The objective of this proposal is to investigate the role of FoxL1+ telocytes during intestinal development through its production of Wnt5a. Wnt5a is a non-canonical Wnt ligand that is essential for development of many organs, including the intestine. The role of Wnt5a in early intestinal development has been examined in Wnt5a-/- mice, which exhibit shortened intestines due to impaired apical basal polarity (ABP) during midgut elongation. However, little is known about how Wnt5a expression is regulated, or its functions during postnatal development. Recently, our lab showed a rare population of mesenchymal cells, the Foxl1+ subepithelial telocytes (FoxL1+ telocytes), to be the essential source of canonical Wnt signals in the stem cell compartment. Furthermore, these FoxL1+ telocytes were found to express Wnt5a along the crypt wall and crypt-distal regions in adult mice. Therefore, I generated FoxL1-Cre; Wnt5af/f mice and FoxL1-CreERT2; Wnt5af/f mice to elucidate expression regulation and function of Wnt5a in FoxL1+ telocytes. This is an innovative approach because all previous Wnt5a studies lacked knowledge of the distinct cell population expressing this gene, which limited the depth of functional studies that could be performed. My model is the first to delete Wnt5a in a cell type-specific manner in the intestine, for an in vivo assessment of Wnt5a function in its biological context.
Aim 1 will determine whether FoxL1 regulates Wnt5a expression to establish ABP during intestinal development. Using FoxL1-Cre; Wnt5af/f mice, I will assess ABP integrity during midgut elongation, and perform western blotting and pulldown assays to elucidate the mechanisms involved. Using FoxL1-/- mice, I will test the hypothesis that FoxL1 binds at the Wnt5a promoter and is a direct regulator of Wnt5a expression by qRT-PCR, ChIP-seq, and luciferase assays.
Aim 2 will use a conditional Wnt5a knockout mouse model, FoxL1-CreERT2; Wnt5af/f, to investigate the role of Wnt5a in neonatal crypt formation, a process that has never been characterized previously. I will use histological methods to see if Wnt5a from FoxL1+ telocytes are required for this process. I will also perform immunohistochemical analyses, western blotting, and qRT-PCR to determine whether Wnt5a antagonizes canonical Wnt/b-catenin signaling during crypt formation. Finally, I will utilize a 3D organoid culture system for an ex vivo analysis of fetal crypt development. Together, these Aims will elucidate the roles of FoxL1 and Wnt5a signaling in regulating mammalian intestinal development, which may point to novel therapeutic targets for intestinal developmental diseases and inflammatory diseases.
The intestine is a highly regenerative organ with much of its functional capacities relying heavily on the structural integrity of the epithelium. Dysregulated signaling networks between the epithelium and the mesenchyme during intestinal development can lead to human disorders such as microvillus inclusion disease and congenital short bowel syndrome. This proposal will deepen our knowledge of how structural integrity is maintained by investigating the role of FoxL1 and Wnt5a.
