SUMMARY The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than twenty genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately eighty percent of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

SUMMARYNotch 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 cellspecific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-J 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.

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.epithelial-mesenchymal cross-talk | field pattern | villus morphogenesis

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.

Epithelial tuft cells are named for their characteristic microtubule bundle located at the cell apex where it is 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, newborn and post-natal 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.