Fig. 3.

Morphological IL2 subtype diversification is controlled by unc-39. (A) The neurites of all six IL2 are labeled by the unc-39 independent marker myIs13 (klp-6prom::GFP). Left: In WT animals, IL2 cell bodies are usually adjacent to the anterior bulb of the pharynx and their processes run in six fascicles alongside. In the unc-39 hypomorphic alleles ot1171 and e257 (both carrying the R203Q change), the lateral IL2 neurons are often out of position, fully overtaking the midline of the anterior bulb (red circled neuron past the blue dash line). Their dendrites join the the dorsal or ventral tracts (red triangles, see SI Appendix, Fig. S4A). Right: Quantification of neurite fasciculation defects (abnormally close tracts). We also considered cell body position defect like anteriorly displaced IL2 (crossing the blue midline) or IL2 soma entering into contact with each other, using NeuroPAL transgene otIs669 in wild type and unc-39^R203Q animals. (B) A cla-1 reporter transgene, that uses the klp-6 driver to label the neurite with mCherry and expresses a GFP-tagged short CLA-1 protein isoform (otIs815) is found in the endings of all six IL2 dendrites (blue segment), but also further along the dendrites (red segment; punctae marked with white triangles) of the lateral IL2 neurons. This second group punctae (along the dendrite) are eliminated in the unc-39^R203Q hypomorph. Upon misexpression of unc-39 these punctae frequently appear in multiple dendrites. See quantifications in SI Appendix, Fig. S5B. (C) Dauer arborization changes as shown by myIs13[klp-6prom::GFP]; worms are SDS selected. Top: In wild-type animals, IL2 neurons display dauer-specific arborization, with increased branching in the dorsoventral subtypes compared to the lateral subtypes. We show dauer pictures of our subtype-specific intregrated cytoplasmic reporter, egas-4(otIs833) Top Left in ventral position, egas-1 (otIs831) Top Right. Bottom: IL2 branching imaged with myIs13 cytoplasmic reporter shows increased branching of the lateral IL2 neurons of unc-39(e257) animals, which is difficult to quantify, while overexpressing unc-39 using the unc-17prom::gfp driver in all IL2 neurons (transgenic lines otEx7887 and otEx7888) strongly suppress this arborization.The backward projecting neurites we saw are labeled with red triangles. See quantifications in SI Appendix, Fig. S5C. (D) Expression of the innexin che-7 reporter allele syb4693 is gained in dauer in the lateral IL2 in WT worms, but not in unc-39^R203Q mutants. (E) An srh-71 GPCR reporter transgene (otIs867) is expressed in all IL2 in wild-type worms and lost from the lateral IL2 when they go into dauer. In unc-39^R203Q mutant dauers (ot1171 and e257 alleles), expression remains in the lateral IL2 neurons while expression is eliminated in wild-type animals.