Figure 8. ceh-34 affects the assembly of pharyngeal circuitry.

(A) ceh-34 null mutants display disorganized axodendritic projections. Axodendritic projections were scored as a whole rather than by individual neuron because with all the pharyngeal neurons being labeled it was difficult to assign specific projections to individual neurons. Projections were classified as defective only when obviously deviating from the wild-type path. Representative pictures and quantification are shown. Reporter gene is ceh-34 (otIs762). Animals were scored at the L1 stage. Statistical analysis was performed using Fisher’s exact test. N is indicated within each bar and represents number of worms. (B) ceh-34 null mutants show disorganized pharyngeal nerve ring presynaptic specializations as visualized with CLA-1 puncta. Representative pictures are shown. Quantification (right panels) shows GFP fluorescent intensity profiles along the anterior posterior axis. Reporter gene is otIs785. Animals were scored at the L1 stage. (C) ceh-34(n4796) hypomorph mutants show axonal defects in NSM (top panel) and I5 (bottom panel). Representative pictures and quantification are shown. For NSM, the ventral, dorsal, and thin projection (not visible in picture) were scored separately (graph on the left) and then data was pulled together to indicate the percentage of worms showing any defect (graph on the right). Reporter genes used are tph-1 (zdIs13) and unc-4 (otEx7503). Animals were scored at the L4 stage. Statistical analysis was performed using Fisher’s exact test. N is indicated within each bar and represents number of neurons or number of worms. (D) ceh-34 affects expression of the axon guidance cue slt-1 (kyIs174). Representative pictures and quantification are shown. Animals were scored at the L1 stage. Statistical analysis was performed using Fisher’s exact test. N is indicated within each bar and represents number of neurons scored. (E) ceh-34 affects expression of CRISPR/Cas9-engineered gfp reporter alleles of rig-3 (syb4763) and rig-6 (syb4729), two Ig superfamily members. rig-3 and rig-6 are expressed in almost all pharyngeal neurons plus many other cells within and outside the pharynx. Worms were scored with a red pan-neuronal marker (otIs355) or a red ceh-34promoter fusion (stIs10447) in the background to facilitate scoring. Number of yellow cells were counted within the pharynx. Animals were scored at the L1 stage. Statistical analysis was performed using unpaired t-test.

Figure 8—figure supplement 1. ceh-34 is required to maintain synapse organization in the pharyngeal nervous system.

Synchronized population of worms at the L1 or young adult stage were transferred onto ethanol or 5-Ph-IAA-coated plates and scored 48 hr later. Worms were expressing TIR1F79G ubiquitously under the rps-28 promoter (cshIs140). The cla-1 reporter gene is cla-1 otIs785. Representative pictures and quantification are shown. Orange bar indicates the region that was quantified. Statistical analysis was performed using unpaired t-test.

Figure 8—figure supplement 2. Axonal defects in slt-1 mutants.

(A) Effects of slt-1 on the I2/I4 neurons. Orange arrows indicate position of pharyngeal nerve ring. Blue arrow indicates ectopic projection. The gur-3 reporter array is nIs780. (B) Effects of slt-1 on the M1 neuron. Orange arrow indicate shortened M1 process, which normally extends to the anterior end of the pharynx. The str-97 reporter array is otIs716. Representative images and quantification are shown. Animals were scored at the L4 stage. Statistical analysis was performed using chi-square test. N is indicated within each bar and represents number of neurons scored.