ctdsp2 Knockout Induces Zebrafish Craniofacial Dysplasia via p53 Signaling Activation

Hemifacial microsomia (HFM) is a rare congenital craniofacial deformity that significantly impacts the appearance and hearing. The genetic etiology of HFM remains largely unknown, although genetic factors are considered to be primary contributors. We previously identified <i>CTDSP2</i> as a potential causative gene in HFM cases. Utilizing CRISPR/Cas9, we knocked out <i>ctdsp2</i> in zebrafish and analyzed the spatiotemporal expression of <i>ctdsp2</i> and neural crest cell (NCC) markers through in situ hybridization (ISH). Craniofacial cartilage and chondrocyte phenotypes were visualized using Alcian blue and wheat germ agglutinin (WGA) staining. Cell proliferation and apoptosis were assessed via immunofluorescence with PH3 and TUNEL. RNA sequencing was performed on <i>ctdsp2</i>^-/- embryos and control siblings, followed by rescue experiments. Knockout of <i>ctdsp2</i> in zebrafish resulted in craniofacial defects characteristic of HFM. We observed abnormalities in NCC apoptosis and proliferation in the pharyngeal arches, as well as impaired differentiation of chondrocytes in <i>ctdsp2</i>^-/- embryos. RNA-Seq analysis revealed significantly higher expression of genes in the p53 signaling pathway in mutants. Furthermore, <i>ctdsp2</i> mRNA injection and <i>tp53</i> knockout significantly rescued pharyngeal arch cartilage dysplasia. Our findings suggest that <i>ctdsp2</i> knockout induces zebrafish craniofacial dysplasia, primarily by disrupting pharyngeal chondrocyte differentiation and inhibiting NCC proliferation through p53 signaling pathway activation.