Ddx3xa Mutations Drive Cardiac Defects in a Zebrafish Model via Dysregulation of Wnt/β-Catenin Signaling

Yu Chen^1,2Mei Lin³Ping Zhu^2,4Xueting Yang³Kexing Yi³Yingshuo Zhang³Xiaoyan Cai³Zhongbei Jiao³Haochen Wang³Wuzhou Yuan³Yongqing Li³Zhigang Jiang³Yuequn Wang³Fang Li^3*Xiushan Wu^2,3*Xiongwei Fan^3*

• ¹Medical Research Center, Guangdong Provincial People's Hospital(Guangdong Academy of Medical Sciences), Southern Medical University, guangzhou, China
• ²Guangdong Provincial Key Laboratory of Pathogenesis, Targeted Prevention and Treatment of Heart Disease, Guangzhou Key Laboratory of Cardiac Pathogenesis and Prevention, Guangzhou, China
• ³The Center for Heart Development, College of Life Science, Hunan Normal University, Changsha, China
• ⁴Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital(Guangdong Academy of Medical Sciences),Southern Medical University, Guangzhou, China

Mutations in the DDX3X gene are the primary cause of DDX3X syndrome, with over 800 diagnosed families worldwide. DDX3X is also recognized as a single-gene driver for rare syndromes associated with epilepsy, autism, and developmental disorders. Clinical studies suggest potential links between DDX3X mutations and various cardiac comorbidities. However, there is no report on whether Ddx3xa knockout leads to cardiac phenotypes or the establishment of zebrafish ddx3xa gene knockout model. Leveraging the high genomic conservation between zebrafish and humans, we generated a ddx3xa knockout model using CRISPR/Cas9 technology. Homozygous knockout (ddx3xa-/-) embryos exhibited developmental delay, trunk malformations, and severe cardiac abnormalities, including pericardial edema, defective cardiac looping, and cardiac contractile dysfunction. Ribonucleic Acid Sequencing (RNA-seq) analysis of ddx3xa-/- zebrafish at 72 hours post-fertilization (hpf) revealed significant enrichment in pathways related to actin cytoskeleton organization, calcium signaling, cardiac and vascular smooth muscle contraction, and Wnt signaling. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (QRT-PCR) and in situ hybridization confirmed dysregulated expression of key cardiac development genes (bmp4, actn2b, tbx5, nppb) and significantly impaired cardiac function. Given the role of Wnt signaling in cardiogenesis, we further analyzed this pathway and found that ddx3xa knockout upregulated the key Wnt/β-catenin transcription factor Tcf/Lef1 (T Cell Factor/Lymphoid Enhancer Factor 1) and disrupted its target genes (bmp4, tbx5) expression. Crucially, treatment of 72 hpf mutant embryos with the Wnt inhibitor IWR-1 partially rescued both the cardiac malformations and the aberrant expression of its target genes. This study provides the first evidence that ddx3xa regulates cardiac morphogenesis by modulating the Wnt/β-catenin signaling pathway, offering direct experimental insight into the mechanisms underlying cardiac comorbidities in DDX3X syndrome. It also highlights the unique value of the zebrafish model for dissecting conserved pathogenic pathways and exploring targeted therapeutic strategies.