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. 2023 Feb 10;14:758. doi: 10.1038/s41467-023-36450-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Quantification of the numbers of ommatidia per eye in female flies. Hh biofunction in eye disks of flies homozygous for transgenic hh (in hh[KO;hh]/hh[KO;hh] flies) is increased over that in w¹¹¹⁸ or heterozygous controls. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. n = 10 for each genotype, F = 13.6. Error bars represent standard deviation. ***p ≤ 0.0004. Source data are provided as a Source Data file. b Inset: Hh produced in the posterior compartment of the wing disc directly patterns the L3-L4 intervein area (orange) and indirectly patterns the L2-L3 intervein area (green). Quantification of L3-L4/L2-L3 intervein areas revealed significantly increased patterning activities of transgenic Hh. Statistical significance was determined by unpaired t-test (two-tailed). n = 13 wings for w¹¹¹⁸ and n = 27 for hh[KO;hh]/hh[KO;hh], ****p < 0.0001. L1-5 indicate the five longitudinal Drosophila wing veins. Source data are provided as a Source Data file. c Segment phenotype changes in hh[KO;hh^R/A]/hh[KO;hh^R/A] larvae resemble those in sfl/sfl larvae that are deficient in HS biosynthesis, and hh[KO;hh^R/E]/hh[KO;hh^R/E] larvae resemble hh^AC/hh^AC larvae that lack hh expression. Segment phenotypes were consistently observed in three independent crosses. Scale bar: 100 μm. c′ Schematic of the self-reinforcing Hh/Wg signaling loop at one of the 14 parasegment boundaries in Drosophila embryos. Due to Hh/Wg interdependency to maintain the loop, depletion of either morphogen will result in embryos that lack a naked cuticle. d Transgenic hh and both hh variants restore eye development in flies homozygous for impaired endogenous hh expression specifically in the eye (hh^bar3/hh^bar3). This demonstrates the largely retained bioactivity of both Hh variants in this system. Scale bar: 100 μm. e Quantification of the numbers of ommatidia per eye of female flies as shown in (d). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. n = 8 for hh^bar3/hh^bar3, n = 10 for hh^bar3/+ and hh[KO;hh]/hh^bar3, and n = 30 for hh[KO;hh^R/A]/hh^bar3 or hh[KO;hh^R/E]/hh^bar3. Error bars represent standard deviation. F = 357. ****p ≤ 0.0001, ns: p = 0.99. Source data are provided as a Source Data file.