Clones em um ensaio comportamental para Mechanosensation de MarcM baseada em<em> Drosophila melanogaster</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

Devido à homologia estrutural e funcional para as células ciliadas do ouvido interno de mamífero, os neurónios que enervam os órgãos dos sentidos de Drosophila externos proporcionam um excelente sistema modelo para o estudo de mechanosensation. Este protocolo descreve um comportamento simples toque na mosca da fruta, que podem ser utilizados para identificar as mutações que interferem com mechanosensation. A estimulação táctil de um macrochaete cerdas sobre o tórax de moscas provoca um reflexo da preparação a partir de qualquer da primeira ou terceira perna. As mutações que interferem com mecanotransdutores (tais como NOMPC), ou com outros aspectos do arco reflexo, pode inibir a resposta de preparação. Uma tela tradicional de comportamentos adultos teria perdido mutantes que têm papéis essenciais durante o desenvolvimento. Em vez disso, este protocolo combina a tela de toque com a análise do mosaico com um marcador de células repressible (MarcM) para permitir apenas regiões limitadas de células mutantes homozigotos para ser gerado e marcado pela exprESSÃO da proteína verde fluorescente (GFP). Ao testar clones MarcM para respostas de comportamento anormal, é possível rastrear um conjunto de mutações letais p-elemento de pesquisa para novos genes envolvidos no mechanosensation que teriam sido perdidas por métodos mais tradicionais.

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 function^1,5. 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 movements^5-7. 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 Drosophila^5,11-13. 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 Drosophila^14,15: 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

1. Cruzes para gerar Flies MarcM Manter moscas em meios de fubá melaço (6,5 g / L de agar; 23,5 g / L de levedura; 60 g / L da farinha de milho; 60 ml / L de melaço; 4 ml / mistura de ácido L; 0,13% Tegosept) ou mídia Drosophila padrão a 22 graus Celsius num ciclo luz / escuro de 12 h. Nota: A Baixa de temperatura padrão é usado para manter MarcM-stocks saudável. Isso amplia o tempo de desenvolvimento entre ovo e idade adulta para 14 dias. Use fêmeas virgens (cerca de 1 – 8…

Representative Results

O sucesso deste protocolo depende em grande medida o rendimento global de moscas testáveis, a eficácia do choque térmico para induzir a recombinação mitótica, e a capacidade de obter uma resposta comportamental robusto em moscas estimuladas em intervalos de 2 min. Dado que cerca de 12,5% de embriões conter os elementos necessários MarcM e estima-se que 16,67% da população adulta que EFeche têm o potencial para a expressão de GFP, que é fundamental para obter um elevado núme…

Discussion

Este protocolo utiliza um ensaio comportamental adulto para o rastreio de mutações que afectam mechanosensation em Drosophila. Uma vez que a colecção de mutantes contém letais mutações P-elemento, que evitarão a rastreio como adultos, este protocolo faz uso de uma técnica genética complexa primeiro descrito por Lee e Luo , (1999) e detalhada como um protocolo por Wu e Luo, (2006) para contornar adulto letalidade. MarcM induz recombinações mitóticas entre cromossomos homólogos para gerar regiões c…

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