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

Abstract

A highly-conserved extracellular matrix protein, Tinagl1, modulates Wnt, integrin, TGF-b, and EGF-R signaling in vitro, but its significance in vivo has 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, tinagl1 mRNA 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/b-catenin signaling that activates the motile cilia transcriptional program is supported by manipulation of Wnt3a and b-catenin levels in tinagl1 knockdown embryos. In addition to ciliopathy-like defects, the tinagl1 knockdown 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.

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“…Tinagl1 is a matricellular protein most commonly associated with embryonic development, including the heart and adrenal glands . Low levels of Tinagl1 expression can be dire for craniofacial development and for female fertility. , As a key matrix component, Tinagl1 has been shown to bind with laminins and integrins and may be modulated by Wnt signaling. , In recent years, research has been done on the role of Tinagl1 expression outside the context of embryonic development, including in cancer development.…”