패트릭 카퍼티는 캐나다의 여러 경화증 협회에서 박사 친교에 의해 지원됩니다.

1 부 : 사전 실험 설정. <ol><li> 한 주 미리, 친구 유충을 생성하는 빠르다 glial 특정 발기인의 통제하에 표현 GFP. 우리의 실험에 대한 우리의 반대 극성 (repo) 발기인 (3, 4)를 사용 glial 세포에 표현 핵 로컬 라이제이션 시퀀스 태그 GFP를 시각. </li><li> 1% 하룻밤 건조 폴리 - L - 라이신 솔루션과 공중에서 10 분 18mm 라운드 커버 전표를 몸으로로 사전에 최소한 1 일 이상 전표를 커버 준비합니다. </li><li> 실험 전날, 70 % 에탄올에서 하룻밤 구성 요소를 몸으로하여 Chamlide 자기 문화 챔버 청소. </li><li> 실험의 날, 무균 기술을 사용 슈나이더의 곤충 중간의 10 ML에 태아 소 혈청, 페니실린 - 스트렙토 마이신 솔루션, 그리고 인슐린을 추가하여 문화 매체를 준비합니다. 작업 농도는 다음과 같습니다 1X 태아 소 혈청, 100U/ml 페니실린, 스트렙토 마이신 0.1mg/ml, 그리고 슈나이더의 곤충 매체 0.2 MG / ML 인슐린 ((5)에서 수정). </li><li> 문화 챔버는 세포 배양 후드의 건조 공기를 허용합니다. 준비 문화 매체와 rinsing하여 문화 챔버에서 잔여 에탄올을 씻으십시오. </li></ol> 2 부 : Drosophila 눈 - 뇌 복합 해부. <ol><li> 몇 분 동안 얼음에 페트리 접시에 차게 문화 매체의 드롭 플라이 약병과 장소의 측면에서 타사 instar 방황하는 유충을 선택합니다. 유충 등골이 오싹한 것은 절개의 용이성을위한 연동 신체 수축 속도가 느린 것을 권장합니다. </li><li> 해부 현미경 아래 Sylgard 코팅 접시에 배지의 드롭에 차게 유충를 놓습니다. </li><li> 해부 현미경에서 단단히 후부 하순부터 방식의 약 3 분의 1을 유충을 파악하기 위해 더몬트 좋은 포셉 한 쌍의을 사용합니다. 유충은 앞쪽에 끝에서 그 입을 갈고리를 extrudes 때까지 기다리십시오. 입 전체 연장시 포셉의 두 번째 쌍의와 후크 파악. 유충을 해부하다하려면, 천천히 반대 방향으로 포셉의 두 쌍을 가져옵니다. 눈 - 뇌 단지, 침샘, 지방 기관 및 imaginal 티슈와 함께 애벌레 몸 가까이 끌어 것입니다. 입들이 눈 - 뇌 복합 찢을 것이다 빠르게 달리 너무 후크 당기지 마십시오. </li><li> 침샘, 지방 기관 및 울트라 미세 클리퍼 가위를 사용하여 imaginal 디스크를 멀리 낸다. 두뇌와 눈 - 디스크의 두 반구는 복부 신경 코드와 구강 갈고리에 연결된 것입니다. </li><li> 유리 슬라이드에 18mm 커버 전표를 놓습니다. 나중에 커버 슬립을 픽업하기 위해 슬라이드의 가장자리를 걸려 커버 슬립 중 하나 가장자리를 남겨주세요. 18mm 커버 슬립에 문화 매체의 드롭을 추가합니다. 포셉로 입 갈고리를 쥐고하여 문화 매체에 눈을 뇌의 복잡한을 전송합니다. 포셉 또는 조직이 손상됩니다 직접 눈 - 뇌 복합 파악하지 마십시오. </li><li> 미세 해부 가위를 사용하여 입 눈 - 뇌 복잡하고 폐기에서 갈고리에서 트림. 입 후크는 조직 현미경 동안 이동하는 원인이 문화 매체에 계약을 계속으로 입 갈고리 제거 라이브 영상 중요합니다. </li><li> 눈 imaginal 디스크 내에 glial 마이 그 레이션을 시각화하기 위해 조심스럽게 광 스토킹, 뇌 및 안구 디스크를 연결하는 얇은 조직을 잘라 (그림 1A 참조) 머리를 밀어 또는 폐기합니다. 광학 스토킹 이내 glia을 시각화하려면, 뇌, 광 스토킹하고, 그대로 눈을 imaginal 디스크 (그림 1E 참조) 두십시오. </li><li> 포셉를 사용하여 coverslip을 선택하고 영구 마커와 관심의 조직 주위 밑에 원을 그려. 이 서클은 4 부의 현미경을위한 조직을 찾는데 도움이됩니다. </li></ol> 3 부 : 자기 문화 챔버의 눈을 imaginal 디스크를 장착. <ol><li> 포셉 사용하여 coverslip의 매달려 가장자리를 파악. 문화의 조직을 방해하지 않고 Chamlide 자기 챔버의 바닥 접시에 coverslip을 전송합니다. </li><li> Chamlide 챔버의 본체에 실리콘 O - 링 놓습니다. 하단 플레이트 위에 본체를 설치합니다. </li><li> 천천히 실로 문화 매체를 추가합니다. 너무 빨리 또는 관심의 조직이 방해되는 문화 매체를 추가하지 마십시오. 완전히 커버 아래의 가스 교환 수 있도록 챔버를 기입하지 마십시오. </li><li> 부드럽게 Chamlide 챔버의 상단에있는 눈에 보이지 않는 덮개를 놓으십시오. </li></ol> 4 부 : 아이 디스크에 glial 세포를 마이 그 레이션의 시각화. <ol><li> 공촛점 또는 형광 현미경의 무대에서 문화 챔버를 놓습니다. 낮은 배율에서 참조로 coverslip에 동그라미를 사용하여 샘​​플을 찾습니다. </li><li> 40x 렌즈를 사용하여 샘​​플에 초점. 조직이 coverslip에 정착하도록 허용합니다. 눈 디스크 또는 3-4시간 이상의 모든 10 - 15 광학 스토킹의 이미지를 캡처합니다. 조직 트위치과 집중을 밖으로 이동할 수 있습니다. 수동으로 이미지를 캡처하기 전에 조직을 중심으로하는 일을 경감됩니다문제가있다. </li></ol> 제 5 부 : 대표 결과 : 제대로 수행, 우리 프로토콜은 우리가 눈 imaginal 디스크 (그림 1 BD)로 광학 줄기에서 마이 그 레이션 GFP - 태그 glial 세포의 이미지 일련의를 수집하는있었습니다. 60 분 동안 라이브 이미지는 야생 타입 눈 imaginal 디스크 이내 glial 핵의 위치 변화를 관찰할 수있는 충분한 동안 glial 세포 마이 그 레이션에 필요한 유전자의 돌연변이에 glial 핵 완전히 광학 스토킹 (화살표를 종료하는 데 실패 그림 1 FH). 우리는 교양 조직의 저하를 관찰하기 전에 240분만큼 기간 동안 교양 눈 - 뇌 단지 있습니다. 240 분 시간이 지점에 따라 우리는 교양 눈 - 뇌 단지 (화살표 그림 2 C)를 둘러싼 문화 매체에 GFP 양성 세포를 관찰하기 시작합니다. 추가 GFP에서 조직의 무결성에 고장을 제안 조직 전반에 걸쳐 확산 장소에 축적됩니다. <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> 그림 1 : 개발 야생 타입과 돌연변이 시각 시스템에서 GFP - 태그 glial 핵의 라이브 영상. A, E) 이미지 차등 간섭 대비 (DIC) 교양 야생 유형과 돌연변이 눈을 imaginal 디스크 (ED)의 현미경을 사용하여 촬영. 야생 타입, glial 세포에서 태어나되고 눈을 디스크에 광학 스토킹 (OS)에서 마이 그 레이션. 머리가 납작하며 눈 imaginal 디스크의 이미지를 용이하게하기 위해 제거되었습니다. BD) (A) 0, 30, 및 60분 시간 포인트는 GFP - 태그 glial 핵은 눈 디스크 내에 마이 그 레이션 공개에 야생 유형 교양 눈 imaginal 디스크의 형광 현미경. FH) (E) 0, 30, 및 60분 시간이 지점은 광학 스토킹 (화살표) 이내에 GFP - 태그 glial 핵의 연기를 보여줍니다에서 돌연변이 눈 - 뇌 복합 형광 현미경. 두뇌 (BR)은 줄기 내에서 glia의 이미지를 용이하게하기 위해 광학 줄기에 붙어 왼쪽으로되었습니다. <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> 그림 2 : 야생 유형 눈 imaginal 디스크에 GFP - 태그 glial 세포막의 라이브 영상. 우리는 성공적으로 240 분 동안 눈을 두뇌 단지을 배양해 있습니다. 조직 교양 초과 240분 신경 쇠약하기 시작합니다. 300분 시간 점 (C)에 비해 눈에 imaginal 디스크를 둘러싼 문화 매체 (ED), 광학 스토킹 (OS), 0 (A) 150 분 (B)에서 두뇌 기억 상실증이 (BR), 무료 GFP 양성 세포의 화살표로 표시.

<ol>
	<li>Aigouy, B., Lepelletier, L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+chain+migration+requires+pioneer+cells.">Glial chain migration requires pioneer cells.</a> J. Neurosci. 28, 11635-11641 (2008).</li><li>Silies, M., Yuva, Y., Engelen, D., Aho, A., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+migration+in+the+eye+disc.">Glial cell migration in the eye disc.</a> J. Neurosci. 27, 13130-13139 (2007).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sepp, K. J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+lacZ+enhancer+trap+lines+to+GAL4+lines+using+targeted+transposition+in+Drosophila+melanogaster.">Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.</a> Genetics. 151, 1093-1101 (1999).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+geometric+order+in+proliferating+metazoan+epitheia.">The emergence of geometric order in proliferating metazoan epitheia.</a> Nature. 442, 1038-1041 (2006).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li></ol>

The authors have nothing to disclose.

이 프로토콜에서는 라이브 현미경을 사용하여 눈을 imaginal 디스크에 glial 세포 이주의 관찰을 설명합니다. 우리의 야생 유형 예를 들어 (그림 1 AD)에서, 우리는 한 시간의 과정을 통해 아이 디스크에 glial 세포의 움직임을 관찰하기 위해 핵 GFP 표지를 사용했습니다. 현재 저희 연구실에서 공부를 아래에 glial 세포 마이 그 레이션에 필요한 후보 유전자에 대한 돌연변이, 우리는 한 시간 동안 (?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
<tr>
<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4458</td>
<td>&nbsp;</td>
<tr>
<td>Insulin solution from bovine pancreas</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Chamlide Magnetic chamber</td>
<td>Tool</td>
<td>Live cell Instrument</td>
<td>CM-R-10</td>
<td>35 mm dish type chamber for 18 mm coverslip</td>
</tr>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>11251-20</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used. </td>
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live imaging of glial cell migration in the drosophila eye imaginal disc

척추 및 무척추 동물 생물 모두 Glial 세포 ensheath하기 위해서는 최종 대상 지역으로 마이 그 레이션과 관련된 뉴런을 지원해야합니다. 최근 진행은 개발 pupal 날개 (1)의 연구에 glial 세포의 라이브 마이 그 레이션을 설명하기 위해 만든되었습니다 있지만<em&gt; Drosophila</em&gt; glial 세포 마이 그 레이션은 일반적으로 고정 조직 검사를 참여했습니다. 운동성이있는 세포의 라이브 현미경 분석의 속도와 성장 방향의 변경을 결정하는 포함하여 철새 과정을 통해 세포 행동을 검사하는 기능을 제공합니다. 유전자 도구의 사용과 결합하여, 라이브 영상은 개발 과정에서 특정 유전자에 대한 더 정확한 역할을 결정하는 데 사용할 수 있습니다. Silies로 이전 작업<em&gt; 외.</em&gt; (2) 고정 조직에서 눈을 imaginal 디스크로, 광 스토킹, 개발 안구와 두뇌를 연결하는 구조에서 발생하는 glia의 마이 그 레이션하는 작업을 설명하고 있습니다. 여기에 glial 세포의 라이브 마이 그 레이션을 조사를위한 프로토콜을 윤곽<em&gt; Drosophila</em&gt; 눈 imaginal 디스크. 우리는 야생 타입과 돌연변이 동물의 glial 세포의 진행에 따라 개발 glia에 GFP를 표현 Drosophila 라인을 활용.

Watch this Scientific Journal Video about Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc at JoVE.com

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

여기 GFP 태그 glial 세포와 결합하여 라이브 미세 분석을 사용하여 개발 Drosophila의 눈을 glial 세포의 마이 그 레이션을 검사하기 위해 프로토콜을 설명합니다.

Drosophila 아이 Imaginal 디스크에 Glial 세포 이미징 마이 그 레이션 라이브

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

live-imaging-glial-cell-migration-drosophila-eye-imaginal

Research

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Biology

10.6K 조회수.  University of British Columbia - UBC. 여기 GFP 태그 glial 세포와 결합하여 라이브 미세 분석을 사용하여 개발 Drosophila의 눈을 glial 세포의 마이 그 레이션을 검사하기 위해 프로토콜을 설명합니다.

비디오: Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

셋째 Instar Drosophila의 유충에서 Imaginal 디스크의 해부

연구

생물학

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

라이브를 떠올리 Drosophila</em형광 염료와 함께> Glial - 신경근육학 융티온

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

의 준비 Drosophila</em공중 드롭 프로토콜을 사용하여 라이브 영상을위한> 태아

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

에있는 아이 Imaginal 디스크의 시각화에 도움을 소설 RFP 리포터 Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

라이브 영상 Drosophila melanogaster 배아 Hemocyte 마이 그 레이션

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

생활의 이미징 셀 모양 변경 Drosophila 태아

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

에 GFP-라벨 단백질의 영상을 라​​이브<em&gt; Drosophila</em&gt; Oocytes

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

모자이크 성인의 라이브 이미징 토마토 / GFP-FLP / FRT 방법<em&gt; 초파리</em&gt; 광 수용체 (photoreceptor)

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

해부와의 설치<em&gt; 초파리</em&gt; 번데기 아이 디스크

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...