Visualization of Proprioceptors in <em>Drosophila</em> Larvae and Pupae

Abstract

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2 . Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16 . The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14 . When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12 . The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system. Multiple ChOs have been described in the adult fly 7 . These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4 . In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly. Video LinkThe video component of this article can be found at https://www.jove.com/video/3846/ ProtocolBefore you start 1. Grow the desired fly cultures. Keep the vials uncrowded (approximately 30 flies per 50 ml vial). Let the flies lay eggs for only one day in each vial. This will provide the larvae sufficient food supply and allow them to reach maximal size before crawling out of the food. Keep the vials in the appropriate temperature until third instar larvae begin to wander on the vial wall. 2. Prepare a fresh stock of phosphate buffered saline (PBS) and PBT (0.1% Tween in PBS). Keep 10 ml of PBS on ice. 3. Prepare 1-5 ml of fresh fixative (4% Formaldehyde in PBT) and keep it on ice. We use bottled 37-38% formaldehyde as a stock solution.

Pleiotropy in enhancer function is encoded through diverse genetic architectures

“…Stage 15 embryos were collected, fixed, and stained using standard protocols with chicken anti-GFP (1:300, Aves Labs), rabbit anti-RFP (1:150, MBL), mouse anti-pGal (1:500, Promega), anti-chicken AlexaFluor 488 (1:250, ThermoFisher), anti-rabbit AlexaFluor 647 (1:150, ThermoFisher) and anti-mouse AlexaFluor 546 (1:400, ThermoFisher). Pupal tissues were dissected, fixed and stained with mouse anti-fBGal and anti-mouse AlexaFluor 546 as described [32].…”

Comprehensive Analysis of a cis-Regulatory Region Reveals Pleiotropy in Enhancer Function

“…Stage 15 embryos were collected, fixed, and stained using standard protocols with chicken anti-GFP (1:300; Aves Labs), rabbit anti-RFP (1:150; MBL), mouse anti-bGal (1:500; Promega), anti-chicken Alexa Fluor 488 (1:250; Thermo Fisher Scientific), anti-rabbit Alexa Fluor 647 (1:150; Thermo Fisher Scientific) and anti-mouse Alexa Fluor 546 (1:400; Thermo Fisher Scientific). Pupal tissues were dissected, fixed, and stained with mouse anti-bGal and anti-mouse Alexa Fluor 546 as described (Halachmi et al, 2012).…”

A Change in ECM Composition Affects Sensory Organ Mechanics and Function

“…To gain an insight into the role of ECM composition in ChO function, we compared the mechanics and function of the ChOs in wild-type (wt) larvae versus larvae homozygous for the prc MB03017 protein null allele (Wilmes et al, 2018). To allow for easy visualization and live imaging of larval ChOs, we used transgenic reporters that drive the cytoplasmic expression of GFP within the cap and ligament cells, and red fluorescent protein (RFP) within the cap-attachment and ligament-attachment cells (Halachmi et al, 2012(Halachmi et al, , 2016) ( Figure 1E).…”

“…The secondary antibodies were Cy3-and Alexa Fluor-647-conjugated anti-mouse/rabbit (Jackson Laboratories, Bar-Harbor, Maine, USA). Larvae were dissected and stained as described in Halachmi et al (2012).…”

“…Imaging Larval ChO Using Scanning Electron Microscopy 3 rd instar larvae were dissected as described in Halachmi et al (2012). Prior to fixation, the longitudinal muscles were torn using fine forceps as described in Tenenbaum and Gavis (2016).…”