一个行为分析对机械感觉MARCM为基础的克隆<em>果蝇</em
Summary
In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.
Abstract
因为结构和功能同源性的哺乳动物内耳的毛细胞，即支配果蝇外部感觉器官的神经元提供机械感觉的研究的优良模型系统。这个协议描述在果蝇中，可用于识别与机械感觉干扰突变简单的触摸行为。一个macrochaete的触觉刺激刷毛上的苍蝇的胸部引出一清洁反射从第一或第三站。与机械传导（如NOMPC）干扰的突变，或者与反射弧的其他方面，可以抑制疏导响应。成人行为的传统画面就会错过这一发展过程中发挥重要作用的突变体。相反，该协议结合镶嵌分析与阻遏细胞标记物（MARCM）触摸屏，以允许纯合突变细胞中，只有有限的区域由EXPR生成并标分裂国家绿色荧光蛋白（GFP）的。通过测试MARCM克隆为异常的行为反应，有可能筛选的致死对元件的突变的集合以寻找涉及机械感觉新基因，将已经错过由更传统的方法。
Introduction
Humans rely on the ability to convert mechanical stimuli from their environment, such as touch, pressure, vibration, or sound waves, into sensory information that can be processed by the nervous system, in a process termed mechanotransduction. Many of the overall mechanistic features of mechanotransduction between humans and invertebrates are the same1, making Drosophila a useful model to study the molecular mechanisms of mechanotransduction. Drosophila melanogaster contain two sets of specialized sensory organs (Type I and II) that are capable of converting mechanical stimuli into action potentials. Type I mechanoreceptors have a neuron with a single dendrite or sensory process, surrounded by three support cells2. The Type I mechanoreceptors include bristle mechanoreceptors, hearing sensitive chordotonal organs (the Johnston’s organ), and campaniform sensilla that convey information about wing beats3. The bristles that cover the dorsal side of the fly are the most abundant and easily accessible of the Type I organs. Similar to the extracellular environment of hair cells associated with hearing and balance in vertebrates, the support cells surrounding fly mechanosensitive neurons secrete a high potassium endolymph that creates an unusual concentration gradient for potassium1. Mechanosensitive neurons, of both the mammalian and fly systems, utilize this high extracellular potassium to depolarize the cell. In response to mechanical stimulation of the macrochaete bristles towards the body wall, the sensory neurons respond with a burst of action potentials driven by this potassium depolarization of the cell membrane4. The Drosophila sensory neurons that innervate bristles resemble the mechanosensitive cells of other organisms, including vertebrate hair cells, in both structure and function1,5. The accessibility of Drosophila external sense organs to experimental manipulation and the abundance of genetic techniques available to researchers make Drosophila an excellent model system to investigate the molecular underpinnings of mechanosensation.
In the fly, stimulation of a single sensory neuron that innervates a bristle leads to an observable behavioral response. Stimulation of different bristles evokes specific, reproducible behavioral responses, depending on the bristle that is stimulated. Upon tactile stimulation, decapitated wild-type flies exhibit a complex grooming reflex wherein they clean the area near the stimulated bristle with a patterned set of leg movements5-7. When homozygous for known single-gene mutations that interfere with learning7 or coordination and locomotor activity5, flies respond abnormally to mechanical stimulation. This grooming reflex is therefore a useful tool to study the effects of single-gene mutations on a specific, replicable behavior.
The robust behavioral response to stimulation of a single macrochaete bristle holds the potential to assist in identifying new genes involved in mechanotransduction. This protocol being used to test a collection of mutant flies for the absence of a behavioral response to indicate that the mutation interferes with mechanosensation. In the mutant collection selected for screening, the mutations cause lethality before adulthood, and therefore would be impossible to test using traditional adult behavior screens. Originally, this collection of lethal p-elements was combined with FRT recombination sites to test cell growth defects in clones. The clones were made specifically in the eye because adult flies can survive in the lab setting without functional vision8. However, removal of all mechanosensation can cause adults to be severely uncoordinated or die before eclosion5. This protocol uses a mosaic approach to circumvent the lethality of the mutations and allow for adult stage testing. A genetic technique called Mosaic Analysis with a Repressible Cell Marker (MARCM)9 is used to generate homozygous mutant cells in a limited number of adult fly sensory organs, while the rest of the organism remains heterozygous. These MARCM flies readily survive until adulthood, yet the bristles are homozygous for the lethal genes of interest.
MARCM allows for regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP), while the rest of the organism remains heterozygous at that particular locus and unmarked9. MARCM combines individual p-element mutations with five common genetic elements: GFP under the control of an upstream-activating sequence (UAS-GFP), Gal80 repressor protein under control of a general promoter (tub-Gal80), Gal4 transcription factor under control of a general promoter (tub-Gal4), the FRT recombinase enzyme expressed through a heat-shock controlled promoter, and a FRT recombination site10. By driving mitotic recombination through a heat-shock activated recombinase, a limited number of cells are made homozygous for the mutation and marked with GFP. GFP expression is repressed in heterozygous cells by the presence of Gal80 on the wild-type copy of the chromosome.
A heat-shock protocol for MARCM was optimized to induce recombination in the fly bristle external sense organs, while much of the organism remains heterozygous, and thus unmarked with GFP. Mosaic flies generated using this protocol contained homozygous mutations most frequently in the post alar or dorsal central bristles on the surface of the notum.
We have tested the utility of this combination of MARCM and the grooming behavior screen with a known mechanosensitive mutant, NOMPC. The ion channel, no mechanoreceptor potential C (NOMPC), is an essential component of the mechanotransduction pathway in Drosophila5,11-13. NOMPC belongs to the transient receptor potential (TRP) superfamily of cation channels5 and satisfies all of the criteria to qualify as a mechanosensitive channel in Drosophila14,15: 1) NOMPC is expressed in the ciliate tips of type 1 sensory neurons of Drosophila 13,16-18, 2) NOMPC null larvae do not have an electrical response to tactile stimulation13, 3) Ectopic expression of NOMPC in touch insensitive cells can induce sensitivity to mechanical stimulation13, 4) heterologous expression of NOMPC in Schneider 2 cells yields a mechanosensitive channel13, and 5) NOMPC adult mutants display defects in their response to mechanical stimulation5. Given this evidence, we predicted that NOMPC mutant clones would show an altered or inhibited grooming response in response to mechanical stimulation of bristles.
A MARCM-stock containing the NOMPC mutation was developed for use in a proof of principle experiment of the grooming assay. Mosaic flies were stimulated at macrochaete bristles containing homozygous NOMPC mutant cells. We expected an inhibition of the grooming response following stimulation of the macrochaete bristle. We found that only 2 of 14 mutant bristle flies tested gave a single response to repeated stimulation; most did not respond to stimulation of the homozygous mutant bristle. Having confirmed that this MARCM-based behavioral assay produces an abnormal grooming reflex in a known mechanosensitive mosaic mutant, this technique can be used in a screen for additional mechanosensitive mutations.
Protocol
Representative Results
Discussion
这个协议利用一个成人行为分析以筛选影响机械感觉在果蝇的突变。因为突变体的集合包含致死对元件的突变，将排除筛选作为成年人，该协议使得使用第一Lee和罗述的复杂的遗传技术的，（1999年）和详细的协议吴，罗，（2006年），以规避成人杀伤力。 MARCM诱导同源染色体之间有丝分裂重组以产生纯合细胞的克隆的区域。因为阻遏蛋白，浴盆-GAL80中有丝分裂重组发生的损失的这些?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者谨感谢布卢明顿股票中心，利群罗，查尔斯·朱克和莉莉和裕侬扬慷慨分享飞股票及以下资金：肉体-URM（以JD和SW），本科福特学院暑期奖学金（申银万国），BD公司暑期研究奖学金（以CL和DL），该蕾妮和Anthony M.马龙，MD '63暑期研究奖学金（以DL），詹姆斯·'75和简Colihan暑期研究奖学金（TO）通过校友/圣十字学院的家长暑期研究基金和Stransky基金会暑期研究奖学金（为TO）。特别感谢生物系和教务处在学院的圣十字对实验室全部工作的支持。
Materials
| Brewers Yeast (25 lb) | MP Biomedicals | ICN90331225 | Fly Food |
| Corn (25lb) | MP Biomedicals | ICN90141125 | Fly Food |
| Agar (1lb) | MoorAgar Inc. | 41004 | Fly Food |
| Tegosept (5kg) | Genesee | 20-259 | Fly Food |
| Molasses (1Gallon) | Sugarmill Brand – Thomsen Food Service | 0 2625 | Fly Food |
| Propionic Acid | Fisher | A258-500 | Fly Food |
| Phosphoric acid | Fisher | A260-500 | Fly Food |
| Drosophila Vials, Narrow (PS) | Genesee | 32-109 | Fly Cultures |
| 6oz Square Bottom Bottle (PP) | Genesee | 32-130 | Fly Cultures |
| Flugs – Plastic Fly Bottles | Genesee | 49-100 | Fly Cultures |
| Rayon Balls, Large | Genesee | 51-100 | Fly Cultures |
| Droso-Filler, Narrow | Genesee | 59-168 | Fly Food Preparation |
| Droso-Filler, Bottles | Genesee | 59-170 | Fly Food Preparation |
| 8A-C / gear driven lab stirrer with c-clamp mount 1/15HP, 700RPM variable speed, 115V, 50/60Hz | Cleveland Mixer | 8A-C | Fly Food Preparation |
| Water jacketed Kettle | Fly Food Preparation | ||
| Diurnal Growth Chamber | Forma Scientific | Temperature and light/dark cycle controlled | |
| Water bath | VWR | For heat shock | |
| MicroScissors | Fine Science Tools | 15000-08 | For removing heads |
| Fluroscence Dissecting Microscope | Zeiss | SteREO Discovery V8 | With GFP cube (KSC295-814D) band pass filter |
| Fluroscence Light Source | Zeiss | X-Cite 120 | Fiber optic light pipe makes this easy to configure |
| Camera for Scope | Zeiss | AxioCam ICc1 | |
| Image acquistion software | Zeiss | ||
| Ice bucket | for cold anthesia | ||
| Homemade cold anthesia tray | for cold anthesia decapitation | ||
| Plastic boxes | for recovery of decaptitated flies in humid environment |
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