Acute knockdown of extracellular matrix protein Tinagl1 disrupts heart laterality and pronephric cilia in zebrafish embryonic development

Abstract

AbstractA highly-conserved extracellular matrix protein, Tinagl1, modulates Wnt, integrin, TGF-β, and EGF-R signalingin vitro,but its significancein vivohas remained in doubt. To bypass possible genetic compensation by an ortholog encoded exclusively in mammalian genomes, we examine Tinagl1 function in zebrafish embryos. In this model,tinagl1mRNA is detected in the developing spinal cord and pronephros. Acute knockdown using either CRISPR/Cas9 somatic mutagenesis or splice-blocking morpholinos reveals left-right (LR) heart looping defects, pronephros dilatations, and ventral body curvature. This constellation of defects characteristically results from the loss of motile cilia function, and we confirm the presence of shortened and fewer cilia in the pronephric duct and in the Kupffer’s vesicle where LR asymmetry is established. A link to known Wnt3a/β-catenin signaling that activates the motile cilia transcriptional program is supported by manipulation of Wnt3a and β-catenin levels intinagl1knockdown embryos. In addition to ciliopathy-like defects, thetinagl1knockdown shows disorganization of longitudinal axon tracts in the spinal cord and defects in motor neuron outgrowth. Together, these results provide evidence that Tinagl1 is important in development, and that zebrafish is an ideal model in which to explore its relationships to cilia and secreted signaling molecules.