Fig. 4.

lapsyn is expressed in glia. a The schematic shows the lapsyn genomic locus, the lapsyn ^ZG1 deletion, and Mi{MIC}lapsyn ^MI01316 and PBac{SAstopDsRed} ^LL00906 insertions. pA polyadenylation site, SA splicing acceptor site. To generate lapsyn ^2xHA-CR13.1, two C-terminal HA epitope tags were inserted into the endogenous locus following a Cas9-induced DSB and HDR with a single-stranded oligo donor nucleotide (ssODN) containing flanking 50-nt homology arms. b For whole animal rescue experiments of lapsyn ^ZG1embryonic lethality, UAS-lapsyn was expressed in neurons (elav ^C155 -Gal4, blue bars, 224 recovered first instar (1L) larvae of 1210 embryos) or in glia (repo-Gal4, green bars, 529 recovered 1L larvae of 2380 embryos). GFP-negative rescue flies were compared to controls carrying CyO Dfd-YFP balancer chromosomes, corresponding to all other surviving progeny (heterozygous, or heterozygous and overexpressing lapsyn, light blue and green bars). Rescue of viability of 1L larvae (left panel) is shown as percentage of expected progeny. Rescue of viability of third instar (3L) larvae, early and late pupae and adults is shown as percentage of surviving progeny in “rescue” and “control” populations followed from the 1L stage onwards. Histograms show data points as means ± standard deviation error bars (n = 3 independent experiments). Unpaired, two-tailed Student’s t-test assuming normality but unequal standard deviation (1L: P = 0.879; 3L: P = 0.1027, P = 0.6796; early pupae: P = 0.006, P = 0.9369; late pupae: P = 0.0042, P = 0.9921; adult: P = 0.0047, P = 0.8668). NS not significant, **P < 0.01. c–h Mi{MIC}lapsyn ^MI01316 is inserted into the first non-coding intron. In 3L larvae, Repo-positive (red) surface glia (sg) and medulla cortex glia (mcg), but not mng (c) are labeled with GFP. mng express GFP at 24, 42, 55, and 72 h APF, and in adults (arrowheads). mng processes are labeled from 55 h APF onwards (f–h, arrows). i, j lapsyn ^2xHA-CR13.1 and the fosmid lapsyn ^fTRG027706 report abundant lapsyn protein localization in mng cell bodies (arrowheads), primary (arrows), and secondary processes (double arrowheads), particularly in layer M5, using anti-HA (red) and GFP labeling (green), respectively. La lamina, Lo lobula, Lop lobula plate, Me medulla. Panels c–j show single optical sections. For genotypes, sample numbers and additional statistical values, see Supplementary Tables 1 and 2. Scale bars, 50 μm