Identification and analysis of regulators of the epithelial to mesenchymal transition using Drosophila wing disc eversion

Abstract

The epithelial to mesenchymal transition (EMT), the process where epithelial cells break free from their adhesive connections and become migratory, is a key mechanism in development and disease. To discover new EMT regulators, wing disc eversion was developed as a model system to enable RNAi screening, in vivo, in Drosophila. During eversion, a population of wing imaginal disc cells, the peripodial cells, lose apico-basal polarity, adherens junctions, breakdown the basement membrane, then invade and migrate over larval cells to reestablish an epithelium. RNAi knockdown using peripodial drivers generates adult phenotypes ranging from thoracic clefts to internalised wings. In a screen of protein/lipid kinases and phosphatases, defects for genes involved in cell polarity, JNK and PI3Kinase signaling, cytokinesis, and novel genes were identified. In a similar screen of putative downstream targets of EMT associated pathways, a strong candidate was identified; the axonal guidance factor, netrinA (netA). Netrins are a family of chemoattractants with roles in migration, cell survival, and epithelial plasticity. A key Netrin receptor is the tumour suppressor DCC (Deleted in Colorectal Cancer), whose loss has been implicated in cancer progression through the promotion of cell survival. Loss of NetA or NetB in peripodial cells produced eversion failure and thoracic clefting. Two roles for Netrin in disc eversion were suggested by this study. Firstly a role in the later stages of thorax closure, in promoting the migration and integrity of the leading edge peripodial cells, post-EMT. In vitro disc culturing of netA RNAi discs revealed a significant reduction in eversion rates, suggesting a second role for NetA in the early stages of disc eversion, likely affecting the EMT. The Drosophila orthologue of DCC, Frazzled (Fra) was shown to antagonize this EMT process. Disc eversion is unaffected by fra RNAi knockdown but is disrupted by fra overexpression. The role of Fra as an antagonist correlates with its cellular localisation. In disc proper epithelia Fra localises to the cell cortex, whereas in peripodial cells Fra is reduced and localises to cytoplasmic puncta. When Fra is overexpressed it becomes enriched at the peripodial cell cortex and eversion is disrupted. The changes in levels and localisation of Fra are dependent on JNK and NetA signalling, both of which are required for disc eversion. Peripodial expression of dominant negative JNK restores Fra to the cell cortex while loss of NetA localizes Fra to the nucleus. Using disc eversion as model, a new EMT signaling pathway involving NetA and Fra was discovered. This study presented a role for NetA in promoting peripodial EMT while Fra was suggested to antagonize it. The work described here suggests that during disc eversion Fra may be involved in epithelial maintenance and that NetA may act as signal to allow EMT in Fra expressing epithelia. A possibility then could be that Fra prevents the progression of cancer not only by modulating cell survival, but also by promoting epithelial state to prevent EMT, thus acting as a suppressor of metastasis.