The authors thank RIKEN for funding. We also thank Cagri Yalgin, Caroline Delandre, and Jay Parrish for discussions on genetic and immunohistochemistry protocols.

1.Preparation of reagents
<ol>
 <li>Prepare Ca++ -free HL3.1 saline28.
 <ol>
 <li>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&deg;C. 
 Note: Ca++ -free solution prevents muscle contraction during dissection.</li>
 </ol>
 </li>
 <li>Make poly-L-lysine (PLL) coverslips.
 <ol>
 <li>Dissolve 100mg PLL in 4.2ml water and make 300&mu;l aliquots in Eppendorf tubes and freeze at -20&de...

<ol>
	<li>Grueber, W. B., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiling+of+the+Drosophila+epidermis+by+multidendritic+sensory+neurons.">Tiling of the Drosophila epidermis by multidendritic sensory neurons.</a> Development. 129, 2867-2878 (2002).</li><li>Crozatier, M., Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+multidendritic+neuron+differentiation+in+Drosophila:+the+role+of+Collier.">Control of multidendritic neuron differentiation in Drosophila: the role of Collier.</a> Dev Biol. 315, 232-242 (2008).</li><li>Hattori, Y., Sugimura, K., Uemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+expression+of+Knot/Collier,+a+transcriptional+regulator+of+the+EBF/Olf-1+family,+endows+the+Drosophila+sensory+system+with+neuronal+class-specific+elaborated+dendritic+patterns.">Selective expression of Knot/Collier, a transcriptional regulator of the EBF/Olf-1 family, endows the Drosophila sensory system with neuronal class-specific elaborated dendritic patterns.</a> Genes Cells. 12, 1011-1022 (2007).</li><li>Jinushi-Nakao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knot/Collier+and+cut+control+different+aspects+of+dendrite+cytoskeleton+and+synergize+to+define+final+arbor+shape.">Knot/Collier and cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape.</a> Neuron. 56, 963-978 (2007).</li><li>Sugimura, K., Satoh, D., Estes, P., Crews, S., Uemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+morphological+diversity+of+dendrites+in+Drosophila+by+the+BTB-zinc+finger+protein+abrupt.">Development of morphological diversity of dendrites in Drosophila by the BTB-zinc finger protein abrupt.</a> Neuron. 43, 809-822 (2004).</li><li>Li, W., Wang, F., Menut, L., Gao, F. 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No conflicts of interest declared.

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. Gal4c155) 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...

<table border="1">
<tr>
<th>Name of the reagent</th>
<th>Company</th>
<th>Catalogue number</th>
<th>Comments (optional)</th>
</tr>
<tr>
<td>SZX16 fluorescence dissection microscope (with GFPHQ filter)</td>
<td>Olympus</td>
<td>SZX16</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Live Insect Forceps</td>
<td>FST</td>
<td>26030-10</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>26mm x 76mm depression slide glass</td>
<td>Toshinriko Co.</td>
<td>T8-R004</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sylgard 184 (or Silpot 184)</td>
<td>Dow Corning</td>
<td>3097358-1004</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Poly-L-lysine</td>
<td>Sigma </td>
<td>P-1524</td>
<td>This product has proven most effective</td>
</tr>
<tr>
<td>DPX mounting medium</td>
<td>Sigma</td>
<td>44581</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Rabbit anti-GFP</td>
<td>Invitrogen</td>
<td>A-11122</td>
<td>Dilution 1:500</td>
</tr>
<tr>
<td>Rat anti-CD8</td>
<td>Caltag </td>
<td>5H10</td>
<td>Dilution 1:200</td>
</tr>
<tr>
<td>Mouse anti-CD2</td>
<td>AbD serotec</td>
<td>MCA443R</td>
<td>Dilution 1:700</td>
</tr>
<tr>
<td>Mouse anti-Fasciclin2</td>
<td>DSHB</td>
<td>1D4</td>
<td>Dilution 1:10</td>
</tr>
</table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

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 differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 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 generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. 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 field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
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-out22,26,27 techniques (summarized in Fig. 1).

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

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

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.

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

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

Research

JoVE Journal

Neuroscience

15.1K Görüntüleme.  RIKEN Brain Science Institute. Dendritik arborization duyusal nöronlar Drosophila Larva periferik sinir sisteminin genel ve nöron nöron farklılaşma sınıfa özel mekanizmaları aydınlatmak için faydalı model vardır. Biz dendritik arborization nöron genetik mozaikleri oluşturmak ve analiz etmek için pratik bir rehber sunuyoruz.