In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes—interstitial cells—by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR–Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals—and, more generally, micronutrients—to energy homeostasis.
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We thank B. Denholm, T. Perry, E. Prince, A. Rodan, E. Rulifson, A. Teleman, A. Vincent and C. Wilson for sharing reagents; M. Ardakani, D. Dormann, C. Ware and C. Whilding for providing imaging advice; M. Brankatschk, G. Juhasz, F. Missirlis, L. Prieto-Godino, H. Gong and all members of the Miguel-Aliaga, Hirabayashi and Cochemé labs for discussions and experimental advice and/or assistance; E. Knust and W. Huttner for supporting the EM work; and T. Ameku, S. Hirabayashi and S. Vernia for providing comments on an earlier version of this manuscript. S.R. thanks A. M. L. Coenen-Stass, I. Al-Khatib and S. Redhai for advice and support. We thank the Bloomington Drosophila Stock Center, Vienna Drosophila Resource Center, the Kyoto Drosophila Genomics and Genetics Resource for flies, and the Developmental Studies Hybridoma Bank for antibodies. This work was funded by an ERC Advanced Grant to I.M.-A. (ERCAdG 787470 ‘IntraGutSex’), NIH funding to N.W.B. (R00DK115879), an ERC Starting Grant to N.W. (ERCStG 335724 ‘VecSyn’), an Imperial Confidence in Concepts grant to I.M.-A., N.W. and S.R., MRC intramural funding to I.M.-A., a BBSRC grant to R.A.B. (BB/L027690/1), SNF funding to L.v.G. (P400PB-180894) and an Equipe FRM label to F.L.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
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.
a, Larval guts stained with anti-Hodor show immunoreactivity in the copper cell (#) and iron cell (*) regions of the gut and the Malpighian tubules (†) in control animals (left), whereas this staining pattern is absent in hodor mutants (right). b, RNAi-mediated hodor knockdown in enterocytes (using mex1-Gal4) substantially reduces Hodor protein levels. c, RNAi-mediated hodor knockdown using hodor-Gal4 reduces protein levels considerably in the copper cell region (#) but does not noticeably reduce levels in the iron cell region (*). d, Expression of GFP in UAS-Stinger-GFP larvae in interstitial cells (#) and Malpighian tubules (†) using hodor-Gal4; note the absence of GFP in the iron cell region (*). e, Staining of iron cells highlighted in green (Fer2LCH>mCD8-GFP) with Hodor antibody illustrating overlap between the two in the anterior portion. f, Expression of lab-Gal4 (visualized in lab>mCD8-GFP larvae) is seen in the copper cells—but not the interstitial cells—of the copper cell region. The panel to the right shows a higher-magnification image of the copper cell region. g, Expression of CtB-Gal4 (visualized in CtB>Stinger-GFP larvae) is confined to the principal cells of Malpighian tubules. h, R2R4-Gal4 (visualized in R2R4>Stinger-GFP larvae) is confined to a subset of enterocytes in the posterior midgut. Note its absence from the copper cell region (#) and the iron cell region (*) as well as from Malpighian tubules (†). See Supplementary Information for sample sizes and full genotypes. Scale bars,1 mm (a, d, f, h); 200 μm (b, e); 300 μm (c); 200 μm (g); 50 μm (f inset).
a, Comparison of embryonic viability between control (w1118), heterozygous (−/+) and homozygous (−/−) hodor mutant larvae; there are no significant differences. b, Developmental progression of larvae lacking hodor compared to control animals (w1118). c, Pupal volume of hodor mutants compared to controls; each data point represents one pupa. d, Wing size measurements in control compared with hodor mutant adults; no significant differences are apparent (see Methods for details of quantification, each data point represents one wing). e, Reduced levels of pAkt relative to total protein in second-instar hodor mutants compared to controls, all raised on a low-yeast diet. pAkt in hodor mutants is comparable to that of wild-type larvae starved for 15 h. f, Reduced food intake is seen in hodor-Gal4-driven hodor knockdown larvae when compared to control larvae. Experiments were performed using second-instar larvae raised on a low-yeast diet. g, Electron micrographs of the junctional region (arrow) between an interstitial cell and a copper cell, showing no obvious defects in first-instar hodor mutants. h, Smurf assay (Methods) on second-instar control larvae and hodor mutants (examples are representative of at least 6 larvae per genotype). No leakage of blue dye from the intestine was seen in either group. i, Overexpression of hodor in interstitial cells using hodor-gal4 does not alter developmental rate in either high- or low-yeast conditions. j, k, Activation or inactivation of Tor signalling in hodor-expressing cells does not affect developmental rate (j) or food intake (k); none of the genetic manipulations is significantly different compared to its respective control. l, Modulation of Rag and Gator1 complex components in the interstitial cells of hodor mutants (from hodor-Gal4) neither rescues nor exacerbates their developmental delay. See Supplementary Information for sample sizes and full genotypes. Scale bars, 0.5 mm (b); 250 μm (d); 500 nm (g); 400 μm (h). 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.
a, The copper cell region (#) of Drosophila larvae is normally acidic (bromophenol blue dye appears yellow/orange) (Methods), but becomes less acidic (purple/blue) upon expression of hodor RNAi in interstitial cells (hodor-gal4) or in hodor mutants. The reduced acidity of hodor mutant midguts can be rescued by re-expressing hodor in hodor-Gal4-expressing cells. Intestinal acidity is also lost by downregulating the gene that encodes the Vha16-1 subunit of the V-ATPase proton pump in copper cells using lab-Gal4. b, Quantification of intestinal acidity. Depletion (by RNAi) or loss of hodor results in a reduction in the number of larvae with acidic middle midguts, as does depletion of the V-ATPase subunit Vha16-1 in copper cells using lab-gal4. c, Larval developmental rate is unaffected when acidity is lost by reducing the activity of V-ATPase within copper cells (using lab-Gal4). d, Electron micrographs of interstitial cells of first-instar larvae, showing a reduction in their characteristic basal infoldings (arrows) in hodor mutants (* denotes basal lamina) relative to control cells. e, hodor-Gal4 driven expression of mCD8-GFP in interstitial cells of control and hodor mutant larvae reveals an increase in luminal volume (*) and interstitial cell volume (insets with quantifications to the right) in first-instar mutant larvae when compared to controls (all raised on a low-yeast diet). See Methods for details of volume quantifications. f, Overexpression of the dominant-negative Shibire ShiK44A in hodor-expressing cells (using hodor-Gal4) reveals an increase in interstitial cell volume in hodor second-instar mutant larvae relative to controls (all raised on a low-yeast diet). LysoTracker staining in green was used to reveal the cytoplasm. Quantifications are shown to the right. Second-instar larvae raised on a low-yeast diet were used for all experiments involving ShiK44A expression. g, This genetic manipulation also results in an increase in the width of the copper cell region (#) but does not affect the subcellular localization of Hodor in interstitial cells (insets). h, Quantification of the copper cell region width in controls, hodor mutant larvae and larvae expressing ShiK44A from hodor-Gal4. i, Expression of ShiK44A in hodor-expressing cells (hodor>ShiK44A) does not alter developmental rate. See Supplementary Information for sample sizes and full genotypes. Scale bars, 500 μm (a); 500 nm (d); 10 μm (e, f); 250 μm (g). For comparisons involving two groups, a non-parametric Mann–Whitney U-test was used. 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.
a, Increased bacterial loads (cfu per larva) in hodor mutants when compared to control larvae. Bacterial loads were assessed in third-instar larvae raised on a high-yeast diet. b, c, Developmental rate of control and hodor mutant larvae in germ-free conditions, or following re-colonization with A. pomorum or L. plantarum in either high- (b) or low-yeast (c) conditions. hodor mutants remain developmentally delayed in germ-free conditions, particularly when reared on a low-yeast diet. Mono-association partially rescues the developmental delay of all larvae in low-yeast conditions, but the difference in developmental rate between control and hodor mutant larvae persists. d, Representative images of FluoZin-3AM (a zinc dye) stainings in the copper cell region of larvae reared in germ-free conditions or bi-associated with A. pomorum and L. plantarum. More zinc is apparent in the copper cell region of high-yeast-fed larvae relative to low-yeast-fed larvae, but this is unaffected by the presence of microbiota. e, Quantification of zinc staining in the copper cell region. See Supplementary Information for sample sizes and full genotypes. Scale bar, 30 μm (d). For comparisons involving two groups, a non-parametric Mann–Whitney U-test was used. 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.
a, Mutational free energy space, in which structural stability is plotted against zinc binding free energy for each double mutant. The E255K/E296F mutant pair (black dot) was selected to increase the free energy of binding but keep the structural stability as low as possible to avoid refolding of the protein. b, Zinc-activated currents from oocytes expressing wild-type Hodor (top) or mutant Hodor(E255K/E296F) (bottom) in response to the indicated concentrations. c, Activation (top) and deactivation (bottom) kinetics of currents elicited by 50 μM ZnCl2 were significantly faster in Hodor(E255K/E296F) (n = 4–5, P < 0.05 for ON, P < 0.001 for OFF, Welch’s t-test). τ, time in seconds. d, Concentration dependence of zinc-activated currents from oocytes expressing Hodor (sigmoidal fit from Fig. 3b in grey) compared with that of oocytes expressing Hodor(E255K/E296F) (red). The estimated EC50 for Hodor(E255K/E297F) was comparable to that of wild-type Hodor (119.90 μM, 95% confidence interval 104.70–137.10 μM), with the only significant difference observed in response to 50 μM ZnCl2 (P < 0.05, two-way ANOVA with post hoc Bonferroni test, n = 5–9). Data represented as mean ± s.e.m., n denotes the number of oocytes. e, Current–voltage (I–V) relationship of zinc-activated currents from uninjected oocytes in response to the indicated concentrations. f, Preference index plotted over time for larvae given a choice between high- and low-yeast diets. Both control and hodor mutant larvae develop a statistically significant preference for a high-yeast diet (positive numbers) after 24 h. g, hodor-Gal4-driven ClopHensor expression in live interstitial cells reveals a reduction in intracellular chloride levels (increased 458 nm/543 nm fluorescence emission ratio) in first-instar larvae raised on a low-yeast diet supplemented with 0.4 mM ZnSO4 compared to larvae raised on a low-yeast diet only. Chloride levels decreased from around 8.6 mM in controls to around 5.7 mM in larvae raised on a ZnSO4-supplemented diet, calculated on the basis of the calibration in Extended Data Fig. 6h. Representative fluorescence images (458 nm) are shown to the left. h, Calibration of the hodor-Gal4 driven fluorescence of ClopHensor in interstitial cells with eight different chloride concentrations (see Methods for details). The calibration graph to the left shows the sigmoidal curve interpolated from individual 458 nm/543 nm ratios obtained at the different chloride concentrations. This graph enables conversion of absorbance ratios to chloride concentration. Images to the right show representative fluorescence signals (at 458 nm) for each concentration. See Supplementary Information for sample sizes and full genotypes. Scale bars, 30 μm (g, h). For comparisons involving two groups, a non-parametric Mann–Whitney U-test was used. 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.
a, Validation of the zinc-sensitive dye, FluoZin-3AM, in adult and larval Malpighian tubules. The tubules of w1118 adults have less zinc than those of wild-type (OrR) adults; levels can be increased by supplementing their adult diet with 1 mM ZnCl2 for 3 days (left three panels). A more modest reduction in zinc levels is observed in larval tubules of second-instar w1118 larvae relative to wild-type OrR larvae (right two panels). b, FluoZin-3AM staining in the middle midgut of second-instar wild-type larvae (OrR, which harbour a wild-type w gene), w mutant larvae (w1118), w mutant larvae with a mini-w transgene (UAS-Rheb/+) and hodor mutant larvae (which are mutant for w but carry mini-w transgenes). # denotes the copper cell region and * denotes the iron cell region. The panels to the right show higher-magnification images of the copper cell region. Zinc levels are higher in the copper cell region of wild-type larvae relative to the other genotypes, which have comparable zinc levels. The bottom panel shows FluoZin-3AM staining of a wild-type (OrR) adult midgut. There is no apparent zinc enrichment in the copper cell region (#). c, Quantification of intestinal zinc in the copper cell region. In both c and d, larvae were raised on a low-yeast diet. d, Wild-type OrR larvae reach the pupal stage significantly faster than w1118 in low-yeast conditions, whereas hodor−/− still causes a significant developmental delay in either a genetic background with an intact w gene (w+;hodor−/−) or when backcrossed eight times into a w mutant background lacking the w gene (w;hodor−/−). e, Heterozygous lines carrying mini-w develop faster than w1118 larvae in low-yeast conditions. Scale bars; 50 μm (a); 500 μm (b); 50 μm (inset). See Supplementary Information for sample sizes and full genotypes. For comparisons involving two groups, a non-parametric Mann–Whitney U-test was used. For cases in which more than two groups were compared, an ordinary one-way ANOVA 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.
a, Quantification of the fraction of Hodor-positive punctae that co-express Rab 5, 7, 11 (all of which are endogenously tagged with YFP), Lysotracker or Lamp1 (endogenously expressed Lamp1–mCherry). b–d, Co-expression analysis reveals limited overlap between Hodor immunoreactivity and the early endosome marker Rab5 (b) or the recycling endosome marker Rab11 (d), whereas more pronounced overlap is apparent with late endosome and lysosome marker Rab7 (c). e, The majority of Lamp1-positive structures co-expressed Hodor on the apical side of interstitial cells (* denotes the intestinal lumen). Larvae were briefly starved (4 h) before dissection in order to visualize lysosomes as punctate structures. f, The endogenously expressed GFP-tagged Vha16-1 subunit of the V-ATPase complex is predominantly localized to the copper cell region (#) within the larval intestine. g, Expression of Vha16-1–GFP is apparent in both the copper cells and, to a lesser extent, the interstitial cells. See Supplementary Information for sample sizes and full genotypes. Scale bars, 10 μm (b–e); 200 μm (f); 30 μm (g). N, nucleus. *P < 0.05, **P < 0.01, ***P < 0.001. Box plots: line, median; box, 75th–25th percentiles; whiskers, minimum to maximum.
a, Representative expression of LysoSensor, LysoTracker, Lamp1–mCherry and hodor-Gal4-driven p62–GFP in the copper cell region of control larvae, larvae in which the V-ATPase subunit Vha44 has been downregulated in interstitial cells (using hodor-Gal4) or hodor mutant larvae. Vha44 knockdown and, to a lesser extent, hodor mutation result in an increase in the number of punctae that are positive for these markers. b, Quantification of the number of punctae that are positive for the abovementioned markers in all three types of larvae shown in a. c, hodor mutants expressing the dual autophagosome/autolysosome marker UAS-GFP-mCherry-Atg8a in all enterocytes (using mex1-Gal4) show regional enrichment of autophagy in both the copper cell region (#) and the iron cell region (*)when compared to an anterior portion of the gut (^). Note the appearance of GFP-positive punctae in the copper cell region (#), which is suggestive of defective autolysosomes unable to quench the GFP signal. d, hodor-Gal4-driven expression of GFP-mCherry-Atg8a in interstitial cells of starved hodor mutants. Large subcellular compartments positive for both GFP and mCherry are apparent. e, Quantification of GFP- and/or mCherry-positive Atg8a-expressing autophagosomes and autolysosomes in the copper cell region of fed or starved controls, and fed or starved hodor mutants (left graph, Atg8a reporter expressed from hodor-Gal4; right graph, Atg8a reporter expressed from mex1-Gal4 in fed hodor mutants). See Supplementary Information for sample sizes and full genotypes. Scale bars, 30 μm (a); 500 μm (c); 45 μm (d). 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.
a, Nucleotide-level maximum likelihood phylogeny of the hodor gene family, highlighting successive duplication events at the base of the Schizophora (orange and red nodes, see Methods for details of phylogenetic reconstruction, and Supplementary Information for a complete gene family tree). Bootstrap support is indicated along individual branches as a percentage of 1,000 rapid bootstraps. b, gRNA target site within exon 2 of the A. gambiae one-to-many orthologue AGAP009616 of fly hodor-like genes, the diagnostic primers used for genotyping and the three frameshift mutants recovered. PAM, protospacer adjacent motif. c, Strategy for the recovery of AGAP009616 mutants. d, Genotyping the progeny of crosses between verified heterozygote males and females revealed that AGAP009616 homozygous mutant adults are inviable. See Methods for details.
Hodor resides in the apical membrane and on the lysosomes of gut interstitial cells (highlighted in blue, adjacent to acid-secreting copper cells (#). Zinc sensing by Hodor promotes chloride transport and Tor signalling within interstitial cells. Hodor/Tor signalling in interstitial cells in turn promotes systemic growth through a neural relay, activating insulin-like signalling and thereby sustaining developmental rate, and by promoting food intake via an as-yet unknown mechanism that is independent of the insulin-producing cells of the brain. The reduced insulin signalling observed in hodor mutants may be secondary to their reduced food intake, hence the dashed arrow.
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Redhai, S., Pilgrim, C., Gaspar, P. et al. An intestinal zinc sensor regulates food intake and developmental growth. Nature 580, 263–268 (2020). https://doi.org/10.1038/s41586-020-2111-5
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