斑马鱼hoxa1a基因调控颅面骨骼发育的功能研究

Functional Study of hoxa1a Regulating Craniofacial Bone Development in Zebrafish

hox基因编码一类高度保守的转录因子家族,人类HOXA1的突变会导致阿萨巴斯卡发育不良综合征(Athabascan brainstem dysgenesis syndrome,ABDS),使人出现因颅骨异常导致的面部畸形和面部麻痹等症状。利用模式生物斑马鱼研究其同源基因hoxa1a的功能机制。首先利用CRISPR/Cas9技术对斑马鱼hoxa1a进行基因编辑,获得了hoxa1a基因突变,T7E1酶切结果显示F0酶切效率平均为70%。F1进一步筛选到两种突变类型,分别是插入了8 bp和删除了7 bp的杂合突变体。杂合子自交得到hoxa1a F2纯合突变体,并且测序验证hoxa1a基因突变成功。5 dpf时,hoxa1a纯合突变体出现颅面发育异常。阿尔新蓝软骨染色和茜素红硬骨染色结果表明,hoxa1a突变体中颅骨发育异常、筛骨板断裂,鳃弓发育出现缺损。成功在斑马鱼中构建ABDS疾病模型,表明hoxa1a突变会造成斑马鱼颅面骨骼发育异常,为其功能机制研究奠定了基础,为人类ABDS疾病的致病机制研究提供了新的思路。

hox genes encode a family of highly conserved transcription factors.Human HOXA1 mutation causes ABDS(athabascan brainstem dysgenesis syndrome),which leads to craniofacial bone deformity induced facial defect and paralysis.In this paper,zebrafish was used to study the functional mechanism of the homologous gene hoxa1 a.Firstly,hoxa1 a gene was edited by using CRISPR/Cas9 technology,which resulted in gene mutation.The T7E1 assay showed F0 digestion efficiency was 70%on average.Then F1 was screened and it was found that hoxa1 a heterozygote generated 8 bp insertion and 7 bp deletion.Furthermore,the heterozygotes were crossed and hoxa1 a homozygous F2 mutant was obtained,which was confirmed by sequencing.At 5 dpf,homozygous mutants of hoxa1 a showed craniofacial dysplasia.The results of alcian blue cartilage staining and alizarin red hard bone staining demonstrated that the hoxa1 a mutant had abnormal skull development,fracture of ethmoid plate,and defect of gill arch development.In this study,ABDS disease model in zebrafish was successfully constructed and the results indicate that hoxa1 a mutation might cause abnormal craniofacial skeletal development,which lays a foundation for its mechanism study and provides a new idea for the pathogenesis of human ABDS disease.