Morfologiske Analyse af Drosophila Larver Perifer sensorisk Neuron dendritter og axoner anvende genetiske Mosaikker
Summary
De dendritiske arborization sensoriske neuroner i det<em> Drosophila</em> Larver perifere nervesystem er nyttige modeller til at belyse både generelle og neuron klasse-specifikke mekanismer neuron differentiering. Vi præsenterer en praktisk guide til at generere og analysere dendritiske arborization neuron genetiske mosaikker.
Abstract
Nervesystemets udvikling kræver den korrekte specifikation af neuron position og identitet, efterfulgt af præcise neuron klasse-specifikke dendritiske udvikling og axonal ledninger. For nylig dendritiske arborization (DA) sensoriske neuroner i det Drosophila larvestadiet perifere nervesystem (PNS) er blevet magtfulde genetiske modeller til at belyse både generelle og klasse-specifikke mekanismer neuron differentiering. Der er fire vigtigste DA neuron klasser (I-IV) 1. De er opkaldt efter stigende dendritceller Arbor kompleksitet, og har klasse-specifikke forskelle i den genetiske kontrol over deres differentiering 2-10. The Da sensoriske system er en praktisk model til at undersøge de molekylære mekanismer bag kontrol af dendritiske morfologi 11-13 fordi: 1) det kan drage fordel af den kraftige genetiske værktøjer til rådighed i bananfluen, 2) DA neuron dendritceller arbor breder sig i kun 2 dimensioner under et optisk Clear larver neglebånd gør det let at visualisere med høj opløsning in vivo, 3) den klasse-specifikke mangfoldighed i dendritiske morfologi letter en sammenlignende analyse for at finde centrale elementer styre dannelsen af simple vs stærkt forgrenede dendritiske træer, og 4) dendritiske arbor stereotype figurer af forskellige DA neuroner lette morfometriske statistiske analyser.
DA neuron aktivitet ændrer produktionen af en larvestadiet bevægelse central mønster generator 14-16. De forskellige DA neuron klasser har forskellige sanser, og deres aktivering udløser forskellige adfærdsmæssige reaktioner 14,16-20. Desuden forskellige klasser sende aksonal fremskrivninger stereotypically i Drosophila larvestadiet centrale nervesystem i ventrale nerve ledning (VNC) 21. Disse fremskrivninger opsige med topografiske fremstillinger af både DA neuron sensorisk modalitet og placeringen i kroppen væggen af dendritiske feltet 7,22, 23. Derfor undersøgelse af DA axonal fremskrivninger kan bruges til at belyse mekanismerne bag topografisk kortlægning 7,22,23, samt ledningsføring af et simpelt kredsløb modulerende larver bevægelse 14-17.
Vi præsenterer her en praktisk guide til at generere og analysere genetiske mosaikker 24 mærkning DA neuroner via MARCM (Mosaik analyse med en Repressible Cell Marker) 1,10,25 og FLP-out 22,26,27 teknikker (opsummeret i Fig. 1).
Protocol
Discussion
Den Drosophila Larvetilstand DA neuron model giver en fremragende genetiske system til at undersøge mekanismerne, der styrer neuron morfologi og kredsløb dannelse. MARCM er generelt bruges til mærkning og for at skabe mutant DA neuron kloner. Til MARCM bruger vi enten en pan-neurale (f.eks Gal4 C155) eller DA neuron-specifik driver. Ved hjælp af en pan-neurale driver det er muligt direkte at bruge flere lagre almindeligt tilgængelige fra offentlige lager centre. Men ved hjælp a…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne takker Riken til finansiering. Vi takker også Cagri Yalgin, Caroline Delandre, og Jay Parrish til diskussioner om genetiske og immunhistokemi protokoller.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| SZX16 fluorescence dissection microscope (with GFPHQ filter) | Olympus | SZX16 | |
| Live Insect Forceps | FST | 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 | P-1524 | This product has proven most effective |
| DPX mounting medium | Sigma | 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 |
References
- Grueber, W. B., Jan, L. Y., Jan, Y. N. Tiling of the Drosophila epidermis by multidendritic sensory neurons. Development. 129, 2867-2878 (2002).
- Crozatier, M., Vincent, A. Control of multidendritic neuron differentiation in Drosophila: the role of Collier. Dev Biol. 315, 232-242 (2008).
- Hattori, Y., Sugimura, K., Uemura, T. 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. Genes Cells. 12, 1011-1022 (2007).
- Jinushi-Nakao, S. Knot/Collier and cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape. Neuron. 56, 963-978 (2007).
- Sugimura, K., Satoh, D., Estes, P., Crews, S., Uemura, T. Development of morphological diversity of dendrites in Drosophila by the BTB-zinc finger protein abrupt. Neuron. 43, 809-822 (2004).
- Li, W., Wang, F., Menut, L., Gao, F. B. BTB/POZ-zinc finger protein abrupt suppresses dendritic branching in a neuronal subtype-specific and dosage-dependent. 43, 823-834 (2004).
- Zlatic, M., Landgraf, M., Bate, M. Genetic specification of axonal arbors: atonal regulates robo3 to position terminal branches in the Drosophila nervous system. Neuron. 37, 41-51 (2003).
- Grueber, W. B., Ye, B., Moore, A. W., Jan, L. Y., Jan, Y. N. Dendrites of distinct classes of Drosophila sensory neurons show different capacities for homotypic repulsion. Curr Biol. 13, 618-626 (2003).
- Grueber, W. B., Jan, L. Y., Jan, Y. N. Different levels of the homeodomain protein cut regulate distinct dendrite branching patterns of Drosophila multidendritic neurons. Cell. 112, 805-818 (2003).
- Moore, A. W., Jan, L. Y., Jan, Y. N. hamlet, a binary genetic switch between single- and multiple- dendrite neuron morphology. Science. 297, 1355-1358 (2002).
- Gao, F. B., Brenman, J. E., Jan, L. Y., Jan, Y. N. Genes regulating dendritic outgrowth, branching, and routing in Drosophila. Genes Dev. 13, 2549-2561 (1999).
- Corty, M. M., Matthews, B. J., Grueber, W. B. Molecules and mechanisms of dendrite development in Drosophila. Development. 136, 1049-1061 (2009).
- Moore, A. W. Intrinsic mechanisms to define neuron class-specific dendrite arbor morphology. Cell Adh. Migr. 2, 81-82 (2008).
- Hughes, C. L., Thomas, J. B. A sensory feedback circuit coordinates muscle activity in Drosophila. Mol. Cell. Neurosci. 35, 383-396 (2007).
- Nishimura, Y. Selection of Behaviors and Segmental Coordination During Larval Locomotion Is Disrupted by Nuclear Polyglutamine Inclusions in a New Drosophila Huntington’s Disease-Like Model. J Neurogenet. 24, 194-206 (2010).
- Song, W., Onishi, M., Jan, L. Y., Jan, Y. N. Peripheral multidendritic sensory neurons are necessary for rhythmic locomotion behavior in Drosophila larvae. Proc. Natl. Acad. Sci. U. S. A. 104, 5199-5204 (2007).
- Hwang, R. Y. Nociceptive neurons protect Drosophila larvae from parasitoid wasps. Curr Biol. 17, 2105-2116 (2007).
- Xiang, Y. Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature. 468, 921-926 (2010).
- Cheng, L. E., Song, W., Looger, L. L., Jan, L. Y., Jan, Y. N. The role of the TRP channel NompC in Drosophila larval and adult locomotion. Neuron. 67, 373-380 (2010).
- Babcock, D. T., Landry, C., Galko, M. J. Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae. Curr Biol. 19, 799-806 (2009).
- Hafer, N., Schedl, P. Dissection of Larval CNS in Drosophila Melanogaster. J. Vis. Exp. (1), e85-e85 (2006).
- Grueber, W. B. Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology. Development. 134, 55-64 (2007).
- Merritt, D. J., Whitington, P. M. Central projections of sensory neurons in the Drosophila embryo correlate with sensory modality, soma position, and proneural gene function. J Neurosci. 15, 1755-1767 (1995).
- Blair, S. S. Genetic mosaic techniques for studying Drosophila development. Development. 130, 5065-5072 (2003).
- Lee, T., Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron. 22, 451-461 (1999).
- Wong, A. M., Wang, J. W., Axel, R. Spatial representation of the glomerular map in the Drosophila protocerebrum. Cell. 109, 229-241 (2002).
- Shimono, K. Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death. Neural Dev. 4, 37-37 (2009).
- Feng, Y., Ueda, A., Wu, C. F. A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae. J Neurogenet. 18, 377-402 (2004).
- Sullivan, W., Ashburner, M., Hawley, R. S. . Drosophila Protocols. , (2000).
- Kaczynski, T. J., Gunawardena, S. Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos. J. Vis. Exp. (46), e2150-e2150 (2010).
- Featherstone, D. E., Chen, K., Broadie, K. Harvesting and preparing Drosophila embryos for electrophysiological recording and other procedures. J Vis Exp. , (2009).
- Medina, P. M., Swick, L. L., Andersen, R., Blalock, Z., Brenman, J. E. A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster. Genetics. 172, 2325-2335 (2006).
- Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D., Brenman, J. E. LKB1 and AMPK maintain epithelial cell polarity under energetic stress. J Cell Biol. 177, 387-392 (2007).
- Brent, J., Werner, K., McCabe, B. D. Drosophila Larval NMJ Immunohistochemistry. J. Vis. Exp. 25, e1108-e1108 (2009).
- Brand, A. H., Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118, 401-415 (1993).
- Sugimura, K. Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons. J Neurosci. 23, 3752-3760 (2003).
- Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y., Goodman, C. S. Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila. Neuron. 22, 719-729 (1999).
- Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites. Dev Biol. 260, 207-225 (2003).
