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. 2019 Aug 8;178(4):901–918.e16. doi: 10.1016/j.cell.2019.07.029

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© 2019 Medical Research Council on behalf of UKRI and Imperial College London

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Figure S7 — Male-Biased Carbohydrate Metabolism Is Genetically Downstream of the JAK-SAT Signaling in Enterocytes of the R4 Region and can be Uncoupled from Larval Growth and Intestinal Proliferation, Related to Figures 5 and 6 and 7

(A) Food intake quantifications based on the number of FlyPAD-monitored sips per male (M) fly following R2 and R5 enterocyte-specific knockdown of the Maltase-A1 (Mal-A1) and CG6484 enzymes. Downregulation of intestinal sugar genes sugar genes in R2 and R5 does not affect male food intake.

(B) Adult wing size quantifications (used as a measurement of body size, (Shingleton et al., 2009, Shingleton et al., 2017)) for male (M) flies following enterocyte-specific knockdown of the following enzymes: CG6484, Aldolase (Ald), Pyruvate dehydrogenase kinase (Pdk), Pyruvate dehydrogenase phosphatase (Pdp), Pyruvate carboxylase (PCB), I’m not dead yet (Indy) and domeless^ΔCYT (dome^ΔCYT). Downregulation of intestinal sugar genes sugar genes or JAK-STAT signaling in R2 and R4 does not reduced male body size.

(C) Intestinal proliferation quantified as the number of pH3-positive cells in male midguts following enterocyte-specific knockdown of the following enzymes: Pdp, PCB, IIndy and for the shutoff (esg^SHOF) mutation. Downregulation of intestinal sugar genes sugar genes in R2 and R4 does not impact male intestinal proliferation.

(D) Food intake quantifications based on the number of FlyPAD-monitored sips per male (M) fly following R2 and R4 enterocyte-specific mis-expression of the JAK-STAT ligand unpaired 3 (udp3) alone or in combination with Mal-A1 downregulation. The increased food intake resulting from upd3 overexpression in male enterocytes can be reduced to wild-type levels by simultaneous downregulation of Mal-A1.

(E) Intestinal proliferation quantified as the number of pH3-positive cells in male midguts following enterocyte-specific mis-expression of the JAK-STAT ligand udp3 alone or in combination with Mal-A1 downregulation. In contrast to its effect on food intake, Mal-A1 downregulation fails to reduce the increased stem cell proliferation observed following upd3 overexpression.

(F) Body size assessments based on adult wing size quantifications for male flies following R2 and R4 enterocyte-specific mis-expression of the JAK-STAT ligand udp3 alone or in combination with Mal-A1 downregulation. Concurrent over-activation of JAK-STAT signaling in ECs (by ectopic upd3 expression) and downregulation of the intestinal sugar gene Mal-A1 reduces food intake without affecting body size.

(G) Food intake quantifications based on the number of FlyPAD-monitored sips per female (F) fly following R2 and R4 enterocyte-specific knockdown of the Mal-A1 and Hexokinase-A (Hex-A) enzymes. Downregulation of intestinal sugar genes does not affect female food intake.

(H) LC-MS quantifications of hemolymph (left graph) and whole-body (right graph) citrate in control males and in males following R2/R4 enterocyte-specific knockdown of the plasma membrane Indy citrate transporter. Intestinal Indy knockdown has no impact on circulating or whole body citrate.

(I) Quantifications of the number of mitotic and meiotic pH3-positive germ cells in control testes and in testes following testis-specific Indy knockdown from the traffic jam (tj)-Gal4 driver line.

(J) Expression pattern of Bag of marbles (Bam) (visualized in green using the Bam^GFP protein reporter) in control testes and in testes following testis-specific Indy knockdown with the tj-Gal4 reporter line. Representative images are shown (DNA is labeled with DAPI in blue).

(K) Quantifications of the number of mitotic and meiotic pH3-positive germ cells in control testes and in testes following testis-specific Indy knockdown from the eyes absent (eya)-Gal4 driver line.

(L, M) Quantification of the CIT8 citrate sensor’s FRET signal in germline stem cells (nanos (nos)-Gal4-positive) (L) or early-stage somatic cells (M) of testes of control males or males with R2/R4 enterocyte-specific knockdown of Maltase-A1 (Mal-A1^RNAi).

(N) The tj-Gal4 and eya-Gal4 reporters are selectively expressed in early-stage and late-stage somatic cells of the testis (respectively), and not in the germ cells (DNA: DAPI, in blue; tj/eya > StingerGFP: GFP, in green; hub cells: Fasciclin 3 (Fas3), in red).

(O) Food intake quantifications based on the number of FlyPAD-monitored sips per male fly following neuronal-specific Indy downregulation (neuronal Synaptobrevin (nSyb) > Indy^RNAi). n denotes the number of flies (A, D, G and H), wings (B and F), midguts (C and E) or testes (I, K, L, M and O).

Scale bars, 200 μm in all images. Asterisks highlighting significant comparisons within female and male datasets are displayed in red and blue boxes respectively. See Table S4 for a list of full genotypes. See also Table S2.