Tinagl1 is a basement membrane protein conserved across animal species having three germ layers; mammals have an additional family member, Tinag. Genetic studies by others in fly and mouse models have not yielded a clear in vivo requirement for Tinagl1. Principally using MO knockdown in zebrafish, we observed defects consistent with a ‘ciliopathy’ spectrum, including small eye, body curvature, L/R randomization of heart looping, hydrocephalus, renal cysts, and craniofacial defects. New results with transient Crispr/Cas9 mosaic knockout and an ENU truncation mutant support heart looping, renal, and craniofacial defects, and mutant analysis is our current focus. With the MOs, we documented shorter and fewer cilia in Kupffer's vesicle and pronephric duct. Tinagl1 morphant pronephros and craniofacial defects were enhanced by co-injection with a Wnt MO but not control MO, and partially rescued by tinagl1 mRNA; ctnnb2 but not egfp mRNA was also demonstrated to partially rescue the pronephric cilia defect. These results are consistent with a Nusse lab demonstration that fruitfly Tinagl1 binds to Wg and is required for its function at a distance from secreting cells in vitro. Overall, our results support a hypothesis that, in zebrafish, Tinagl1 is broadly required for cilia function and may act via Wnt/beta-catenin signaling shown by others to function upstream of the motile cilia regulator foxj1a in pronephros and KV. We plan to test whether Tinagl1 can be linked to cilia regulation in mammalian cells, and whether it physically interacts with zebrafish Wnt(s) and activates downstream target genes or regulates PCP components in either model. Zebrafish may be a particularly favorable model for studying Tinagl1: it has only the tinagl1 gene, so compensatory expression of Tinag is removed as an issue, yet it has good conservation of the organ systems in which Tinagl1 is highly expressed in mammals. In contrast, flies express modified cilia only in sperm and sensory neurons, and have more divergent organs, offering less scope for comparative studies and detection of context-specific effects. Interestingly, a zebrafish laminin mutant is reported to show short KV cilia able to initially establish L/R pattern, but later signal diffusion across the midline results in L/R randomization of abdominal organs. Mechanisms for basement membrane Tinagl1 and laminin roles in cilia regulation may involve signaling at focal adhesions in addition to promoting signaling of growth factors such as Wnts.