Drosophila eugracilis - Akt.

Gene Model for Akt in the D. eugracilis (DeugGB2) assembly (GCA_000236325.2).

The microRNA miR-33 is a pleiotropic regulator of metabolic and developmental processes in Drosophila melanogaster.

BACKGROUND: miR-33 family members are well characterized regulators of cellular lipid levels in mammals. Previous studies have shown that overexpression of miR-33 in Drosophila melanogaster leads to elevated triacylglycerol (TAG) levels in certain contexts. Although loss of miR-33 in flies causes subtle defects in larval and adult ovaries, the effects of miR-33 deficiency on lipid metabolism and other phenotypes impacted by metabolic state have not yet been characterized. RESULTS: We found that loss of miR-33 predisposes flies to elevated TAG levels, and we identified genes involved in TAG synthesis as direct targets of miR-33, including atpcl, midway, and Akt1. miR-33 mutants survived longer upon starvation but showed greater sensitivity to an oxidative stressor. We also found evidence that miR-33 is a negative regulator of cuticle pigmentation and that miR-33 mutants show a reduction in interfollicular stalk cells during oogenesis. CONCLUSION: Our data suggest that miR-33 is a conserved regulator of lipid homeostasis, and its targets are involved in both degradation and synthesis of fatty acids and TAG. The constellation of phenotypes involving tissues that are highly sensitive to metabolic state suggests that miR-33 serves to prevent extreme fluctuations in metabolically sensitive tissues.

Regulation of cuticle pigmentation in drosophila by the nutrient sensing insulin and TOR signaling pathways.

BACKGROUND: Insect pigmentation is a phenotypically plastic trait that plays a role in thermoregulation, desiccation tolerance, mimicry, and sexual selection. The extent and pattern of pigmentation of the abdomen and thorax in Drosophila melanogaster is affected by environmental factors such a growth temperature and access to the substrates necessary for melanin biosynthesis. This study aimed to determine the effect of nutritional status during development on adult pigmentation and test whether nutrient sensing through the Insulin/IGF and target of rapamycin (TOR) pathways regulates the melanization of adult cuticle in Drosophila. RESULTS: Flies reared on low quality food exhibit decreased pigmentation, which can be phenocopied by inhibiting expression of the Insulin receptor (InR) throughout the entire fly during mid to late pupation. The loss of Insulin signaling through PI3K/Akt and FOXO in the epidermis underlying the developing adult cuticle causes a similar decrease in adult pigmentation, suggesting that Insulin signaling acts in a cell autonomous manner to regulate cuticle melanization. In addition, TOR signaling increases pigmentation in a cell autonomous manner, most likely through increased S6K activity. CONCLUSION: These results suggest that nutrient sensing through the Insulin/IGF and TOR pathways couples cuticle pigmentation of both male and female Drosophila with their nutritional status during metamorphosis.

The microRNA miR-8 is a positive regulator of pigmentation and eclosion in Drosophila.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally silence gene expression by binding to target mRNAs. Previous studies have identified the miRNA miR-8 as a pleiotropic regulator of Drosophila development, controlling body size and neuronal survival by targeting multiple mRNAs. In this study we demonstrate that miR-8 is also required for proper spatial patterning of pigment on the adult abdominal cuticle in females but not males. RESULTS: Female adult flies lacking miR-8 exhibit decreased pigmentation of the dorsal abdomen, with a pattern of pigmentation similar to wild type flies grown at higher temperatures. This pigmentation defect in miR-8 mutants is independent of the previously reported body size defect, and miR-8 acts directly in the developing cuticle to regulate pigmentation patterning. The decrease in pigmentation in miR-8 mutants was more pronounced in flies grown at higher temperatures. We also found that loss of miR-8 dramatically affected the ability to eclose at higher temperatures. CONCLUSION: Loss of miR-8 increased the sensitivity of Drosophila to higher temperatures for both pigmentation patterning and the ability to eclose. Together, these data suggest that miR-8 acts as a buffer to stabilize gene expression patterns in the midst of environmental variation.

The microRNA miR-8 is a conserved negative regulator of Wnt signaling.

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.

Wnt signaling inhibits adipogenesis through beta-catenin-dependent and -independent mechanisms.

Wnt signaling has been reported to block apoptosis and regulate differentiation of mesenchymal progenitors through inhibition of glycogen synthase kinase 3 and stabilization of beta-catenin. The effects of Wnt in preadipocytes may be mediated through Frizzled (Fz) 1 and/or Fz2 as these Wnt receptors are expressed in preadipocytes and their expression declines upon induction of differentiation. We ectopically expressed constitutively active chimeras between Wnt8 and Fz1 or Fz2 in preadipocytes and mesenchymal precursor cells. Our results indicated that activated Fz1 increases stability of beta-catenin, inhibits apoptosis, induces osteoblastogenesis, and inhibits adipogenesis. Although activated Fz2 does not influence apoptosis or osteoblastogenesis, it inhibits adipogenesis through a mechanism independent of beta-catenin. An important mediator of the beta-catenin-independent pathway appears to be calcineurin because inhibitors of this serine/threonine phosphatase partially rescue the block to adipogenesis caused by Wnt3a or activated Fz2. These data supported a model in which Wnt signaling inhibits adipogenesis through both beta-catenin-dependent and beta-catenin-independent mechanisms.

T-cell factor 4N (TCF-4N), a novel isoform of mouse TCF-4, synergizes with beta-catenin to coactivate C/EBPalpha and steroidogenic factor 1 transcription factors.

We have cloned T-cell factor 4N (TCF-4N), an alternative isoform of TCF-4, from developing pituitary and 3T3-L1 preadipocytes. This protein contains the N-terminal interaction domain for beta-catenin but lacks the DNA binding domain. While TCF-4N inhibited coactivation by beta-catenin of a TCF/lymphoid-enhancing factor (LEF)-dependent promoter, TCF-4N potentiated coactivation by beta-catenin of several non-TCF/LEF-dependent promoters. For example, TCF-4N synergized with beta-catenin to activate the alpha-inhibin promoter through functional and physical interactions with the orphan nuclear receptor steroidogenic factor 1 (SF-1). In addition, TCF-4N and beta-catenin synergized with the adipogenic transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) to induce leptin promoter activity. The mechanism by which beta-catenin and TCF-4N coactivated C/EBPalpha appeared to involve p300, based upon synergy between these important transcriptional regulators. Consistent with TCF-4N's redirecting the actions of beta-catenin in cells, ectopic expression of TCF-4N in 3T3-L1 preadipocytes partially relieved the block of adipogenesis caused by beta-catenin. Thus, we propose that TCF-4N inhibits coactivation by beta-catenin of TCF/LEF transcription factors and potentiates the coactivation by beta-catenin of other transcription factors, such as SF-1 and C/EBPalpha.

Wnt signaling protects 3T3-L1 preadipocytes from apoptosis through induction of insulin-like growth factors.

Ectopic expression of Wnt-1 in 3T3-L1 preadipocytes stabilizes beta-catenin, activates TCF-dependent gene transcription, and blocks adipogenesis. Here we report that upon serum withdrawal, Wnt-1 causes 3T3-L1 cells to resist apoptosis through a mechanism that is partially dependent on phosphatidylinositol 3-kinase. Although activation of Wnt signaling by inhibition of GSK-3 activity or ectopic expression of dominant stable beta-catenin blocks apoptosis, inhibition of Wnt signaling through expression of dominant negative TCF-4 increases apoptosis. Wnt-1 stimulates 3T3-L1 preadipocytes to secrete factors that increase PKB/Akt phosphorylation at levels comparable with treatment with 10% serum. With DNA microarrays, we identified several secreted antiapoptotic genes that are induced by Wnt-1, notably insulin-like growth factor I (IGF-I) and IGF-II. Consistent with IGFs mediating the antiapoptotic effects of Wnt-1 in preadipocytes, conditioned medium from Wnt-1 expressing 3T3-L1 cells was unable to promote protein kinase B phosphorylation after the addition of recombinant IGFBP-4. Thus, we demonstrated that Wnt-1 induces expression of antiapoptotic genes in 3T3-L1 preadipocytes such as IGF-I and IGF-II, which allows these cells to resist apoptosis in response to serum deprivation.