<em>Drosophila</em> Pupal Abdomen Immunohistochemistry

Abstract

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies [1][2][3] . During pupation, which spans approximately 96 hours (at 25 °C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4 . Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells-LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy. Video LinkThe video component of this article can be found at https://www.jove.com/video/3139/ Protocol Day 1 Before you start:A healthy population of flies should be maintained using standard culturing protocols: remove adults from bottles or vials after 3-4 days of egglay and allow development to proceed at a constant temperature until wandering 3 rd instar larvae initiate pupariation. The larval/pupal transition is marked by the formation of the prepupae (considered 0 hours after puparium formation-APF). Immobile pupae are distinguished from older pupae by their white coloration and from larvae that have not yet begun pupariation by their oblong,rounded shape and protrusion of the anterior spiracles. You will need:• A paint brush for collecting pupae • A humid chamber for culturing pupae: A Petri dish lined with wetted paper towel and covered with filter paper marked for the various time points of collection, genotype or sex of the pupae • 1X Phosphate buffered saline (PBS) for washing pupae Collection, culturing and staging pupae 1. Using a wetted paintbrush gently remove 0hr APF pupae from culture bottles/vials and place them in the lid of the humid chamber. 2. Using the paintbrush and 1X PBS gently wash the pupae to remove debris from the pupa case 3. If nece...

<em>Ex vivo</em> Culture of <em>Drosophila</em> Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

“…Alternatively, it is possible to identify male L3 larvae using a stereo-microscope, as the testes can be identified as translucent round organs embedded in the lateral fat body tissues of the intact larva 23,34 , and to collect them in fresh culture vials that can be used to pick pre-pupae for staging and dissections. It is also possible to identify male pre-pupae 35 .…”

Methods for studying planar cell polarity

“…The pupal case can be left as a stabilizing structure until final mounting. Wang and Yoder used deoxycholic acid as detergent in the initial fixation step, and omitted permeabilizing agents to minimize cell loss (Wang & Yoder, 2011). In addition, rocking during incubation was minimized.…”

Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex

“…The pOneStrepCTCFattB construct to rescue CTCF loss of function was assembled by placing the N-terminal OneStrEP-tag (IBA) fused in frame with the 2.6-kb CTCF ORF under control of the ubiquitous promoter of the Ubi-p63E gene (Butcher et al 2004 Patel (1994) and Wang and Yoder (2011). To control for immunostaining variability, homozygous (mutant) and heterozygous (control) embryos and larval nerve cords were stained simultaneously in the same vial.…”