Ptk7 promotes non-canonical Wnt/PCP-mediated morphogenesis and inhibits Wnt/β-catenin-dependent cell fate decisions during vertebrate development

Abstract

Using zebrafish, we have characterised the function of Protein tyrosine kinase 7 (Ptk7), a transmembrane pseudokinase implicated in Wnt signal transduction during embryonic development and in cancer. Ptk7 is a known regulator of mammalian neural tube closure and Xenopus convergent extension movement. However, conflicting reports have indicated both positive and negative roles for Ptk7 in canonical Wnt/β-catenin signalling. To clarify the function of Ptk7 in vertebrate embryonic patterning and morphogenesis, we generated maternal-zygotic (MZ) ptk7 mutant zebrafish using a zinc-finger nuclease (ZFN) gene targeting approach. Early loss of zebrafish Ptk7 leads to defects in axial convergence and extension, neural tube morphogenesis and loss of planar cell polarity (PCP). Furthermore, during late gastrula and segmentation stages, we observe significant upregulation of β-catenin target gene expression and demonstrate a clear role for Ptk7 in attenuating canonical Wnt/β-catenin activity in vivo. MZptk7 mutants display expanded differentiation of paraxial mesoderm within the tailbud, suggesting an important role for Ptk7 in regulating canonical Wnt-dependent fate specification within posterior stem cell pools post-gastrulation. Furthermore, we demonstrate that a plasma membrane-tethered Ptk7 extracellular fragment is sufficient to rescue both PCP morphogenesis and Wnt/β-catenin patterning defects in MZptk7 mutant embryos. Our results indicate that the extracellular domain of Ptk7 acts as an important regulator of both non-canonical Wnt/PCP and canonical Wnt/β-catenin signalling in multiple vertebrate developmental contexts, with important implications for the upregulated PTK7 expression observed in human cancers.

Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish

“…In contrast, canonical Wnt signaling is diminished. As has been recently reported for other Wnt inhibitors (Flowers et al, 2012; Hayes et al, 2013; Liu et al, 2013), the reduced expression of direct Wnt targets in slc35c1 expressing embryos is consistent with an inhibitory function of Slc35C1 on Wnt signaling. Reduced intensity of nuclear β-Catenin in the “core” region (future dorsal) and the immediate Wnt target boz prior to formation of the dorsal organizer are consistent with inhibition of maternal Canonical Wnt signaling.…”

“…Limiting fucosylation in mice is known to cause developmental defects including early embryonic death (Du et al, 2010; Smith et al, 2002), neuronal migration disorders (Ohata et al, 2009), skeletal abnormalities and other defects (Hayes et al, 2013; Ma et al, 2006). However, the consequence of excess fucosylation has not been examined.…”

The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo

“…In summary, hArf1 Q71L -injected embryos demonstrate significant disruptions in axial morphogenesis, phenotypes typically observed in core PCP signalling mutants 31,32 , and we thus conclude that Arf1 could play a conserved role in PCP establishment from Drosophila to vertebrates.…”

“…For example, dorsal ectoderm cells undergoing PCP-directed CE movements elongate along the ML axis as they migrate towards the embryo midline, and PCP-core factor function is required for this elongation 31,32 . Consistent with a conserved function of Arf1 in vertebrate PCP, the length-to-width ratio (LWR) of dorsal ectoderm cells in hArf1 Q71L -injected embryos was significantly reduced compared with that of WT Arf1 cells (Fig.…”

“…31 ) using myoD and krox20 antisense RNA probes generated by in vitro transcription (DIG RNA Labeling Kit, Roche).…”