Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Summary

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Abstract

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation. There are four main DA neuron classes (I-IV)¹. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation^2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology^11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.

DA neuron activity modifies the output of a larval locomotion central pattern generator^14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses^14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)²¹. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field^7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping^7,22,23, as well as the wiring of a simple circuit modulating larval locomotion^14-17.

We present here a practical guide to generate and analyze genetic mosaics²⁴ marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)^1,10,25 and Flp-out^22,26,27 techniques (summarized in Fig. 1).

Protocol

1.Preparation of reagents

1. Prepare Ca⁺⁺ -free HL3.1 saline²⁸.
   1. In mM: 70 NaCl, 5 KCl, 20 MgCl₂ , 10 NaHCO₃ , 5 HEPES, 115 sucrose, and 5 trehalose; pH 7.2. Filter sterilize and store at 4°C.
      Note: Ca⁺⁺ -free solution prevents muscle contraction during dissection.
2. Make poly-L-lysine (PLL) coverslips.
   1. Dissolve 100mg PLL in 4.2ml water and make 300μl aliquots in Eppendorf tubes and freeze at -20°C.
   2. Before coating coverslips, first thaw an aliquot, bring it up to 10ml in ddH₂O and add 20μl of Kodak Photo-Flo; the final PLL concentrat....

Discussion

The Drosophila larval DA neuron model provides one excellent genetic system to investigate mechanisms that control neuron morphology and circuit formation. MARCM is generally used for labeling and for generating mutant DA neuron clones. For MARCM we use either a pan-neural (e.g. Gal4^c155) or DA neuron-specific driver. Using a pan-neural driver it is possible to directly use several stocks widely available from public stock centers. However using a DA-neuron specific driver can be adv.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SZX16 fluorescence dissection microscope (with GFPHQ filter)	Olympus Corporation	SZX16
Live Insect Forceps	Fine Science Tools	26030-10
26mm x 76mm depression slide glass	Toshinriko Co.	T8-R004
Sylgard 184 (or Silpot 184)	Dow Corning	3097358-1004
Poly-L-lysine	Sigma-Aldrich	P-1524	This product has proven most effective
DPX mounting medium	Sigma-Aldrich	44581
Rabbit anti-GFP	Invitrogen	A-11122	Dilution 1:500
Rat anti-CD8	Caltag	5H10	Dilution 1:200
Mouse anti-CD2	AbD Serotec	MCA443R	Dilution 1:700
Mouse anti-Fasciclin2	DSHB	1D4	Dilution 1:10