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Fig. S12 The lhx9 morpholino does not induce apoptosis and can be rescued by lhx9 overexpression. Tg(hcrt:mRFP) embryos were stained with acridine orange at 24 hpf to quantify apoptotic cells (A-D). There was no increase in apoptosis in embryos injected with lhx9 morpholino (C) compared to embryos injected with the lhx9 mismatch control morpholino (B) or wild-type embryos (A), suggesting that the reduced number of Hcrt neurons in lhx9 morphants is not due to apoptosis. Acridine orange labeled fewer cells in embryos injected with the p53 morpholino (D), presumably because apoptosis that normally occurs during development was suppressed. (E) Mean ± s.e.m. number of apoptotic cells in the white boxed region. At least 4 embryo brains were analyzed for each condition. **, p<0.01 compared to WT by one-way ANOVA followed by Bonferroni’s correction for multiple comparisons. (F) To rescue the morpholino phenotype, Tg(hcrt:EGFP) embryos were co-injected with the lhx9 morpholino and the hs:lhx9 plasmid. Following heat shock at 24 hpf, lhx9-overexpressing cells located in the endogenous hcrt expression domain also expressed hcrt (white arrowheads), indicating that lhx9 overexpression can rescue the lhx9 morpholino phenotype. (G) Mean ± s.e.m. number of endogenous and rescued hcrt cells per brain. Following rescue, the total number of hcrt-expressing cells is similar to the number observed in wild-type embryo brains (see Fig. 2G). hcrt expression in rescued cells was weaker than in endogenous Hcrt neurons, presumably because the rescued cells only received a pulse of lhx9 while endogenous Hcrt neurons continuously express lhx9. Three embryo brains with ectopic lhx9 expression in the endogenous Hcrt region were quantified. Anterior views of 26 hpf embryos are shown. Scale = 10 µm.