Selected Oral Presentation: Blueprint for Intestinal Villus Development

Abstract

Finger-like projections called villi are the functional units of the small intestine that carry out the absorptive role of this organ. Each villus is covered by an endodermally derived absorptive epithelium and contains a mesodermally derived core of supporting tissues, including tightly coupled vascular and lymphatic networks. In the mouse, villus development is a late fetal event that begins at E14.5, with the formation of regularly spaced clusters of mesenchymal cells. Cluster formation requires Hedgehog ligands that are secreted by the epithelium and act on the underlying mesenchymal cells to promote their clustering. Clusters form directly beneath the pseudostratiified epithelium and associate closely with the basement membrane. Cluster formation begins proximally and dorsally, spreading ventrally and distally over the next 36 hours. This spreading cluster field is patterned in a manner consistent with a self-organizing Turing field that is controlled by Bmp signaling within the mesenchyme. As clusters grow, epithelial cells in contact with the forming clusters begin to change shape, becoming shorter and wider, resulting in the emergence of the first villus domes. As villi lengthen, clusters remain tightly associated with the tips of the emerging villi, becoming part of the villus core12. Since every villus contains vascular elements, we explored the means by which these important elements become associated with each villus. Here, we demonstrate that the earliest nascent mesenchymal clusters are closely associated with endothelial sprouts. Indeed, when vascular development is inhibited, clusters and villi fail to form, providing a direct functional link between the vascular system and villus growth. To explore this link further, we altered vascular pattern and examined the effects of this perturbation on villus growth. Notch signaling is known to be a critical regulator of vascular development, controlling the balance of tip (angiogenic sprouts) versus stalk cells. Indeed, we found that global inhibition of Notch signaling by treatment of intestinal explants with the gamma-secretase inhibitor, DAPT, altered villus development and pattern. To investigate this effect more precisely in vivo, we used tissue-specific genetic recombination to alter Notch signaling in the endothelium and analyzed the effect of altered vasculature on cluster formation and villus emergence. To reduce Notch signaling in vascular cells, we genetically deleted the required Notch co-factor Rbpj or activated the transgenic expression of a dominant negative form of the required co-factor Mastermind-like (MAML). In both cases, we observed increased vascular sprouting, accompanied by formation of larger clusters and the presence of multiple clusters within large branched villi. In contrast, increased Notch signaling in the endothelium was generated by transgenic overexpression of the Notch intracellular signaling domain, NICD. This manipulation led to decreased endothelial sprouts, a reduction in the size and number of clusters, and diminished villus development. Together, these results demonstrate that vascular sprouts provide critical cues to the forming mesenchymal clusters. The contact between mesenchymal clusters and vascular sprouts directs the outgrowth and patterning of villi. Furthermore, the continued association between mesenchymal clusters and vascular elements insures that each villus is endowed with an associated vasculature that is essential for its absorptive function.