Fig. 5: Zfh1 regulates wing dimorphism independent of the IIS pathway.
From: The transcription factor Zfh1 acts as a wing-morph switch in planthoppers
a Number of females with different wing morphs upon double-gene knockdown. The LW ratio is compared between two groups using Pearson χ2 test (dsInR1 + dsFoxO vs dsInR1 + dsGfp: **** P = 5.0866E-47, χ2 = 207.131, df = 1; dsAkt + dsFoxO vs dsAkt + dsGfp: ****P = 4.763E-45, χ2 = 198.359, df = 1; n.s. non-significant). b The FoxO::HA protein level in FoxO::HA mutants following dsRNA treatments. Treatment with dsZfh1 only lowers the level of FoxO proteins. c Construction of Zfh1::HA mutants using CRISPR/Cas9-mediated homology-directed repair. The target site for Cas9, protospacer-adjacent motif (PAM), and exons 1–5 are indicated. An ssDNA donor contains a left homology arm, a HA-tag (HA), and a right homology arm. The primers of Zfh1-KI-F and Zfh-KI-R are designed for Sanger sequencing. d Confirmation of Zfh1::HA mutants by western blot. Zfh1::HA fusion protein was detected by an HA antibody. e Confirmation of Zfh1::HA mutants by Sanger sequencing. PCR products amplified from genomic DNA with Zfh1-KI-F and Zfh-KI-R were exposed to Sanger sequencing. The HA sequence is in blue. f The Zfh1::HA protein level in the context of perturbation of the IIS pathway in Zfh1::HA mutants. β-actin is used to show equal protein loading in (b), (d), and (f), and the experiments were repeated three times independently with similar results. g Relative expression of components of the IIS pathway in dsZfh1-treated BPHs. The experiments were repeated five times with similar results (dots or circles). Data are presented as mean ± SEM. Two corresponding columns are compared using two-tailed unpaired t-test (n.s. non-significant). h Morphology of an InR2-null mutant (InR2E4)43. i dsZfh1 increases the forewing size of InR2E4 mutants. InR2E4 nymphs were microinjected with dsGfp (InR2E4::dsGfp) or dsZfh1 (InR2E4::dsGfp), and then emerged adults were collected for wing size measurement. The whiskers of box plot represent the quantile percentile, from bottom to top are minima, 25%, median, 75%, and maxima, respectively. Two corresponding boxes are compared using two-tailed unpaired t-test (*P = 0.0222 for male and ****P = 5.70008E−05 for female). Source data are provided as a Source Data file.
