Fig. 3. Eye-disc-specific hh variant expression from clones in moving and static sources.

a The cartoon shows reiterated cell-to-cell Hh signaling from a clonal source (Supplementary Fig. 5) moving at a particular pace to drive photoreceptor differentiation across the eye primordium. Red arrows denote Hh spread, blue arrows denote the movement of the morphogenetic furrow (MF), t₀-t₊₂ indicates early and later stages. The asterisk and dashed area indicate the ocellar field (as schematically shown in b). Clones homozygous for hh[KO] do not express hh, which severely impairs eye development, but hh or hh variant cDNA expression from both alleles restores eye development. Scale bar: 100 μm. Despite the observed restoration of eye development, quantification of ommatidia numbers per eye of female flies reveals significantly impaired activities of both hh variants. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. Error bars represent standard deviation. n = 6 (hh), n = 32 (hh[KO]), n = 19 (hh[KO;hh]), n = 18 (hh[KO;hh^R/A]), and n = 22 (hh[KO;hh^R/E]), F = 1474, ****p ≤ 0.0001. Ctrl: FRT82B donor line. Source data are provided as a Source Data file. b More severely affected development of another eye disc area (as shown in the cartoon adapted from⁵²) that uses the same Hh signaling components, but differs from the system described in (a) in that it depends on Hh signaling over a relatively long distance (tens of micrometers⁵²) from a static source (indicated by red bent arrows). The asterisk in (a) marks this ocellar region in the disc. Clones homozygous for hh[KO] do not express hh in this area and strongly impair the development of one or more of the three camera-type eyes called anterior and posterior ocelli (aOC and pOC, red circle, the hh expression domain will become the interocellar region (iOR), Supplementary Fig. 5). Clones homozygous for hh[KO;hh^R/E] also impaired their development. The average number of ocelli per hh[KO] fly: 0.44 ± 0.6 ocelli, n = 16. hh[KO;hh^R/E]: 1 ± 0.8 ocelli, n = 24. The normal number of three ocelli/fly was observed for all other genotypes. Error bars represent standard deviation. No sex bias was observed in any assay. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test, F = 99. n = 6 (hh), n = 16 (hh[KO]), n = 20 (hh[KO;hh]), n = 18 (hh[KO;hh^R/A]), and n = 24 (hh[KO;hh^R/E]). ****p ≤ 0.0001, ns: p > 0.99. Scale bar: 100 μm. Source data are provided as a Source Data file. Bottom: Fly genotype used in these experiments, the X denotes no hh (hh[KO]), hh (hh[KO;hh]), or hh variants.