Figure 3: Lgr3 is expressed in a subpopulation of CNS neurons.

(a) CRISPR/Cas9-mediated sfGFP knock-in strategy used to generate the Lgr3 protein reporter allele ag5 (named as sfGFP::Lgr3), which does not contain indels in the exon 2 region, and alleles ag6-9, which contain PTC+ indels (Supplementary Fig. 4). Two thin black lines indicate the sites of the CRISPR gRNAs used. (b) Lgr3 protein scheme depicting the approximate localization of the sfGFP insertion and the protein truncations caused by the PTC+ indel mutations. (c) Sum of confocal z-stack slices of the CNS of a third instar larva stained with anti-GFP (green) to show sfGFP::Lgr3 expression (green) and with anti-nc82 (magenta) and DAPI (blue) counterstains to show the synapses (neuropil) and nuclei, respectively. Arrows point to two bilateral pairs of PIL neurons (top), to the MIL neurons in the midline of the VNC (middle), and the distal VNC pair (bottom). Arrowheads point to the proximal projections of the PIL neurons. sfGFP::Lgr3 is also expressed in ∼170 other cell bodies, but at a lower level than in PIL and MIL neurons. f, oesophageal foramen, seg, subesophageal ganglion. See also Supplementary Fig. 4c for controls on the specificity of this staining pattern. (d) Box plots (see Methods) showing pupariation time of (N) larvae obtained from 11 egg layings. Whiskers are 5 and 95% percentiles. Dots, outliers. P<0.0001, Kruskal–Wallis test. Genotypes sharing the same letter (blue) are not statistically different at α=0.01, Conover post hoc test. Degrees of freedom, H and C values for the Kruskal–Wallis tests are df=7, H=397.84 and C=1.00. Scale bar, 50 μm.