Met is required to prevent CaPs from expressing an inappropriate neurotransmitter. For each condition we examined eight spinal hemisegments per embryo. (a-c″) Embryos at 26 hpf labeled with antibodies to GABA (red) and zn1 plus znp1 (green); (a′,b′,c′) only the green channel is shown; (a″,b″,c″) only the red channel of the micrographs shown in (a,b,c) is shown. CaPs expressed GABA in embryos injected with translation blocking (b-b″) or splice blocking (c-c″) met MOs, but not in controls (a-a″). Note that some CaPs in met MO-injected embryos also have ectopic axons within the spinal cord (arrows). (d-e″) Embryos at 26 hpf labeled with riboprobes to gad1 (green) and islet2 (red). (d′,e′) Only the green channel is shown; (d″,e″) only the red channel of the micrographs shown in (d,e) is shown. In met MO-injected embryos, islet2-positive CaPs also express gad1 (e-e″), whereas these two genes are not co-expressed in CaPs of control embryos (d-d″). (f-g″) Embryos at 26 hpf labeled with antibodies to GABA (red) and zn1 plus znp1 (green). (f′,g′) Only the green channel is shown; (f″,g″) only the red channel of the micrographs shown in (f,g) is shown. CaPs do not express GABA in embryos co-injected with met MO and mMet mRNA (f-f″), but do express GABA in embryos co-injected with met MO and lacZ mRNA (g-g″). (h,i) Embryos at 26 hpf labeled with antibodies to GABA (red) and Islet (green). CaPs co-express Islet and GABA in met MO-injected embryos (i) but not in controls (h). n = 80 embryos in (a); n = 128 embryos in (b); n = 24–32 embryos each for (c,e,f,g); n = 20 embryos for (h); n = 35 embryos for (i). The phenotype was seen in 70% of embryos injected with the splice-inhibitor MO and 80% of embryos injected with translation-blockers; in affected embryos all segments showed the phenotype. Rescue was seen in 90% of embryos injected with mMet mRNA and 0% of embryos injected with lacZ mRNA. Scale bars, 10 μm.
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