ABSTRACT
Apoptosis is crucial during the morphogenesis of most organs and tissues, and is utilized for tissues to achieve their proper size, shape and patterning. Many signaling pathways contribute to the precise regulation of apoptosis. Here we show that Jun N-terminal Kinase (JNK) activity contributes to the coordinated removal of interommatidial cells via apoptosis in the Drosophila pupal retina. This is consistent with previous findings that JNK activity promotes apoptosis in other epithelia. However, we found that JNK activity is repressed by Cindr (the CIN85 and CD2AP ortholog) in order to promote cell survival. Reducing the amount of Cindr resulted in ectopic cell death. Increased expression of the Drosophila JNK basket in the setting of reduced cindr expression was found to result in even more severe apoptosis, whilst ectopic death was found to be reduced if retinas were heterozygous for basket. Hence Cindr is required to properly restrict JNK-mediated apoptosis in the pupal eye, resulting in the correct number of interommatidial cells. A lack of precise control over developmental apoptosis can lead to improper tissue morphogenesis.
Subject(s)
JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Apoptosis/physiology , Body Patterning/physiology , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Epithelium/enzymology , Epithelium/metabolism , Gene Expression Regulation, Developmental/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Microfilament Proteins/metabolism , Morphogenesis , Pupa/metabolism , Retina/cytology , Retina/enzymology , Retina/metabolism , Signal TransductionABSTRACT
Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4-driven UAS-α-catenin:GFP are used to visualize cell boundaries in the eye epithelium (1-3). After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.
