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To identify differentiating secretory cells early in embryogenesis, we used the pan-secretory monoclonal antibody 2F11 at 74?hpf, which is the first time this marker is present in the intestinal epithelium. During this period other markers of secretory cell differentiation are not yet expressed. We find increases in secretory cells when Notch signaling is inhibited between 30 and 34?hpf but are not statistically significant. The p value generated in the T test is just above the level of significance with application of the Bonferroni correction (p=0.029 with a level of significance pselleck inhibitor inhibition from 34 to 48?hpf ( Fig. 6C). Inhibition of Notch signaling between 64 and 74?hpf increases secretory cell numbers however, at 74?hpf most of the epithelium VAV2 appears to express the 2F11 epitope. Since embryos were fixed just after DAPT inhibition, we thought that this might be affecting 2F11 expression. To allow additional time between DAPT inhibition and fixation, we again inhibited Notch signaling with DAPT but allowed the embryos to develop until 96?hpf. At 96?hpf, we find that 2F11 is expressed in cells with characteristic secretory cell shape and no longer throughout the entire epithelium. Comparison of Notch inhibition between 64 and 74?hpf and DMSO control embryos demonstrates a significant increase in secretory cells at 96?hpf ( Fig. 6D). Therefore, high throughput screening upon Notch inhibition with DAPT between 30�C34?hpf and 64�C74?hpf, we find increases in cells expressing ascl1a and deltaD as well as increases in the overall number of intestinal epithelial secretory cells. Embryos which underwent Notch inhibition during 30�C34 and again during 64�C74?hpf were grown to 5?dpf and analyzed for changes in secretory cell numbers. In addition to the 2F11 antibody, we utilized markers of differentiated secretory cells for enterochromaffin and goblet cells. 5HT was used to identify enterochromaffin cells while wheat germ agglutinin (WGA) was used to identify mucin in goblet cells. Embryos exposed to DAPT only during the 30�C34?hpf or 64�C74?hpf period did not have significant changes in the number or type of secretory cells at 5?dpf. We hypothesized that there may be a compensatory affect within the epithelium after Notch inhibition by DAPT is relieved, resulting in subsequent restoration of the wild type secretory to enterocyte ratio. To determine whether increased numbers of secretory cells could be retained until the end of embryogenesis, Notch inhibition using DAPT was continued until 5?dpf. We began DAPT exposure beginning around the times when we observe increases in ascl1a, deltaD, or secretory cell numbers at 74?hpf (30�C60?hpf) or when Notch signaling appears to not be active (34?hpf).