Limits...
TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

Ghosh A, Rideout EJ, Grewal SS - PLoS Genet. (2014)

Bottom Line: When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth.This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle.Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and Clark H. Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, Health Research Innovation Center, Calgary, Alberta, Canada.

ABSTRACT
The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

Show MeSH

Related in: MedlinePlus

Reduction of Imp-L2 levels or removal of one copy of foxo (foxo25/+) partially rescues dMef2>TIF-IA IR induced body growth defect and developmental delay.(Aโ€“B) Representative images of larvae of indicated genotypes. The images were captured when control larvae (dMef2>+) reached wandering third instar stage. The larval body areas were measured and analyzed: A) dMef2>TIF-IA IR larvae were 33.9% (+/โˆ’1) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, foxo/+ were 57.8% (+/โˆ’3) of control larvae size (P<0.01 vs dMef2>TIF-IA IR larvae). B) dMef2>TIF-IA IR larvae were 31.4% (+/โˆ’4.6) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, Imp-L2 IR were 45.7% (+/โˆ’1.5) of control larvae size (P<0.05 vs dMef2>TIF-IA IR larvae). Scale bar-500 ยตm (C) Developmental timing of larvae of indicated genotypes from hatching to pupation. Mean time to pupation for each genotype: dMef2>+, 6.7 days; dMef2>TIF-IA IR, 8.7 days; dMef2>TIF-IA IR, foxo25/+, 8.1 days (*Pโ€Š=โ€Š0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test); dMef2>TIF-IA IR, Imp-L2 IR, 7.9 days (*Pโ€Š=โ€Š0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214618&req=5

pgen-1004750-g007: Reduction of Imp-L2 levels or removal of one copy of foxo (foxo25/+) partially rescues dMef2>TIF-IA IR induced body growth defect and developmental delay.(Aโ€“B) Representative images of larvae of indicated genotypes. The images were captured when control larvae (dMef2>+) reached wandering third instar stage. The larval body areas were measured and analyzed: A) dMef2>TIF-IA IR larvae were 33.9% (+/โˆ’1) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, foxo/+ were 57.8% (+/โˆ’3) of control larvae size (P<0.01 vs dMef2>TIF-IA IR larvae). B) dMef2>TIF-IA IR larvae were 31.4% (+/โˆ’4.6) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, Imp-L2 IR were 45.7% (+/โˆ’1.5) of control larvae size (P<0.05 vs dMef2>TIF-IA IR larvae). Scale bar-500 ยตm (C) Developmental timing of larvae of indicated genotypes from hatching to pupation. Mean time to pupation for each genotype: dMef2>+, 6.7 days; dMef2>TIF-IA IR, 8.7 days; dMef2>TIF-IA IR, foxo25/+, 8.1 days (*Pโ€Š=โ€Š0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test); dMef2>TIF-IA IR, Imp-L2 IR, 7.9 days (*Pโ€Š=โ€Š0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test).

Mentions: The findings presented here suggest that TIF-IA function in muscle is required for normal nutrient-dependent systemic insulin signaling and growth. Hence, upon knockdown of TIF-IA in muscle, we saw reduced growth and delayed development. To further implicate a role for reduced insulin signaling in these effects, we tested whether restoring insulin signaling to some degree could have any effect on the phenotypes we observed. To achieve this we examined partial loss of negative regulators of insulin signaling. We first tested the effects of reducing foxo gene dosage. We found that the decrease in larval growth seen in dMef2>TIF-IA IR larvae was partially reversed in larvae that were heterozygous for a loss-of-function mutation in foxo (foxo25) (Figure 7A). We next examined the effects of reducing the levels of Imp-L2, whose expression was increased in dMef2>TIF-IA IR larval muscle. We found that co-expression of a UAS-Imp-L2 inverted repeat (IR) line with the UAS-TIF-IA IR in muscle, also partially reversed the growth defects seen with expression of UAS-TIF-IA IR alone (Figure 7B). Loss of one copy of foxo (foxo25/+) alone or expression of UAS-Imp-L2 IR alone in the muscle had no effects on larval size (Figure S7). When we measured developmental timing, we also saw that both the delayed larval development and reduced numbers of pupating larvae seen in dMef2>TIF-IA IR larvae were partially reversed in larvae that either were heterozygous for foxo25, or which co-expressed UAS-Imp-L2 IR in the muscle (Figure 7C). These experiments provide genetic evidence that muscle TIF-IA function is required for normal larval growth and development at least in part by maintaining systemic insulin signaling.


TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

Ghosh A, Rideout EJ, Grewal SS - PLoS Genet. (2014)

Reduction of Imp-L2 levels or removal of one copy of foxo (foxo25/+) partially rescues dMef2>TIF-IA IR induced body growth defect and developmental delay.(Aโ€“B) Representative images of larvae of indicated genotypes. The images were captured when control larvae (dMef2>+) reached wandering third instar stage. The larval body areas were measured and analyzed: A) dMef2>TIF-IA IR larvae were 33.9% (+/โˆ’1) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, foxo/+ were 57.8% (+/โˆ’3) of control larvae size (P<0.01 vs dMef2>TIF-IA IR larvae). B) dMef2>TIF-IA IR larvae were 31.4% (+/โˆ’4.6) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, Imp-L2 IR were 45.7% (+/โˆ’1.5) of control larvae size (P<0.05 vs dMef2>TIF-IA IR larvae). Scale bar-500 ยตm (C) Developmental timing of larvae of indicated genotypes from hatching to pupation. Mean time to pupation for each genotype: dMef2>+, 6.7 days; dMef2>TIF-IA IR, 8.7 days; dMef2>TIF-IA IR, foxo25/+, 8.1 days (*Pโ€Š=โ€Š0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test); dMef2>TIF-IA IR, Imp-L2 IR, 7.9 days (*Pโ€Š=โ€Š0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214618&req=5

pgen-1004750-g007: Reduction of Imp-L2 levels or removal of one copy of foxo (foxo25/+) partially rescues dMef2>TIF-IA IR induced body growth defect and developmental delay.(Aโ€“B) Representative images of larvae of indicated genotypes. The images were captured when control larvae (dMef2>+) reached wandering third instar stage. The larval body areas were measured and analyzed: A) dMef2>TIF-IA IR larvae were 33.9% (+/โˆ’1) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, foxo/+ were 57.8% (+/โˆ’3) of control larvae size (P<0.01 vs dMef2>TIF-IA IR larvae). B) dMef2>TIF-IA IR larvae were 31.4% (+/โˆ’4.6) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, Imp-L2 IR were 45.7% (+/โˆ’1.5) of control larvae size (P<0.05 vs dMef2>TIF-IA IR larvae). Scale bar-500 ยตm (C) Developmental timing of larvae of indicated genotypes from hatching to pupation. Mean time to pupation for each genotype: dMef2>+, 6.7 days; dMef2>TIF-IA IR, 8.7 days; dMef2>TIF-IA IR, foxo25/+, 8.1 days (*Pโ€Š=โ€Š0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test); dMef2>TIF-IA IR, Imp-L2 IR, 7.9 days (*Pโ€Š=โ€Š0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test).
Mentions: The findings presented here suggest that TIF-IA function in muscle is required for normal nutrient-dependent systemic insulin signaling and growth. Hence, upon knockdown of TIF-IA in muscle, we saw reduced growth and delayed development. To further implicate a role for reduced insulin signaling in these effects, we tested whether restoring insulin signaling to some degree could have any effect on the phenotypes we observed. To achieve this we examined partial loss of negative regulators of insulin signaling. We first tested the effects of reducing foxo gene dosage. We found that the decrease in larval growth seen in dMef2>TIF-IA IR larvae was partially reversed in larvae that were heterozygous for a loss-of-function mutation in foxo (foxo25) (Figure 7A). We next examined the effects of reducing the levels of Imp-L2, whose expression was increased in dMef2>TIF-IA IR larval muscle. We found that co-expression of a UAS-Imp-L2 inverted repeat (IR) line with the UAS-TIF-IA IR in muscle, also partially reversed the growth defects seen with expression of UAS-TIF-IA IR alone (Figure 7B). Loss of one copy of foxo (foxo25/+) alone or expression of UAS-Imp-L2 IR alone in the muscle had no effects on larval size (Figure S7). When we measured developmental timing, we also saw that both the delayed larval development and reduced numbers of pupating larvae seen in dMef2>TIF-IA IR larvae were partially reversed in larvae that either were heterozygous for foxo25, or which co-expressed UAS-Imp-L2 IR in the muscle (Figure 7C). These experiments provide genetic evidence that muscle TIF-IA function is required for normal larval growth and development at least in part by maintaining systemic insulin signaling.

Bottom Line: When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth.This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle.Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and Clark H. Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, Health Research Innovation Center, Calgary, Alberta, Canada.

ABSTRACT
The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

Show MeSH
Related in: MedlinePlus