zyx-1 mutants are unable to maintain mechanosensory synapses. (A) Timecourse of PLM synapse development in wild-type and zyx-1(gk190) animals at 22.5°C. The mechanosensory neurons were double labeled with GFP::RAB-3 and cytosolic mRFP. Images are maximal projections of multiple image planes taken in widefield epifluorescence configuration. The left panels show animals in a roughly ventral orientation, whereas the larger animals in the right panels are in a lateral orientation. Arrows mark the position of PLM branches. PVM is born at ∼8 hph, and thus was absent from the earlier time points. Scale bars: 10 µm for 2-16 hph; 20 µm for 24-48 hph. (B) Quantification of PLM processes that had initiated branches, formed full branches reaching the VNC, and formed synapses at various time points after hatching. n=24-192 for individual time points. A detailed breakdown of this dataset is given in supplementary material Fig. S4. Strains used were NM3361 and NM3413.

Breakage and retraction of PLM collateral branches. Epifluorescence images of live L4 zyx-1 animals. (A-C) Examples of retracting branches (open arrowheads) that sometimes included a retraction bulb (see C). (D-I) mRFP-positive PLM remnants (arrows) were often present in L3, L4 and young adult zyx-1 animals. They usually consist of one very bright spot (but occasionally multiple closely spaced spots, see D) that were positioned between the processes of PVM (lower brighter process marked by double arrow) and AVG (upper brighter process marked by double arrow), which demarcate the most ventral and most dorsal regions of the VNC. Most remnants were mRFP positive and GFP negative, but a minority were double positive (see E,F). Occasionally, two distinct widely spaced remnants were seen (H,I; the less bright remnant is out of the plane of focus). Dashed arrows mark PLM processes; white arrowheads label large PLM synapses; and asterisks label presumed distal ends of severed PLM branches (in A,B). Gut granules (examples marked with chrevrons) are also visible in many images. Strains used were NM3361 and NM3413. Scale bar: 10 µm.

zyx-1 mutant synapses retract during development. (A) Timecourse of PLM synapse structure of representative animals. Individual wild-type or zyx-1(gk190) animals were immobilized in 10 mM sodium azide at 12 hph, imaged at 100 ×, recovered on an NGM plate, and then reimaged in similar fashion at 24 hph and 48 hph. The zyx-1 examples illustrate that PLM synapse loss is stochastic. In the top example, one synapse is lost while the other grows. Furthermore, they illustrate that PLM synapses can be lost both early (bottom) as well as late (top) in development. Scale bar: 10 µm. (B) Temperature shift of zyx-1(gk190) animals showing the PLM synapses in L4 larvae as a function of the time of shift (from permissive to non-permissive temperature, or vice versa). n=127-253 animals for shifted time points and n=95-173 for unshifted controls. Strains used were NM3361 and NM3413.

ZYX-1 functions cell-autonomously to regulate mechanosensory synapse development. (A) The expression of full-length ZYX-1A cDNA in presynaptic mechanosensory neurons (Pmec-7::mCherry::zyx-1::GFP) rescues the zyx-1 mutant phenotype, whereas muscular or interneuronal expression does not. PLM synapses formed per animal are displayed for wild type, zyx-1, and zyx-1 mutants expressing zyx-1::GFP fusions under the control of distinct promoters. Also shown are rescues by a complete zyx-1 genomic construct and a genomic construct only capable of expressing zyx-1b from the P4 promoter. (B) The expression of ZYX-1 C-terminal LIM domains in mechanosensory neurons rescues the PLM synapse defects of zyx-1 mutants. PLM synapses formed per animal are displayed for wild type, zyx-1, and zyx-1 mutants expressing various domains of ZYX-1 under the control of the mec-7 promoter. (A,B) Error bars indicate mean ± s.e.m. n=58-897. (C) Subcellular localization of ZYX-1 LIM domain fusions. The structure of various ZYX-1 domain constructs is depicted alongside the localization pattern observed in PLM and PVM, and the phenotypic consequences of expression of the construct in touch receptor neurons. Many LIM domain constructs were able to traffic to the nucleus, but the rescuing activity of the constructs was independent of subcellular localization. Scale bar: 10 μm.