Functional and Genetic Analyses Unveil the Implication of hoxa4a in Zebrafish Craniofacial Development

Microtia–atresia is a rare craniofacial malformation primarily affecting the first and second pharyngeal arches, leading to the deformity of the auricle and atresia of the external ear canal. Its etiology is heterogenous and largely unknown, including both genetic and environmental factors. The HOXA4 gene has been identified as potentially pathogenetic for microtia–atresia in three twin families. A hoxa4a mosaic knockdown zebrafish model was constructed using CRISPR/Cas9. hoxa4a was expressed in the mandible during early development in zebrafish, while the F0 mosaic knockdowns exhibited craniofacial malformations with abnormal chondrocyte morphologies. Specifically, hoxa4a knockdown reduced cranial neural crest cell proliferation while increasing apoptosis, markedly downregulating chondrogenic markers sox9a and col2a1a. Consequently, pharyngeal arch chondrocytes exhibited disorganized arrangement and morphological abnormalities, resulting in mandibular hypoplasia. Our findings provide important insights into the role of hoxa4a in zebrafish mandibular development and the pathology of microtia–atresia caused by HOXA4 gene mutations in humans.