Border cell migration during Drosophila melanogaster oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, or Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. Here we show that the metastasis suppressor gene homolog Nm23/awd is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominantnegative dynamin. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion.

The expression levels of the metastasis suppressor gene Nm23 have been shown to correlate positively or inversely with prognosis in different cancer cohorts. This indicates that Nm23 may be needed at different expression levels and may function differently in various tissues. Here we report a novel epithelial function of the Drosophila melanogaster homolog of human Nm23, abnormal wing discs (awd). We show a dynamic expression pattern of the Awd protein during morphogenesis of the Drosophila follicle cells during oogenesis. Loss-of-function awd mutant cells result in the accumulation and spreading of adherens junction components, such as Drosophila E-cadherin, beta-catenin/Armadillo, and alpha-spectrin, and the disruption of epithelial integrity, including breaking up of the epithelial sheet and piling up of follicle cells. In contrast, overexpression of awd diminishes adherens junction components and induces a mesenchymal-cell-like cell shape change. The gain-of-function phenotype is consistent with a potential oncogenic function of this metastasis suppressor gene. Interestingly, we demonstrate that the epithelial function of awd is mediated by Rab5 and show that the Rab5 expression level is downregulated in awd mutant cells. Therefore, awd modulates the level and localization of adherens junction components via endocytosis. This is the first demonstration of an in vivo function of Nm23 family genes in regulating epithelial morphogenesis.

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.