Visualization of PCP Defects in the Eye and Wing of Drosophila melanogaster

Abstract

"Tissue" or "planar" polarity is a characteristic of many epithelial tissues and is not only required for proper cell alignment, but in many instances is absolutely essential for normal function. Planar cell polarity (PCP) is the polarization of cells within the plane of an epithelium in a direction perpendicular to the axis of the apico-basal polarity. The oriented hair alignment in mammalian skin (feather in birds or scales in fish) and highly organized stereocilia bundles in the vertebrate inner ear are examples of such tissue organization. PCP was first described in Drosophila, with non-canonical Wnt signaling (also called PCP signaling, see Chapter 10, Volume 1) shown to be critical for its establishment. Two of the best characterized PCP models in Drosophila are the developing wing and the eye, where the graded activity of the Frizzled (Fz) receptor determines proximo-distal orientation of the wing hairs and mirror-imaged patterning of ommatidia, respectively. In this chapter, we describe simple methods to visualize PCP defects in the Drosophila eye and wing, in both developing and adult tissues. These methods include confocal immunofluorescent analysis of larval or pupal tissues, stained with antibodies specific to PCP components or cytoskeleton markers, and light microscopy of the adult eye and wing.

Methods for studying planar cell polarity

“…DPX is a medium which hardens and provides long term storage. It can be used as a replacement for xylene-based media such as diluted Canada balsam (Arbouzova & McNeill, 2008). Euparal is another commonly selected mounting medium that hardens to provide long-term preservation of samples.…”

“…Euparal can be diluted with ethanol to slow hardening and to lower viscosity. Commonly the sample is dehydrated through exposure to ethanol before mounting or, in the case of water based media, rehydrated (Arbouzova & McNeill, 2008; Stern & Sucena, 2012a; Stern & Sucena, 2012b). This list of media is certainly not exhaustive.…”

“…Storing in a mix of 30% glycerol and 70% absolute ethanol has been suggested to be ideal, as glycerol does not evaporate and protects the tissue (Stern & Sucena, 2012a). This mixture has also been suggested as a dehydration solution before mounting in DPX and Canada balsam (Arbouzova & McNeill, 2008). …”

“…The orientation of ommatidia is often studied in thin sections of eye, as described in detail in (Arbouzova & McNeill, 2008; Gaengel & Mlodzik, 2008; Jenny, 2011; Mishra & Knust, 2013). Each ommatidium comprises eight photoreceptors whose arrangement can then be judged without staining based on the position and appearance of their light-harvesting organelles, the rhabdomeres (Gaengel & Mlodzik, 2008).…”

“…The heads are then embedded in plastic, and sectioned using a microtome. The sections can be stained with toluidine blue for improved contrast (Arbouzova & McNeill, 2008; Gaengel & Mlodzik, 2008; Jenny, 2011). The sections can be mounted between a microscope slide and a coverslip in medium such as DPX.…”