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

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.

Figure 1. Nutrition-TOR signaling maintains TIF-IA mRNA and protein levels in larvae.

(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved larvae compared to fed larvae. (B) Immunoblot indicates TIF-IA protein levels were reduced in tor^ΔP larvae compared to wild-type (WT) larvae, at 72 hr AEL. (C) Immunoblot indicates TIF-IA protein levels were unchanged between WT and s6k null (s6k^L1) larvae, at 72 hr AEL. In all immunoblots, β tubulin levels indicate loading control. (D) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved larvae compared to fed larvae. Data normalized to β tubulin. (^* P = 3.46×10⁻⁶, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in 24 hr starved larvae compared to fed larvae, at 72 hr AEL. Data normalized to β tubulin. (^* P = 4.47×10⁻⁵, Student's t-test). (F) qPCR indicates TIF-IA mRNA levels were reduced in tor larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (^* P = 0.01, Student's t-test). (G) qPCR indicates pre-rRNA levels were reduced in tor^ΔP larvae compared to yw (control) larvae, at 72 hr AEL. Data normalized to actin. (^* P = 0.04, Student's t-test). All error bars indicate SEM.

Figure 2. TIF-IA function is required in muscle to maintain overall body growth and development.

(A) Immunoblot indicates TIF-IA protein levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. β tubulin levels indicate loading control. (B) qPCR indicates TIF-IA mRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (^* P = 0.009, Student's t-test). (C) qPCR indicates pre-rRNA levels were reduced in 24 hr starved muscle compared to fed muscle, at 72 hr AEL. Data normalized to β tubulin. (^* P = 0.0059, Student's t-test). (D) qPCR indicates TIF-IA mRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to control larval muscle (dMef2>+), at 72 hr AEL. Data normalized to β tubulin. (^* P = 0.0022, Student's t-test). (E) qPCR indicates pre-rRNA levels were reduced in dMef2>TIF-IA-IR muscle compared to dMef2>+ (control) larval muscle, at 72 hr AEL. Data normalized to β tubulin. (^* P = 0.015, Student's t-test). (F) Developmental timing from larval hatching to pupation of dMef2>+ and dMef2>TIF-IA IR animals, n = 134, n - number of larvae assessed per genotype, (mean time to pupation: dMef2>+, 6.1 days and dMef2>TIF-IA, 7.8 days, ^* P<0.05, Mann-Whitney U test). (G) Representative images of dMef2>+ (top) and dMef2>TIF-IA IR (bottom) larvae. Numbers at the bottom of the panel indicates hours AEL. (H) Representative images of dMef2>+ and dMef2>TIF-IA IR pupae, scale bar-200 µm. All error bars indicate SEM.

Figure 3. Fat-specific and lymph gland-specific TIF-IA inhibition has modest effects on growth and development.

(A) Developmental timing from larval hatching to pupation of r4>+ and r4>TIF-IA IR animals, n = 158, n - number of larvae assessed per genotype, (mean time to pupation: r4>+, 6.4 days vs. r4>TIF-IA IR, 6.7 days, ^* P<0.05, Mann-Whitney U test). (B) Representative images of r4>+ (top) and r4>TIF-IA IR (bottom) larvae. Numbers at the bottom of the panel indicates hours AEL. (C) Pupal volume of r4>+ (n = 100) and r4>TIF-IA IR pupae (n = 28), n - number of pupae per genotype, (P = 0.92, Student's t-test). (D) Developmental timing from larval hatching to pupation of ppl>+ and ppl>TIF-IA IR animals, n = 120, n - number of larvae assessed per genotype, (mean time to pupation: ppl>+ 6.1 days vs. ppl>TIF-IA IR 6.1 days, not significant, Mann-Whitney U test). (E) Pupal volume of ppl>+ (n = 41) and ppl>TIF-IA IR pupae (n = 44), (P = 0.92, Student's t-test). (F) Developmental timing from larval hatching to pupation of hml>+ and hml>TIF-IA IR animals, n = 192, n - number of larvae assessed per genotype, (mean time to pupation: hml>+, 7.2 days vs. hml>TIF-IA IR, 6.9 days, ^* P<0.05, Mann-Whitney U test). (G) Developmental timing from larval hatching to pupation of pxn>+ and pxn>TIF-IA IR animals, n = 103, n - number of pupae counted per genotype, (mean time to pupation: pxn>+, 7.8 days vs. pxn>TIF-IA IR, 7.8 days, not significant, Mann-Whitney U test). All error bars indicate SEM.

Figure 4. TOR activity in muscle is required and sufficient to promote body growth and TIF-IA inhibition in muscle blocks Rheb induced body growth.

(A) Pupal volume of dMef2>+ and dMef2>TOR^TED pupae, n = 29, n - number of pupae per genotype, (^* P = 2.47×10⁻⁷, Student's t-test). (B) Pupal volume of dMef2>+ and dMef2>Tsc1,Tsc2 pupae, n>55, n - number of pupae per genotype, (^* P = 1.08×10⁻⁵¹, Student's t-test). (C) Pupal volume of dMef2>+ and dMef2>slif^Anti pupae, n>80, n - number of pupae per genotype, (^* P = 5.14×10⁻¹⁰, Student's t-test). (D) Pupal volume of dMef2>+ and dMef2>Rheb pupae, n>38, n - number of pupae per genotype, (^* P = 0.003, Student's t-test). (E-F) Representative figures of larvae and pupae of indicated genotypes, scale bar-500 µm. (G) dMef2>Rheb animals showed increased pupal volume (White bar, ^* P<0.0001, One-way ANOVA and Tukey's post test) compared to dMef2>+ control. Muscle specific inhibition of TIF-IA (dMef2>TIF-IA IR) reduced pupal volume, with respect to dMef2>+ control (Grey bar, ^• P = 0.0003, One-way ANOVA and Tukey's post test). TIF-IA knockdown in muscle abrogated the Rheb-induced increase in pupal volume (Blue bar, ^⧫ P<0.0001, One-way ANOVA and Tukey's post test), n - number of pupae per genotype. All error bars indicate SEM.

Figure 5. Muscle-specific TIF-IA inhibition reduces systemic insulin signaling.

(A–C) Representative fat body images indicating FOXO (red) subcellular localization in (A) dMef2>+ (Fed), (B) dMef2>+ (Starved) and (C) dMef2>TIF-IA IR larvae, scale bar-500 µm. (D) Quantification indicating mean (N∶C, Nuclear∶Cytoplasmic) ratio of pixel intensity per fat body cell of dMef2>+ (Starved) (Grey bar, ^** P<0.001, One-way ANOVA and Tukey's post test) and dMef2>TIF-IA IR (White bar, ^* P<0.001, One-way ANOVA and Tukey's post test) animals, compared to fed control (dMef2>+) animals. 21 cells/genotype were scored. (E) qPCR indicates 4EBP mRNA levels were increased in dMef2>TIF-IA IR larvae compared to dMef2>+ control (^* P = 0.002, Student's t-test). Data normalized to β tubulin mRNA. (F) Immunoblots indicate phospho Akt (Ser505), Akt and βtubulin levels in control (dMef2>+) and TIF-IA IR (dMef2>TIF-IA IR) larvae. (G) dMef2>TIF-IR larvae had reduced dILP3 mRNA (^* P = 0.0003, Student's t-test) and dILP5 mRNA (^* P = 0.015, Student's t-test) levels but dILP2 mRNA (P = 0.14, Student's t-test) levels were unaltered, compared to dMef2>+ control. Data normalized to β tubulin mRNA. (H–I) Representative images of larval brain insulin producing cells (IPC) at 96 hr AEL, indicating dILP2 protein accumulation of (H) dMef2>+ and (I) dMef2>TIF-IA IR animals, scale bar-20 µm. (J) Quantification showing mean pixel intensity/IPC cluster of dMef2>+ (n = 16) and dMef2>TIF-IA IR (n = 16) animals, n – number of IPC cluster assessed per genotype, images quantified with Image J software, (^* P = 1.21×10⁻¹⁰, Student's t-test). (K) qPCR indicates Imp-L2 mRNA levels were induced in dMef2>TIF-IA IR larval muscle compared to dMef2>+ (control), (^* P = 0.025, Student's t-test). Data normalized to β tubulin mRNA. All error bars indicate SEM.

Figure 6. TIF-IA overexpression in muscle can partially reverse the effects of starvation on FOXO-dependent genes.

(A) Data present mean +/− SEM values from qPCR analysis of InR mRNA levels in fed and starved larvae of dMef2>+ and dMef2>TIF-IA animals. Starvation increased InR mRNA levels, compared to fed controls (^* P<0.0001, One-way ANOVA and Tukey's post test). Overexpression of TIF-IA in muscle significantly suppressed this starvation-mediated InR induction (^* P<0.0001, One-way ANOVA and Tukey's post test). Data normalized to β tubulin mRNA. (B) Data present mean +/− SEM values from qPCR analysis of 4EBP mRNA levels in fed and starved larvae of dMef2>+ and dMef2>TIF-IA animals. Starvation increased 4EBP mRNA levels, compared to fed controls (^* P = 0.0003, One-way ANOVA and Tukey's post test). Overexpression of TIF-IA in partially suppressed the starvation mediated 4EBP mRNA induction, although not to a statistically significant level (P = 0.125, One-way ANOVA and Tukey's post test). Data normalized to β tubulin mRNA. All error bars indicate SEM.

Figure 7. 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.

(A–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% (+/−1) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, foxo/+ were 57.8% (+/−3) of control larvae size (P<0.01 vs dMef2>TIF-IA IR larvae). B) dMef2>TIF-IA IR larvae were 31.4% (+/−4.6) of control (dMef2>+) larvae size. dMef2>TIF-IA IR, Imp-L2 IR were 45.7% (+/−1.5) of control larvae size (P<0.05 vs dMef2>TIF-IA IR larvae). Scale bar-500 µm (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 (^* P = 0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test); dMef2>TIF-IA IR, Imp-L2 IR, 7.9 days (^* P = 0.05 vs. dMef2>TIF-IA IR larvae, Mann-Whitney U test).