Gbb/BMP signaling is required to maintain energy homeostasis in Drosophila

Abstract

The coordination of animal growth and development requires adequate nutrients. During times of insufficient food, developmental progression is slowed and stored energy is utilized to ensure that cell and tissue survival are maintained. Here, we report our finding that the Gbb/BMP signaling pathway, known to play an important role in many developmental processes in both vertebrates and invertebrates, is critical in the Drosophila larval fat body for regulating energy homeostasis. Animals with mutations in the Drosophila BMP-5,7 orthologue, glass bottom boat (gbb), or in its signaling components, display phenotypes similar to nutrient-deprived and Tor mutant larvae. These phenotypes include a developmental delay with reduced overall growth, a transparent appearance, and altered total lipid, glucose and trehalose levels. We find that Gbb/BMP signaling is required in the larval fat body for maintaining proper metabolism, yet interestingly, following nutrient deprivation larvae in turn show a loss of BMP signaling in fat body cells indicating that Gbb/BMP signaling is a central player in homeostasis. Finally, despite strong phenotypic similarities between nutrient-compromised animals and gbb mutants, distinct differences are observed in the expression of a group of starvation responsive genes. Overall, our results implicate Gbb/BMP signaling as a new pathway critical for positive regulation of nutrient storage and energy homeostasis during development.

Figure 1. gbb mutants exhibit abnormal growth and altered fat body morphology

A. gbb null larvae (gbb¹/gbb¹) are transparent and slightly smaller compared to wild-type larvae of the same developmental stage. B. (Left panels) Phalloidin staining of dissected fat bodies reveals a difference in the size of fat body cells between wild-type and gbb mutants. (Right panels) The distribution of fat body cell sizes is shown for third instar wild-type and gbb null larvae, where the average cell size is approximately 50% smaller than wild-type fat bodies. (n>450). C. High magnification Nomarski images of dissected wild type and gbb mutant fat bodies. Variation in the size and distribution of lipid droplets (arrow) is apparent in gbb mutant tissue. Cell out line - dotted line. D. The body mass of thrid instar larvae is reduced for gbb and Tor mutants, as well as for wild type larave that have been deprived of nutrients. E. The size of crawling third instar larval wing imaginal discs is approximately 30% smaller in wild-type larvae deprived of nutrients for 12 hours compared with discs from fed wild-type larvae. Wing discs from crawling gbb¹/gbb³ larvae are 50% reduced in size compared with control discs. (n>10) * and ** p<0.0001 compared with wild-type fed and control discs, respectively (student's t-test).

Figure 2. gbb mutant larvae have reduced metabolic stores

A. Total lipids are reduced in early third instar fed gbb null and starved wild-type larvae. B. After normalization for larval weight, fed gbb null larvae and starved wild-type larvae display reduced TAG levels relative to fed wild-type larvae. C. Glucose and trehalose levels are decreased in wild-type larvae starved for 12 hours and gbb mutant larvae. *p<0.05 compared to wild-type (student's t-test). D. There is no significant change in the protein:mass ratio in gbb mutant larvae compared to wild-type larvae. Tor mutant larvae exhibit a reduced protein:mass ratio. E. GC-MS analysis indicates that early third instar fed gbb null and wild-type larvae starved for 12 hours have lower levels of short-chain fatty acids compared to wild-type fed larvae. Percent of each fatty acid type in wild-type fed larvae is given in parentheses. F,G. Short-chain fatty acids are also reduced in Tor mutant larvae.

Figure 3. Loss of BMP signaling leads to alterations in lipid metabolism in the fat body

Midguts and fat bodies from wandering third instar larvae fed Bo-C₁₂ (green) and stained with Hoechst (blue) and Phalloidin (red). A,B. Third instar wild-type (A) and gbb¹/gbb³ mutant (B) guts ingest Bo-C₁₂ from the food. (A′,B′) Magnification to 300% of regions of the midgut lumen (L) and epithelium (E), indicated by white box, illustrates the increased uptake of Bo-C₁₂ by gbb mutants in the epithelium. C. Lipid droplets in fat body cells of wandering third instar wild-type larvae have little Bo-C₁₂ fluorescence following continuous feeding of low levels of Bo-C₁₂. D-I. Wild-type larvae starved for 12 hours (D), gbb¹/gbb³ (E), Mad¹²/Mad⁷ (F), sax⁵/Df(2R)H23 (G), and Tor^k17004/Tor^k17004 (I) mutant fat bodies exhibit elevated levels of Bo-C₁₂ fluorescence within lipid droplets indicative of increased uptake from ingested Bo-C₁₂, likely in response to lower overall metabolic stores (see also text and Figure S2). Scale bar = 47.62μm. n> 20 for each genotype. All images were taken with identical confocal settings.

Figure 4. BMP signaling in the fat body is required for proper lipid metabolism

A. RT-PCR shows that gbb mRNA is expressed in isolated larval fat body tissue and wing imaginal discs, a tissue where gbb expression has previously been established (Khalsa et al., 1998). B. The clarity of 47% of gbb null larvae is rescued by expression of gbb in the fat body (FBGal4 gbb¹/UASgbb9.9 gbb¹). The number of transparent larvae observed is given as a ratio of total larvae examined. *p<0.01 (Chi square analysis compared to gbb¹/gbb¹). C. pMad accumulates in the nuclei of wild-type fat bodies (top row) but not in gbb¹/gbb³ mutant fat bodies (middle row) or in approximately 65% of fat bodies from wild-type larvae deprived of nutrients for 12 hours (bottom row). n>12. Scale bar = 20μm. D,E. Knockdown of endogenous gbb expression in the fat body by RNAi leads to an increase in Bo-C₁₂ fluorescence (green) (E) compared to control fat bodies (D). F. Fat body expression of gbb in gbb null larvae that were fed Bo-C₁₂ restores proper lipid metabolism to little to no Bo-C₁₂ fluorescence levels. G. FLPout clones (outlined by dotted white line and marked by the presence of nuclear GFP) overexpressing dad produce higher levels of Bo-C₁₂ fluorescence in lipid droplets (example outlined by dotted yellow line). Scale bar = 47.62μm. H. Control FLPout clone (outlined by dotted white line and marked by the presence of nuclear GFP) in third instar larvae not fed Bo-C₁₂ food shows lack of GFP clone marker in lipid droplets. Scale bar = 20μm.

Figure 5. gbb mutants show alteration in the expression of some starvation-responsive genes

A. mnd expression in early third instar larvae is reduced in wild-type larvae deprived of nutrients for 12 hours as well as in fed gbb null larvae. A further reduction in transcript levels occurs when gbb null larvae are starved for 12 hours. B. lip3 expression is increased in starved wild-type larvae and in fed gbb null larvae. Depriving gbb mutants of nutrients further increases the elevated lip3 mRNA levels. C. RfaBp mRNA levels are elevated in fed and starved gbb null larvae but are not altered in nutrient-deprived wild-type larvae. D. CG31217 (low density LDL receptor) expression is elevated in fed and starved gbb mutants relative to wild-type fed larvae. E. bmm lipase expression does not increase in gbb mutants as in wild-type starved larvae. However, bmm transcript levels slightly increase in starved gbb mutants. F. dilp2 mRNA levels are elevated in starved wild-type larvae as well as fed and starved gbb mutant larvae. In each RT-PCR sample band intensity of gene is normalized to band intensity of actin. *p<0.05 compared to wild-type fed larvae; ** p<0.05 compared to wild-type and gbb¹/gbb¹ fed larvae (student's t-test).

Figure 6. Gbb/BMP signaling regulates energy homeostasis

The model schematizes the proposed action of Gbb/BMP signaling in the fat body to regulate energy homeostasis. gbb is expressed in fat body cells and Gbb/BMP signaling is active within this tissue through the phosphorylation and translocation of pMad to the nucleus. Gbb signaling influences the transcription of genes involved in amino acid uptake (mnd), sugar metabolism (dilp2), and lipid hydrolysis (lip3) and transport (RfaBp and CG31217 (LDL receptor)). Interestingly, in addition to its role in promoting nutirent storage BMP signaling is itself responsive to the level of metabolic stores. When nutrint levels are low the accumulation of pMad in fat body nuclei is lost.