Loss of wbp11 causes multi-system developmental defects: a zebrafish model of VACTERL association

VACTERL association is a congenital disorder characterized by the non-random co-occurrence of vertebral, anal, cardiac, tracheo-esophageal, renal, and limb anomalies. WBP11 has been identified as a candidate causative gene; however, existing heterozygous Wbp11 knockout mice recapitulate only a subset of the patient phenotypes, most notably lacking the cardiac defects, which limits a comprehensive understanding of the pathogenic mechanisms. Whether WBP11 loss of function is sufficient to induce the full multi-system spectrum of VACTERL association in a vertebrate model remains unexplored.

We established a wbp11 knockdown zebrafish model by microinjecting a translation-blocking morpholino (wbp11-MO) at the optimized concentration of 0.25 mM. Knockdown efficiency was validated by Western blot. Bioinformatics analysis of public single cell and expression atlas datasets was combined with whole-mount in situ hybridization to characterize the spatiotemporal expression of wbp11. Phenotypic defects across cardiac, vascular, spinal, fin, and renal systems were assessed by stereomicroscopy, transgenic reporter lines (Tg(myl7:EGFP) and Tg(flila:EGFP)), and whole mount in situ hybridization. Underlying molecular changes were interrogated by transcriptome sequencing (RNA-seq) coupled with differential alternative splicing analysis (rMATS) and quantitative real-time PCR validation. Rescue experiments were performed by co-injection of capped wbp11 mRNA.

wbp11 is evolutionarily conserved and maternally expressed, peaking at the gastrula stage with elevated signals in the head, skeletal muscle, and heart. wbp11-MO injection reduced Wbp11 protein levels, induced a 70% overall malformation rate, and caused a general delay in embryonic development. wbp11-deficient larvae exhibited multi-system developmental defects that closely mirror the core clinical features of VACTERL association: cardiac malformations with pericardial edema and impaired heart looping; vascular defects including underdeveloped subintestinal vessels and reduced cranial vasculature; spinal curvature with aberrant notochord structure; hypoplasia of the pectoral, pelvic, anal, and caudal fins; and transcriptional dysregulation of kidney developmental markers. Transcriptomic and alternative splicing analyses revealed significant dysregulation of genes associated with cardiac function, angiogenesis, skeletal patterning, fin development, and kidney development, accompanied by widespread splicing aberrations.

This study establishes the first wbp11-knockdown zebrafish model that partially recapitulates the key organ phenotypes of human VACTERL association notably including the cardiac defects absent in the mouse model and thereby provides a powerful platform for dissecting the splicing dependent molecular mechanisms underlying this disorder and for exploring potential therapeutic interventions.