Somatic receptor mutation indicates that Cimetidine acts through hrh2b in zebrafish. A: Examples of gels used to assess the efficiency of injected CrRNA by RFLP are shown. Each lane represents one embryo. In uninjected controls, a complete band shift is observed, indicating activity of the indicated restriction enzyme, whereas in 8 haCR-injected animals almost no digest occurs, indicating successful mutation of the recognition site. B: A schematic timeline of the experimental design, combining somatic mutation with drug treatment is depicted. C: Lateral view of the spinal cord injury sites in somatic mutant larvae with and without Cimetidine treatment are shown. D: Quantification of the overall GFP fluorescence of the cimetidine-treated somatic receptor mutants relative to DMSO-treated control indicate that in condition in which hrh2b haCRs were injected the effect of Cimetidine is abolished. (uninjected condition, Mann-Whitney tests, p* = 0.0373; hrh2a CrRNA condition, t-test, p*** = 0.0005). E: Cimetidine treatment leads to an increase in the relative hrh2b mRNA expression in lesioned wild-type larvae (50 treated or control larvae were pooled per measurement; Kruskal-Wallis test, p* = 0.0242 with Dunn's multiple comparisons test: injured, DMSO vs injured, cimetidine p* = 0.0324). hrh2a mRNA expression is unaffected by cimetidine treatment. Scale bars: 100µm.
|
