Ann S. Grosse

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.

Paracrine Hedgehog Signaling in Stomach and Intestine: New Roles for Hedgehog in Gastrointestinal Patterning

BACKGROUND & AIMS Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine, and colon. METHODS Hedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed. RESULTS Hedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include the following: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the 2 layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; and after birth, the muscularis mucosa and villus smooth muscle consist primarily of Hedgehog-responsive cells and Hh levels actively modulate villus core smooth muscle. CONCLUSIONS These studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus, and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association.

Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi

In the adult intestine, an organized array of finger-like projections, called villi, provide an enormous epithelial surface area for absorptive function. Villi first emerge at embryonic day (E) 14.5 from a previously flat luminal surface. Here, we analyze the cell biology of villus formation and examine the role of paracrine epithelial Hedgehog (Hh) signals in this process. We find that, before villus emergence, tight clusters of Hh-responsive mesenchymal cells form just beneath the epithelium. Cluster formation is dynamic; clusters first form dorsally and anteriorly and spread circumferentially and posteriorly. Statistical analysis of cluster distribution reveals a patterned array; with time, new clusters form in spaces between existing clusters, promoting approximately four rounds of villus emergence by E18.5. Cells within mesenchymal clusters express Patched1 and Gli1, as well as Pdgfrα, a receptor previously shown to participate in villus development. BrdU-labeling experiments show that clusters form by migration and aggregation of Hh-responsive cells. Inhibition of Hh signaling prevents cluster formation and villus development, but does not prevent emergence of villi in areas where clusters have already formed. Conversely, increasing Hh signaling increases the size of villus clusters and results in exceptionally wide villi. We conclude that Hh signals dictate the initial aspects of the formation of each villus by controlling mesenchymal cluster aggregation and regulating cluster size.

Gastric tuft cells express DCLK1 and are expanded in hyperplasia

Epithelial tuft cells are named after their characteristic microtubule bundles located at the cell apex where these are exposed to the luminal environment. As such, tuft cells are found in multiple organs, including the gastrointestinal (GI) tract where the apical “tuft” is hypothesized to detect and transmit environmental signals. Thus, the goal of our study was to characterize gastric tuft cells during GI tract development, then subsequently in the normal and metaplastic adult stomach. GI tracts from mouse embryos, and newborn and postnatal mice were analyzed. Tuft cells were identified by immunohistochemistry using acetylated-α-tubulin (acTub) antibody to detect the microtubule bundle. Additional tuft cell markers, e.g., doublecortin-like kinase 1 (DCLK1), were used to co-localize with acTub. Tuft cells were quantified in human gastric tissue arrays and in mouse stomachs with or without inflammation. In the developing intestine, tuft cells in both the crypts and villi expressed all markers by E18.5. In the stomach, acTub co-localized with DCLK1 and other established tuft cell markers by E18.5 in the antrum, but not until postnatal day 7 in the corpus, with the highest density of tuft cells clustered at the forestomach ridge. Tuft cell numbers increased in hyperplastic human and mouse stomachs. In the adult GI tract, the tuft cell marker acTub co-expressed with DCKL1 and chemosensory markers, e.g.,TRPM5. In summary, tuft cells appear in the gastric antrum and intestine at E18.5, but their maximal numbers in the corpus are not achieved until after weaning. Tuft cell numbers increase with inflammation, hyperplasia, and metaplasia.

Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development

Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor.

WNT5a in tongue and fungiform Papilla development.

Fungiform papillae are complex taste organs that develop in a pattern on anterior tongue in rodent embryos. Several intrinsic secreted molecules are important for papilla development and patterning, including sonic hedgehog, bone morphogenetic proteins, Noggin, epidermal growth factor, and WNTs. Recent data about roles of WNTs in regulation of tongue and fungiform papilla development lead to new insights about the importance of tissue and timing contexts when studying the effects of morphogenetic proteins. WNT/β‐catenin signaling is required for formation of fungiform papillae, but not for determining tongue size and shape. In contrast, WNT5a apparently is important for tongue outgrowth, but not papilla development. Preliminary data from WNT5a mutant mice separate genetic programs for papilla number from those for tongue shape and size.

250 Ezrin, an Apical Surface Protein, Functions in a Distinct Type of Cell Division in the Early Intestinal Epithelium to Aid Expansion of Luminal Surface

Intestinal villi provide an enormous surface area for nutrient absorption. Significant loss of intestinal surface area can compromise intestinal function, causing intestinal failure. Though short-term treatments for this life-threatening condition are available, all patients need lifelong monitoring for growth and nutritional status, and would benefit from treatments that can directly increase intestinal surface area. In mice, a large increase in surface area occurs with villus development, which begins at embryonic day (E)14.5, when the thick pseudostratified epithelium with a flat luminal surface is converted to a columnar epithelium covering a field of emerging villi. Though it has long been thought that epithelial remodeling occurs by formation and fusion of secondary lumina, recent work in our laboratory showed that secondary lumina do not exist (Grosse et al., Development 138:4423, 2011). Seeking an alternative mechanism for luminal expansion, we found a unique type of cell division that is triggered specifically at E14.5, which we have named an e-division (lumen extending division). We propose that in an e-division, new apical surface is deposited at the cytokinetic plane such that the two daughter cells segregate onto adjacent villi. The e-division is distinct from cell divisions occurring before E14.5, which we have named g-divisions (girth building divisions); g-divisions do not involve deposition of new apical surface between daughter cells. Our data (lineage tracing, 3D reconstruction, and SEM) suggest that in mice deficient in the apical surface protein Ezrin, e-divisions fail stochastically, resulting in fused villi. We are modeling e-divisions in vitro using MDCK and Caco2 cell lines, which form luminal surfaces during the first cell division when plated in a 3D matrix. Using RNA interference, we have found that reducing Ezrin expression compromises lumen formation in our 3D cyst assay, providing a mechanistic explanation for the presence of fused villi in vivo. Further understanding of the process of villus development will improve in vitro bioengineering of intestinal surface, potentially yielding novel therapies for those with intestinal failure.