1. Sexually Dimorphic unc-6/Netrin Expression Controls Sex-Specific Maintenance of Synaptic Connectivity 
Peter Weinberg, Matthew Berkseth, David Zarkower, Oliver Hobert 
Current Biology 
Volume 28, Issue 4, Pages e3 (February 2018)
DOI: /j.cub
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2. Current Biology 2018 28, 623-629.e3DOI: (10.1016/j.cub.2018.01.002)
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3. Figure 1
UNC-6/Netrin and Its Receptor UNC-40/DCC Are Required to Maintain PHB>AVG Synaptic Connections
(A) Phasmid neurons PHA and PHB are connected to interneuron targets in a sexually dimorphic manner [1, 2], and this dimorphism arises through the pruning of synapses in a sex-specific manner during late larval stages [3].
(B) Imaging sexually dimorphic PHB>AVG synapses with GRASP [3]. Each individual green fluorescent dot is a synaptic density; red fluorescence marks the processes of the connected neurons (see STAR Methods); and arrowheads indicate en passant synapses. Quantification of these wild-type data is shown in (C).
(C) Dot plots that quantify the male-specific PHB>AVG synapses (as shown in B) in wild-type, unc-6, and unc-40 animals (visualized with GRASP array otIs614 for wild-type versus unc-6 comparison; otEx6913 for wild-type versus unc-40 comparison). Data for ced-5 are shown in Figure S1. Data for adults (not shown) are the same as shown here for L4. Quantifications are shown as beeswarm plots, with each dot representing a single animal, the blue line representing the median, and each black line representing the upper and lower quartiles. Comparisons were done with the Wilcoxon rank-sum test (∗∗p < 0.01, ∗∗∗p < 0.001; n.s., not significant). Between 40% and 50% of males or hermaphrodites display axon guidance defects of PHB and AVG, such that the red fluorescent protein (RFP)-labeled PHB/AVG processes (see B) are not neighboring one another. Synapses were only scored in animals in which RFP-labeled PHB and AVG are directly adjacent to one another.
(D) Hermaphrodite-specific PHA>AVG synapses (visualized with otEx6347 and otIs630, which label synapses with iBLINC [3, 30]) are not affected in unc-6 mutants.
(E) The PHB>AVA synaptic connection at the first larval stage. This connection is defective in unc-6 mutants already in the first larval stage, arguing for a developmental role of unc-6.
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4. Figure 2 Dimorphic Transcription of unc-6 in the AVG Neuron
(A) unc-6 fosmid reporter expression (otIs638; schematically shown in Figure S2) in both sexes at various stages. Expression levels were binned into 3 categories, normal (bright), dim, and not detectable (off); for statistical comparison, normal and dim expression were binned together. Cellular sites of expression were identified by characteristic position and morphology of cells (AVG has an unusually large nucleus), as well as by co-labeling with an AVG-specific landmark reporter (inx-18).
(B) Non-dimorphic, broad expression of an unc-40 fosmid reporter construct (otIs647; schematically shown in Figure S2) in the tail region of the worm (where the PHA>AVG synapse is generated; see Figure 1A). Expression in early stages shows no obvious differences (not shown). The red arrows indicate PHB neurons (identified by dye filling) in which UNC-40 is expected to function.
(C) smFISH showing unc-6 expression at different larval stages and, as control for probe specificity, in unc-6 (ev400) mutant animals, which due to a premature stop codon and nonsense-mediated mRNA decay are not expected to contain unc-6 mRNA. For the beeswarm plot quantification, each dot represents a single animal with the number of individual mRNA molecules in AVG indicated. Comparisons in (A) and (C) were done with the Wilcoxon rank-sum test (∗∗p < 0.01; ∗∗∗p < 0.001).
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5. Figure 3
unc-6 Is Required and Sufficient to Induce Synapse Maintenance
Non-dimorphic, constitutive expression of unc-6 under control of the inx-18prom driver (otEx6914) in wild-type and unc-6 mutant animals shows rescue of the unc-6 mutant synapse phenotype (PHB>AVG synapses, visualized with otIs614) and sufficiency of unc-6 function. Three extrachromosomal lines were first generated and analyzed in a wild-type background (upper panel), and one line (otEx6914) was then crossed into a unc-6 mutant background (lower panel). Quantifications are shown as beeswarm plots, with each dot representing a single animal, the blue line representing the median, and each black line representing the upper and lower quartiles. Comparisons were done with the Wilcoxon rank-sum test (∗∗p < 0.01, ∗∗∗p < 0.001).
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6. Figure 4
TRA-1 and LIN-11 Control unc-6 Expression in an Intersectional Manner
(A) Degradation of TRA-1 specifically in the AVG neuron of hermaphrodites, via ectopic expression of fem-3 with the inx-18prom driver (otIs606), results in the upregulation of unc-6 fosmid reporter expression (otIs638). For statistical comparison, normal and dim expression were binned together.
(B) The 7th intron of unc-6 contains cis-regulatory information that is sufficient for sexually dimorphic unc-6 expression; this dimorphic expression requires four predicted TRA-1 binding sites.
(C) lin-11 is required for the non-sex-specific expression of the unc-6 fosmid reporter (otIs638) in pre-sexual maturation stages. Quantification is shown for effect in the AVG neuron (the RIF neuron is similarly affected).
(A and C) ∗∗∗p <
(D) In the context of the nervous system, the gfp-tagged tra-1 locus (see Figure S2 for details) produces GFP::TRA-1 exclusively in hermaphrodite neurons, beginning at the third larval stages. Note that in the context of the retrovesicular ganglion (RVG), expression in AVG is most pronounced (AVG was identified by characteristic position and nuclear size). Asterisks indicate autofluorescence of gut tissue.
(E) Schematic summary of results.
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