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. 2015 Jul 28;4:e06930. doi: 10.7554/eLife.06930

Figure 3. Systemic JH secreted after mating acts directly in the intestinal epithelium to drive reproductive remodelling.

Circulating juvenile hormone (JH) is elevated after mating in the haemolymph of female flies (A, p = 0.02 at 24 hr, p = 0.002 at 48 hr, t-test with Holm's correction). Increased tissue renewal (B, B′) and SREBP activation (C, C′, quantified in D, p < 0.001, Mann–Whitney test) are apparent following a 3-day dietary supplementation with JH analogue (JHa). JHa treatment is sufficient to increase mitoses (E, p < 0.001, negative binomial GLM) and size (H, p < 0.001, t-test) of the posterior midgut. Conversely, when the endogenous JH source is genetically ablated by means of Aug21 > NiPp1 (Yamamoto et al., 2013), the proliferation and size increase that follow mating are abolished, although they can be reinstated by feeding JHa (proliferation F, p < 0.001 between Aug21/+ and Aug21 > NiPp1 mated, p < 0.001 between Aug21 > NiPp1 and NiPp1/+ mated, p < 0.001 between Aug21 > NiPp1 and Aug21 > NiPp1 + JHa mated; all relevant comparisons between virgins are not significant, negative binomial GLM with Holm's correction; gut diameter I, p = 0.002 between Aug21/+ and Aug21 > NiPp1 mated, p < 0.001 between Aug21 > NiPp1 and NiPp1/+ mated, p < 0.001 between Aug21 > NiPp1 and Aug21 > NiPp1 + JHa mated; all relevant comparisons between virgins are not significant, t-test with Holm's correction). Downregulation of either gce or Met in adult progenitors abrogates post-mating proliferation (G, p < 0.001 between esgReDDM/+ and esgReDDM > gce RNAi mated, p < 0.001 between esgReDDM/+ and esgReDDM > Met RNAi mated, negative binomial GLM with Holm's correction) and gut size increase (J, p < 0.001 between esgReDDM/+ and esgReDDM > gce RNAi mated, p < 0.001 between esgReDDM/+ and esgReDDM > Met RNAi mated, t-test with Holm's correction). The upregulation of bgm reporter upon mating is abolished by the downregulation of gce, but not Met, in ECs using the EC-specific driver Mex-Gal4 (K–K′′′′). See Table 1 for full genotypes.

DOI: http://dx.doi.org/10.7554/eLife.06930.006

Figure 3.

Figure 3—figure supplement 1. Intestinal JH signalling is relayed through Kr-h1 and underlies mating-dependent intestinal growth and gene expression phenotypes.

Figure 3—figure supplement 1.

JHa application in virgin females results in a net growth of the gut, as shown by the increase in caudal-marked cells (A, p = 0.004, t-test). The JH signalling pathway can be targeted using RNAi constructs against the receptors Met and gce, which decrease transcript abundance compared to a tubts control when expressed globally in larvae for 3 hr at 29°C (B, p = 0.002 for Met, p = 0.005 for gce, paired one-tailed t-test). Consequently, using esgReDDM to specifically knockdown gce in adult intestinal progenitor cells abolishes the proliferative effect of JHa application (C, C′), but does not reduce the number of progenitors after 7 days of downregulation (D, p > 0.05, t test). Progenitors in which gce is downregulated can still proliferate normally to replenish a gut damaged by a 24 hr application of the toxin paraquat (E, E′ with quantification of mitoses in F, p < 0.001 for both esgReDDM/+ and esgReDDM > gce RNAi, p > 0.05 for all other relevant comparisons, t test with Holm's correction) and the number of progenitors is not reduced by this treatment (G, p = 0.04 between esgReDDM/+ untreated control and esgReDDM > gce RNAi untreated control, p > 0.05 for all other relevant comparisons, t test with Holm's correction). The transcription factor Kruppel homolog 1 (Kr-h1), a well-established effector of JH responses, is transcriptionally upregulated after 3 days of mating (H, p = 0.02 in w1118, p = 0.02 in OregonR, paired two-tailed t-test). Kr-h1 function is necessary and sufficient for the re-sizing of the gut after mating, as its downregulation in intestinal progenitors through RNA interference using esgReDDM prevents the increase in proliferation (I, p < 0.001 between esgReDDM/+ and esgReDDM > Kr-h1 RNAi mated, negative binomial GLM with Holm's correction) and gut size (J, p < 0.001 between esgReDDM/+ and esgReDDM > Kr-h1 RNAi mated, t-test with Holm's correction) typically observed after 7 days of mating, while overexpression of Kr-h1 constructs from the same cells recapitulates the effect of mating in virgins (proliferation, I, p < 0.001 between esgReDDM/+ and esgReDDM > Kr-h1GS virgin, p < 0.001 between esgReDDM/+ and esgReDDM > Kr-h1UAS virgin, negative binomial GLM with Holm's correction; gut size J, p < 0.001 between esgReDDM/+ and esgReDDM > Kr-h1UAS virgin, t-test with Holm's correction). RNAi constructs against Kr-h1 and SREBP are effective in downregulating these genes; they decrease transcript abundance compared to a tubts control when expressed globally in larvae for 3 hr at 29°C (K, p = 0.02 for Kr-H1, p < 0.001 for both SREBP constructs, paired one-tailed t-test). Downregulating gce constitutively from ECs using Mex-Gal4 significantly suppresses the transcriptional increase of the lipid metabolism gene bgm upon mating, as indicated by the intensity ranking of a gce reporter (L, p = 0.004 between gce RNAi/+ and Mex > gce RNAi, p = 0.02 between Mex > gce RNAi and Mex/KK control, Mann–Whitney test with Holm's correction; relevant comparisons with Met RNAi are not significant). See Table 1 for full genotypes.
DOI: http://dx.doi.org/10.7554/eLife.06930.007