程中在多个组织中表达，包括胚胎头部中胚层，迁徙后额鼻神经c细胞，以及发育中的腭和鼻结构的上皮和间充质细胞。而Six2-/-小鼠表现出颅底缺损，但没有重现FND4患者的额鼻表型，Six1-/-Six2-/-双突变小鼠表现出严重的颅面缺损，包括中线面部裂开。FND4患者和Six1-/-Six2-/-突变小鼠的复杂表型表明，Six2在颅面形态发生的多个阶段在不同的细胞类型中起着至关重要的作用。在这里，我们报告了使用CRISPR/Cas9介导的基因组编辑，携带Six2基因（Six2f等位基因）的外显子1侧翼的一对loxP位点插入的小鼠的产生。我们表明，Six2f等位基因正常工作，并在体内通过Cre介导的重组有效灭活。此外，我们显示Six2f/f;Wnt1-Cre小鼠概括了Six2-/-小鼠的颅底缺损，但没有新生儿致死性。这些结果表明，Six2f/f小鼠能够系统地研究发育和疾病中特定类型和阶段的Six2功能。
