Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression.

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid ß-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid ß-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid ß-oxidation.

Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction.

Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. Here we use chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, we identified wingless (wg), a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), as a potential Repo target gene. We demonstrate that Repo regulates wg expression in vivo and that local glial cells secrete Wg at the NMJ to regulate glutamate receptor clustering and synaptic function. This work identifies Wg as a novel in vivo glial-secreted factor that specifically modulates assembly of the postsynaptic signaling machinery at the Drosophila NMJ.

The Wnt pathway regulates wing morph determination in Acyrthosiphon pisum.

Wing dimorphism occurs in insects as a survival strategy to adapt to environmental changes. In response to environmental cues, mother aphids transmit signals to their offspring, and the offspring either emerge as winged adults or develop as wingless adults with degeneration of the wing primordia in the early instar stage. However, how the wing morph is determined in the early instar stage is still unclear. Here, we established a surgical sampling method to obtain precise wing primordium tissues for transcriptome analysis. We identified Wnt as a regulator of wing determination in the early second instar stage in the pea aphid. Inhibiting Wnt signaling via knockdown of Wnt2, Wnt11b, the Wnt receptor-encoding gene fz2 or the downstream targets vg and omb resulted in a decreased proportion of winged aphids. Activation of Wnt signaling via knockdown of miR-8, an inhibitor of the Wnt/Wg pathway, led to an increased proportion of winged aphids. Furthermore, the wing primordia of wingless nymphs underwent apoptosis in the early second instar, and cell death was activated by knockdown of fz2 under the wing-inducing condition. These results indicate that the developmental plasticity of aphid wings is modulated by the intrinsic Wnt pathway in response to environmental challenges.

Lmpt regulates the function of Drosophila muscle by acting as a repressor of Wnt signaling.

BACKGROUND: LIM domain is considered to be important in mediating protein-protein interactions, and members of the LIM protein family can co-regulate tissue-specific gene expression by interacting with different transcription factors. However, its exact function in vivo remains unclear. Our study demonstrates that the LIM protein family member Lmpt may act as a cofactor that interacts with other transcription factors to regulate cellular functions. METHODS: In this study, we generated Lmpt knockdown Drosophila (Lmpt-KD) using the UAS-Gal4 system. We assessed the lifespan and motility of Lmpt-KD Drosophila and analyzed the expression of muscle-related and metabolism-related genes using qRT-PCR. Additionally, we utilized Western blot and Top-Flash luciferase reporter assay to evaluate the level of the Wnt signaling pathway. RESULTS: Our study revealed that knockdown of the Lmpt gene in Drosophila resulted in a shortened lifespan and reduced motility. We also observed a significant increase in oxidative free radicals in the fly gut. Furthermore, qRT-PCR analysis indicated that knockdown of Lmpt led to decreased expression of muscle-related and metabolism-related genes in Drosophila, suggesting that Lmpt plays a crucial role in maintaining muscle and metabolic functions. Finally, we found that reduction of Lmpt significantly upregulated the expression of Wnt signaling pathway proteins. CONCLUSION: Our results demonstrate that Lmpt is essential for motility and survival in Drosophila and acts as a repressor in Wnt signaling.

The bHLH Protein Nulp1 is Essential for Femur Development Via Acting as a Cofactor in Wnt Signaling in Drosophila.

BACKGROUND: The basic helix-loop-helix (bHLH) protein families are a large class of transcription factors, which are associated with cell proliferation, tissue differentiation, and other important development processes. We reported that the Nuclear localized protein-1 (Nulp1) might act as a novel bHLH transcriptional factor to mediate cellular functions. However, its role in development in vivo remains unknown. METHODS: Nulp1 (dNulp1) mutants are generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal. Expression of Wg target genes are analyzed by qRT-PCR. We use the Top-Flash luciferase reporter assay to response to Wg signaling. RESULTS: Here we show that Drosophila Nulp1 (dNulp1) mutants, generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal, are partially homozygous lethal and the rare escapers have bent femurs, which are similar to the major manifestation of congenital bent-bone dysplasia in human Stuve- Weidemann syndrome. The fly phenotype can be rescued by dNulp1 over-expression, indicating that dNulp1 is essential for fly femur development and survival. Moreover, dNulp1 overexpression suppresses the notch wing phenotype caused by the overexpression of sgg/GSK3ß, an inhibitor of the canonical Wnt cascade. Furthermore, qRT-PCR analyses show that seven target genes positively regulated by Wg signaling pathway are down-regulated in response to dNulp1 knockout, while two negatively regulated Wg targets are up-regulated in dNulp1 mutants. Finally, dNulp1 overexpression significantly activates the Top-Flash Wnt signaling reporter. CONCLUSION: We conclude that bHLH protein dNulp1 is essential for femur development and survival in Drosophila by acting as a positive cofactor in Wnt/Wingless signaling.

Gene duplication, lineage-specific expansion, and subfunctionalization in the MADF-BESS family patterns the Drosophila wing hinge.

Gene duplication, expansion, and subsequent diversification are features of the evolutionary process. Duplicated genes can be lost, modified, or altered to generate novel functions over evolutionary timescales. These features make gene duplication a powerful engine of evolutionary change. In this study, we explore these features in the MADF-BESS family of transcriptional regulators. In Drosophila melanogaster, the family contains 16 similar members, each containing an N-terminal, DNA-binding MADF domain and a C-terminal, protein-interacting, BESS domain. Phylogenetic analysis shows that members of the MADF-BESS family are expanded in the Drosophila lineage. Three members, which we name hinge1, hinge2, and hinge3 are required for wing development, with a critical role in the wing hinge. hinge1 is a negative regulator of Winglesss expression and interacts with core wing-hinge patterning genes such as teashirt, homothorax, and jing. Double knockdowns along with heterologous rescue experiments are used to demonstrate that members of the MADF-BESS family retain function in the wing hinge, in spite of expansion and diversification for over 40 million years. The wing hinge connects the blade to the thorax and has critical roles in fluttering during flight. MADF-BESS family genes appear to retain redundant functions to shape and form elements of the wing hinge in a robust and fail-safe manner.