Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23

Marilena Ignesti,Serena Duchi,Valeria Cavaliere,Tien Hsu,Julie A Woolworth,Giuseppe Gargiulo,Gouthami Nallamothu,Marilena Barraco

doi:10.1186/1741-7007-12-12

Abstract

BackgroundThe Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. In this study, we analyzed the role of Awd in controlling Notch signaling during development.ResultsTo study the awd gene function we used genetic mosaic approaches to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. Our results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments we show that Notch receptor accumulates in early endosomes in awd mutant follicle cells. A trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation.ConclusionsIn this report we demonstrate that awd is essential for Notch signaling via its endocytic role. In addition, we identify the endocytic step at which Awd function is required for Notch signaling and we obtain evidence indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.

Highlights

The Drosophila abnormal wing discs belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis
To test the notion that the Notch signaling deficiency in awd mutant cells is the result of defective endocytosis, we examined the localization of Notch receptor in awd follicle cell clones
Since in this report we show that Notch signaling cannot occur in the follicle cell without awd function, we conclude that, at least in follicle cells, endocytosis is a requisite process for ligand-dependent Notch signaling

Summary

Introduction

The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. In other cancer cohorts, those of ovarian cancers, up-regulated Nm23 levels have been correlated with poor prognosis [9,10], suggesting an oncogenic function These discrepancies have so far been difficult to reconcile because the exact cellular function of Nm23 has remained unclear, several molecular activities have been assigned to the Nm23 family of proteins. During our analyses of awd function in the follicular epithelium, we noted a proliferation abnormality in awd mutant cells that is reminiscent of the Notch signaling defect. This observation prompted us to revisit the original ‘abnormal wing discs’ phenotype, which led to the discovery of the classic ‘notched wing’ phenotype in flies carrying mosaic awd mutant clones. Elucidating the Notch signaling defect in awd mutant cells should shed light on the awd action in the endocytic pathway

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Conclusion