A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.
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.
Before you start
1. Dissection and Fixation of Third Instar Larvae
2. Dissection and Fixation of Pupae
Part I
Part II
3. Immunostaining of Larvae and Pupae
4. Mounting of Larvae
5. Mounting of Pupae
6. Representative Results
An example of immuno-stained ChOs of third instar larva is shown in Figure 1. This example shows a nicely stretched abdominal segment in which seven of the eight ChOs are clearly visible. Neurons are labeled with MAb22C10 (1:20, obtained from the Developmental Studies Hybridoma Bank at the University of Iowa); the cap, ligament, cap-attachment and ligament attachment cells are labeled with anti-αTub85E (1:10, Klein et al., 2010). Secondary antibodies for fluorescent staining were Cy3, or Cy5-conjugated anti-mouse/rabbit (1:200, Jackson Immunoresearch Laboratories). Samples were viewed using confocal microscopy (LSM 510, Zeiss).
A cluster of wing ChOs at the ventral radial vein of a 35 hr-old pupa is shown in Figure 2.
Figure 1. (A) Schematic illustration of the six cell types that constitute a single Ch organ: cap attachment (CA), cap (C), scolopale (S), neuron (N), ligament (L) and ligament attachment (LA). The LCh5 organ, in which five ChOs are clustered, is stretched across the group of lateral transverse muscles (LT1-4). The lateral longitudinal muscle (LL1), ventral longitudinal muscles (VL1-4) and lateral oblique muscle (LO1) are also illustrated. Tendon cells are illustrated as brown spheres (Taken from Klein et al., 2010). (B) One abdominal segment of a third instar larva viewed at a 10X magnification. Seven of the eight ChOs present in each abdominal segment are evident: five lateral organs that together form the pentascolopidial organ (LCh5), a single lateral organ (LCh1) and one of two ventral ChOs (VChB). The neurons (N) of the ChOs are labeled with the neuronal marker MAb 22C10 (red). The ligament (L), cap (C) and attachment cells (LA, CA) are labeled with anti-αTub-85E (blue). The cap and ligament cells are additionally labeled with a ChO-specific GFP reporter harboring a regulatory element from the dei locus (Nachman et al., unpublished data).
Figure 2. The wing chordotonal organs at the ventral radial vein are viewed at a 40X magnification. The neurons (N) are marked with the neuronal marker 22C10 (Red, B). The ligament (L) and cap (C) cells are co-labeled with anti-αTub85E antibodies (Blue, C) and a ChO-specific GFP reporter transgene (Nachman et al., unpublished data).
The protocol described in this video provides a way to visualize proprioceptive ChOs during larval and pupal stages. Studies about the structure and development of proprioceptive ChOs have been so far restricted mainly to embryonic stages and to larval imaginal discs. Thus, many aspects of later stages of development, of both larval and adult ChOs, remain largely unknown. The described protocol, combined with common genetic tools in Drosophila, can be utilized to identify and study new genes and pathways that play a role in later stages of ChOs development. This protocol was optimized for the visualization of ChO, but it can be adapted for the staining of other tissues, such as pupal wings 6.
The authors have nothing to disclose.
The authors would like to thank the Developmental Studies Hybridoma Bank at the University of Iowa for sending us antibodies. This work was supported by a grant (No. 29/08) from The Israel Science Foundation. A.S. is also supported by a research grant from the DFG (Deutsche Forschungsgemeinschaft).
Name of the reagent/tool | Company | Catalogue number | Comments |
Dumont #5 (or #55) forceps, biologie tip | F.S.T | 11252-20 or 11252-10 (or similar forceps) |
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Austerlitz stainless steel insect pins, minutiens 0.1mm | Roboz Surgical Instrument Co | RS-6083-10 | |
Sylgard 184 silicone elastomer kit | DOW Corning Corporation | 240.4019862 | |
Vannas micro scissors (straight, 7.5 cm, blade 3mm) | AS Mdeizintechnik GmbH | 11-590-00 | Vannas Spring Scissors with identical specifications can be purchased from Roboz Surgical Instrument Co. |
Orbital shaker – Rotamax-120 | Heidolph | N/A | |
Dako Fluorescent Mounting Medium | Dako Cytomation, Glostrup, Denmark | DK-5302392 | |
X10 PBS formulation | 2 gr/lit KCl, 2 gr/lit KH2PO4, 80 gr/lit NaCl, 21.7 gr/lit Na2HPO4.7H2O |
The quality of PBS is critical for the success of this protocol | |
PBT | X1 PBS + 0.1% Tween 20 |