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Fig. 4. 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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