Wnt/PCP signaling is implicated in gut development, but the underlying mechanisms are unclear and even controversial. This proposal aims to determine how Wnt/PCP signaling controls endoderm convergence and extension (C&E)?an early step in the gut formation. We and others found that Glypican 4 (Gpc4, a heparan sulfate proteoglycan that can regulate Wnt signaling) and VanGogh-like 2 (Vangl2, a core Wnt/PCP protein) are required for endoderm C&E. In contrast with a previous report, we found that endodermal cells elongate mediolaterally and that their anterior edges are enriched with Vangl2-GFP. In gpc4 or vangl2 mutants, endodermal cell polarity is lost and endoderm C&E are impaired, indicating endoderm cells exhibit planar polarity that depends on Wnt/PCP signaling. Additionally, our data suggest that the Wnt5b ligand forms a gradient along the posterior-anterior axis, which can act as an instructive signal for endodermal polarity. Furthermore, Gpc4 and Vangl2 affect different aspects of endoderm morphogenesis; and genetic interaction assays support a model in which Gpc4 and Vangl2 act in distinct Wnt/PCP signaling branches to control endoderm C&E, with Gpc4 regulating the Wnt5b gradient and Vangl2 acting independently of Wnt5b. However, despite this knowledge, how endoderm C&E are regulated by Wnt/PCP signaling is unclear. The objective of this proposal is to elucidate how Gpc4 regulates gut morphogenesis and the crosstalk between the germ layers, by employing in vivo imaging with biosensors, embryonic and genetic manipulations, cell biology, as well as biochemical tools. Specifically, we will 1) define the distinct roles of Gpc4 and Vangl2, in regulating Wnt/PCP signaling during endoderm C&E; 2) dissect the mechanisms by which Wnt5b signaling is regulated by Gpc4 for endoderm C&E; and 3) how endodermal-expressing Gpc4 regulates non-cell autonomous functions in other germ-layers. Thus, the proposed studies are expected to provide the first detailed understanding of the function of two Wnt/PCP-signaling branches in regulating endoderm morphogenesis and illuminate how Gpc4 influences Wnt/PCP signaling during both mesoderm and endoderm C&E. Since Wnt/PCP signaling is implicated in many developmental processes as well as congenital defects and diseases, our findings will likely have a broad impact.
The proposed work will improve our understanding of the regulation of Wnt/PCP signaling in the development of the digestive system. This will shed light into the causes that contribute to common digestive birth defects such as atresia , intestinal malrotation, and congenital short bowel. In addition, our study will also have significant broad implications for other congenital defects and diseases such as spinal bifida and cancer progression that are caused by disruption of Wnt/PCP signaling.
