a, Design of enterocyte-specific RNAi-screen and generation of hodor mutant. Distribution of the categories of genes targeted for intestinal knockdown and number of genes and lines tested in each round of the genetic screen. b, Larval gut expressing UAS-Stinger-GFP under the control of mex1-Gal4, showing expression in all enterocytes, including those in the copper cell region (#) and the iron cell region (*). There is no expression in the Malpighian tubules (†). c, Flies carrying UAS-RNAi targeted against candidate genes were crossed to those carrying mex1-Gal4 to achieve enterocyte-specific knockdown in the resulting larval progeny, which were placed on either high- or low-yeast food and allowed to develop into pupae. d, Results from the first round of the RNAi screen using mex1-Gal4 with plots showing the average time to pupariation after egg laying. Blue stars represent four different control lines crossed to mex1-Gal4. Linear models for these control lines (analysed together) are displayed as dashed lines with a 90% prediction interval shown in dotted lines; knockdown of genes B (CG11340) and F (CG4797) frequently led to a delay to pupariation. See Source Data for the lines and genes that the specific letters correspond to, and Supplementary Information for details of—and reasons for—the percentage deviation data display. e, Strategy for generating hodor mutants using pTVcherry vector51 to direct homologous recombination. Candidate recombinants were recovered after several crosses, identified on the basis of viability and eye colour. f, PCR verification of integration of pTVcherry construct at the hodor locus, no band is seen in w1118 controls (1,3), but a correctly sized band of 3–4 kbp (arrowheads) is seen in hodor+/− (2,4). g, Real-time quantitative PCR of control and hodor mutant larvae relative to gapdh, showing the absence of hodor transcripts in the mutant. h, Larval survival in low-yeast conditions when hodor is knocked down in all enterocytes using mex1-Gal4. i, RNAi targeting a different segment of the hodor transcript also causes a developmental delay when expressed with mex1-Gal4. j, Limiting expression of hodor RNAi to interstitial cells and principal cells of the Malpighian tubules (using hodor-Gal4) causes a significant delay to development. See Supplementary Information for sample sizes and full genotypes. Scale bar, 1 mm (b). For cases in which more than two groups were compared, an ordinary one-way ANOVA test was performed with a Tukey post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. Box plots: line, median; box, 75th–25th percentiles; whiskers, minimum to maximum. Source Data