A <em>Drosophila</em> Model to Study Wound-induced Polyploidization

Abstract

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it contains more than the diploid copy of its chromosomes, which is a result of endoreplication or cell fusion. In tissue repair, wound-induced polyploidization (WIP) has been found to be a conserved healing strategy from fruit flies to vertebrates. WIP has several advantages over cell proliferation, including resistance to oncogenic growth and genotoxic stress. The challenge has been to identify why polyploid cells arise and how these unique cells function. Provided is a detailed protocol to study WIP in the adult fruit fly epithelium where polyploid cells are generated within 2 days after a puncture wound. Taking advantage of D. melanogaster's extensive genetic tool kit, the genes required to initiate and regulate WIP, including Myc, have begun to be identified. Continued studies using this method can reveal how other genetic and physiological variables including sex, diet, and age regulate and influence WIP's function.. This method increases the suitability of the adult D. melanogaster abdominal epithelium as a model to study the role and regulation of polyploidy in wound repair.

Wound-induced polyploidization is dependent on integrin-yki signaling

“…S1A " type="url"/> – ). We assayed for endocycle entry using the thymidine analog EdU as an S phase marker, as cells undergo successive S phases with each endocycle during WIP ( Bailey et al, 2020 ; Losick et al, 2016 ). At 2 dpi, we observed 178 EdU + nuclei on average for the control around the wound site as previously reported, whereas mys knockdown reduced the mean number of EdU + epithelial nuclei to 74 ( mys RNAi#2 ) and 40 ( mys RNAi#3 ) ( …”

“…To confirm that epithelial ploidy was reduced, we directly measured nuclear ploidy in uninjured (−) and 3 dpi (+) epithelia. Our previous studies have shown that the uninjured epithelial nuclei are diploid (2C) and therefore can be used as an internal control to measure changes in the fly epithelial cell ploidy ( Bailey et al, 2020 ; Losick et al, 2013 ). As expected at 3 dpi, we observed that 41% of epithelial nuclei were polyploid compared to only 4% in uninjured control epithelial cells ( Fig.…”

“…Adult female flies were injured, dissected, fixed and stained as recently reported ( Bailey et al, 2020 ). Tissues were stained with antibodies (manufacturer and dilutions) as follows: mouse anti-FasIII (DSHB 7G10, 1:50), chicken anti-βgal (Abcam ab9361, pre-absorbed, 1:1000), rabbit anti-RFP (MBL PM005, 1:2000), rabbit anti-Grh (1:300) ( Losick et al, 2016 ), and rabbit anti-Phospho-Fak (Tyr397) (Thermo Fisher Scientific 44-624G, 1:100).…”

“…Drosophila epithelial nuclear ploidy was performed as recently reported ( Bailey et al, 2020 ). All samples were imaged under the same conditions and settings with a 300 µm×300 µm area used for analysis.…”

Blocking cell fusion inhibits age-induced polyploidy and maintains epithelial organization in Drosophila

“…Epithelial ploidy was calculated according to (Bailey et al, 2020). Samples from 5d and 40d old flies were dissected and stained as described above, then imaged with the same exposure time.…”