Live imaging and conditional disruption of native PCP activity using endogenously tagged zebrafish sfGFP-Vangl2

Abstract

Tissue-wide coordination of polarized cytoskeletal organization and cell behaviour, critical for normal development, is controlled by asymmetric membrane localization of non-canonical Wnt/planar cell polarity (PCP) signalling components. Understanding the dynamic regulation of PCP thus requires visualization of these polarity proteins in vivo. Here we utilize CRISPR/Cas9 genome editing to introduce a fluorescent reporter onto the core PCP component, Vangl2, in zebrafish. Through live imaging of endogenous sfGFP-Vangl2 expression, we report on the authentic regulation of vertebrate PCP during embryogenesis. Furthermore, we couple sfGFP-Vangl2 with conditional zGrad GFP-nanobody degradation methodologies to interrogate tissue-specific functions for PCP. Remarkably, loss of Vangl2 in foxj1a-positive cell lineages causes ependymal cell cilia and Reissner fiber formation defects as well as idiopathic-like scoliosis. Together, our studies provide crucial insights into the establishment and maintenance of vertebrate PCP and create a powerful experimental paradigm for investigating post-embryonic and tissue-specific functions for Vangl2 in development and disease.

Genetically encoded affinity reagents (GEARs): A toolkit for visualizing and manipulating endogenous protein functionin vivo

“…Nanog is a maternally deposited transcription factor that has pioneering activity in the embryo and regulates genome activation, localizing to the nucleus 29 . Vangl2, a core component of the planar cell polarity pathway, is localized to the membrane 30 . Importantly, the biological function of Nanog and Vangl2 30 are unperturbed upon tagging the N-terminus of the protein (Figure S1 B-E).…”

“…Vangl2, a core component of the planar cell polarity pathway, is localized to the membrane 30 . Importantly, the biological function of Nanog and Vangl2 30 are unperturbed upon tagging the N-terminus of the protein (Figure S1 B-E). We reasoned that nuclear or membrane translocation of the EGFP-GEARs would provide a robust readout for binding in vivo .…”

“…Injection of the ALFAgrad into ALFA -nanog homozygous embryos resulted in arrest of epiboly in 100% of the embryos phenocopying the maternal-zygotic nanog (MZ nanog ) mutant phenotype (Figure 3F-I) 39,40 . Furthermore, ALFA- vangl2 embryos injected with ALFAgrad fully recapitulated the MZ vangl2 convergent-extension phenotype 30 (Figure S3C-G). Importantly, the pool of maternal proteins is also targeted by these degron reagents, enabling robust clearance of proteins that establish the earliest events of embryogenesis.…”

Rbm8a deficiency causes hematopoietic defects by modulating Wnt/PCP signaling

“…Non-canonical Wnt/PCP signaling controls cell migration during convergence and extension movements that drive zebrafish embryonic axis elongation (Hammerschmidt et al, 1996;Heisenberg et al, 2000;Jussila et al, 2022;Love et al, 2018;Whitfield et al, 1996;Yang and Mlodzik, 2015). Hallmarks of defective convergence and extension include reduced length of the anteroposterior axis and increased width of the neural plate and somites (Hammerschmidt et al, 1996;Heisenberg et al, 2000;Jessen et al, 2002;Kilian et al, 2003;Marlow et al, 2002;Pierpont et al, 2018;Solnica-Krezel et al, 1996;Topczewski et al, 2001;Williams and Solnica-Krezel, 2020).…”

“…Non-canonical Wnt/PCP signaling controls cell migration during convergence and extension movements that drive zebrafish embryonic axis elongation 83,85,89,[91][92][93] . Hallmarks of defective convergence and extension include reduced length of the anteroposterior axis and increased width of the neural plate and somites 51,60,61,89,90,[93][94][95][96] .…”