Genetic interactions between pac1 and otpb. (A,C-H) High-resolution micrographs of embryos (at 52 hpf, lateral view, anterior to the left) that were subjected to in situ hybridization with an isotocin (IT)-directed probe, followed by immunostaining with an anti-tyrosine hydroxylase (TH) antibody. The two prominent clusters of dopaminergic (DA) neurons (Gr. 2 and Gr. 3-6), stained in brown, as well as isotocinergic (IT) neurons, stained in purple, are indicated. (B) Bar chart showing average cell counts of isotocinergic (IT), dopaminergic Group 2 (DA Gr. 2) and Groups 3-6 (DA Gr. 3-6). Error bars indicate s.d. The number of embryos (n) is shown above. In order to represent unilateral cell number (as shown in the micrographs), neurons were counted on both sides of the brain and the total number was divided by two. Statistical analysis for all treatments was done by ANOVA test. Wild-type (WT) embryos were injected with antisense morpholinos directed against either otpb (otpbMO; C,D,E and see Fig. S7 in the supplementary material) or pac1 (pac1MO; F,G,H). (C-H) Embryos were injected with in vitro synthesized capped mRNA encoding either Otpb (D,G; at 7-15 pg per embryo) or a mutant form of PAC1 (denoted pac1*) containing a single E239Q base substitution that renders it constitutively active (E,H; at 200pg per embryo). Injected pac1* and otpb RNAs were constructed without their respective morpholino-binding site, thus enabling a genuine gene-function complementation rather than competition for morpholino binding. The amount of injected mRNA in Otpb and PAC1 gain-of-function experiments was determined by titrating the minimal doses of mRNA that could rescue otpb and pac1 morphant phenotype, respectively. Embryos that showed abnormal brain morphology were omitted from our analysis. Scale bar: 50 μm.
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