The experiment was repeated for a third time, conducted above 29 days with three concentrations of DAPT (1, three, and 9 g/g). The pattern of significant focus-dependent inhibition of regeneration in each 863405-60-1 spines and tube toes was LY354740 maintained for 1 and 3g/g, but the animals dealt with with 9g/g DAPT died at 9 dpa (spine and tube feet measurements are demonstrated in S5 Table). There was substantial down-regulation of expression of Notch target genes hey (.sixty .02 relative fold alter, p < 0.05, one-way Fig 3. Spine and tube feet regeneration following treatment with DAPT. Regeneration (% of full-length, uncut appendages) in spines (A) and tube feet (B) after treatment with 0 (black bars), 1 g/g (grey bars), and 3 g/g (white bars) DAPT. Data are means s.e.m., n = 4 individuals (except tube feet from 0 g/g, 29 dpa, n = 3 due to mortality prior to measurement). Significant reduction in regeneration with concentration of DAPT (arcsine-transformed, Oneway ANOVA, post-hoc MRT, p<0.05).gataC (0.49 0.06 relative fold change, p < 0.05, Kruskal-Wallis), and hes (0.72 0.07 relative fold change, p = 0.05, Kruskal-Wallis), in tube feet sampled 24 hours after final treatment of 3 g/g (29 dpa). There was no clear down-regulation and high inter-individual variability of gcm in the same samples (Fig 4).The expression of stem cell marker genes, piwi and vasa, was demonstrated in mRNA isolated from untreated, homeostatic tube feet and spines (Table 1). mRNA levels, estimated by Fig 4. Inhibition of Notch signaling in regenerating sea urchins treated with DAPT. Tube feet sampled 24 hours after final treatment with DAPT, 29 days post amputation. Gene expression (qRT-PCR) of selected Notch target genes (hey, gataC, hes, gcm), compared with the geometric mean of three most stable control genes (cyclophilin7, rpl8, profilin), data are geomeans s.e.m., n = 3 animals, significant down-regulation (p < 0.05).Table 1. Gene expression of stem cell markers in sea urchin tube feet and spines. Data are means s. e.m., n = 6 individuals. Gene Tube feet vasa piwi ubiquitin cyclophilin7 rpl8 profilin qRT-PCR cycle threshold (Ct), of vasa were within the Ct range of control genes (ubiquitin, cyclophilin7, rpl8, profilin), and piwi mRNA was well within detection range. Immunohistochemical analysis of tube feet demonstrated the presence of the Vasa protein throughout the epidermis of the stalk and disc but absence in the muscle and connective tissue layers that line the lumen (Fig 5). Immunohistochemical analysis of other tissues showed Vasa-positive cells in esophagus and radial nerve, and in a sub-population of coelomocytes (circulating immune cells), but little staining in muscle from Aristotle’s lantern (Fig 5).