Name: imaging subcellular structures in the living zebrafish embryo
Uploaded: 2016-04-02T14:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Imaging Subcellular Structures in the Living Zebrafish Embryo at JoVE.com

P.E. is supported by the Deutsche Forschungsgemeinschaft (DFG) Research Training Group 1373 and the Graduate School of the Technische Universit&#228;t M&#252;nchen (TUM-GS). G.P. was supported by TUM-GS. L.T. is supported by an EMBO fellowship (EMBO ALTF 108-2013). D.P.'s work on zebrafish was supported by the DFG through the Sonderforschungsbereich "Molecular Mechanisms of Neurodegeneration" (SFB 596); the Center for Integrated Protein Sciences (Munich) and the European Community's Seventh Framework Programme (FP7/2007-2013) under Grant agreement no. 200611 (MEMOSAD). He is currently a New York Stem Cell Foundation-Druckenmiller Fellow and was supported by a fellowship from the German Academy of Sciences Leopoldina. L.G. is supported by funding from the DFG through SFB 870 "Assembly and Function of Neuronal Circuits", Project A11. 
We are grateful to Kristina Wullimann for maintaining our fish facility, Yvonne Hufnagel for technical support and Thomas Misgeld for comments on the manuscript. We are grateful to R. K&#246;ster (Technische Universit&#228;t Braunschweig) for providing the M1 Medusa vector (pSKmemmRFP:5xUAS:H2B-CFP:5xUAS:Centrin2-YFP) from which we cloned out Centrin-YFP and S.C. Suzuki and T. Yoshimatsu (University of Washington) for providing the 14xUAS:MA-cerulean cassette which we used to generate the reporter construct to make CentrinFish. We further thank S.C. Suzuki and T. Yoshimatsu (University of Washington) for the Otx2:Gal4 transgenic line, A. Sagasti for the Sensory:Gal4-VP16 construct (UCLA) and M. Nonet (Washington University in St. Louis) for the pCold Heart Tol2 vector. We acknowledge Bettina Schmid, Alexander Hruscha and Christian Haass (German Center for Neurodegenerative Diseases Munich - DZNE) for contributing to the development of MitoFish.

The authors have nothing to disclose.

여기, 우리는 찬란 태그 미토콘드리아, 중심체와 제브라 피쉬 배아에서 생체 내에서 특정 세포 유형의 세포막에 GAL4-UAS 발현 시스템의 다양성을 보여줍니다. 다른 세포 내 소기관 또는 구조 라벨 많은 형광 융합 단백질은 공개 된 문헌에서 발견 될 수 있고, 각각의 실험실 상업적 소스 또는 비상업적 플라스미드 기탁 (예 Addgene)로부터 얻을 수있다. 새로운 형광 융합 단백질을 디자인?...

생체 내 이미징에서 가장 생리 학적 맥락에서 세포 행동의 직접 시각화를 제공합니다. 제브라 피쉬 배아, 자신의 신속하고 외부 개발 및 형광 라벨을 허용 유전 도구의 풍부한 배열의 투명성은 모든 주요 발달 이벤트의 역학을 규명하기 위해 생체 현미경의 사용 증가에 기여했다. 제브라 피쉬의 신경 시스템 개발의 이미징 연구는 예를 들어 크게 신경 전구 세포의 행동과 그 이후의 이동, 분화 및 회로 통합 1-8 포함한 자손의 운명에 대한 우리의 지식을 확장합니다. 스테이지는 이제 이러한 셀룰러 동작을 기초 세포 내 역학 조사를하도록 설정된다. 사실, 제브라 피쉬는 이미 생체 내 세포 생물학위한 도구로 악용되고있다. 골지 (15), 미토콘드리아 9-11을 시각화 할 수있게되었습니다 2,8,12-14 중심체, 미세 소관 (4)과 액틴 세포 골격 (16)은 17 엔도 좀과 생체 내에서 제브라 피쉬 배아에서 다른 세포 내 구조 중, 1,18 혀끝 멤브레인의 구성 요소가 복잡한. 지금까지 이러한 세포 소기관의 기능에 대해 알려진 내용의 대부분은 배양 된 세포에 자신의 행동을 연구에서 비롯됩니다. 시험 관내 연구는 세포 생물학에 엄청난 통찰력을 산출하고 있지만, 문화의 세포는 완전히 생체 내 상황의 복잡성을 대변하지 않습니다 때문에 반드시 기능과 생체 내에서 세포 내 소기관의 역 동성을 반영하지 않습니다. 제브라 피쉬 배아는 세포 내 역학 조사에 생체 대안의 생존을 제공합니다. 척추 동물로서, 제브라 피쉬는 포유류에서 발견되는 상동 많은 기관계 (예, 신경 망막)을 갖는다. 또한, 피쉬 배아 점점 인간 질환 (19, 20)을 모델링하는데 사용되는 </s위로&gt;, centrosomal 기능 (예를 들어, 소두증 (21)과 Leber 씨의 선천성 흑암 22)와에 미토콘드리아 기능 (예, 파킨슨 병 23, 10, 24 및 바르트 증후군 25 퇴행성 신경)과 관련이있는 책임을 포함. 세포와 세포 내 수준에서 생체 내 이미징 이러한 경우 이러한 병리 적 상태의 기초가되는 세포 생물학의 더 나은 이해를 허용 할 것이다. 여기에 설명 된 방법의 전체 목표는 생체 광학 현미경에 사용 제브라 피쉬 배아 세포 내 소기관 및 기타 구조를 조사하기 위해 포괄적 인 가이드를 제공하는 것이다. 생체 내에서 세포 내 구조를 시각화 및 추적에 관련된 전체 작업 흐름을 설명한다 - 유전자 표지 접근 방식에서 일시적으로 발생 표현하고 안정적인 형질 전환 어류, 그리고 마지막으로 넓은 필드와 공 초점 현미경을 사용하여 영상화하기에. 이러한 시저의 각 동안edures이 많은 제브라 피쉬 실험실에서 사용되는 설명 프로토콜 최적화 및 세포 내 구조의 역학을 조사하기위한 간소화된다. 여기에 설명 된 일의 두 가지 특정 양태 언급 보증 먼저, 특정 세포 타입에서 유전자 라벨 소기관 여러 구성에서 GAL4-UAS 발현 시스템의 용도. 둘째, 생체 내에서 이미지 세포 내 구조에 넓은 필드와 공 초점 현미경의 직접적인 비교. 제브라 피쉬의 유전 라벨 세포 기관 및 기타 세포 내 구조에 대한 현재 전략 중 하나를 만들 프로모터 요소를 직접 융합 단백질 9,14,15. 시험 관내 전사 출장 RNA 결과의 발현을 유도 출장 mRNA의 -1,4,8- 또는 DNA 기반 구조의 사용 신속하고 폭 넓은 표현, 즉 그러나 조직 특이하지 않다. 캡핑 RNA 희석 또는 열화로 또한 발현 수준은 시간이 지남에 따라 감소. RNA의 따라서 사용을 기반으로(포스트 수정을 일반적으로 최대 3 일) 개발의 나중 단계에서 세포 기관의 역학 제한을 검토 구성한다. 이러한 제한은 표현의 공간 및 시간 제어가 특이 적 프로모터 요소에 의해 결정되는 DNA 작 제물을 사용하여 극복 될 수있다. DNA 기반 구조는 유전자 발현 수준에 GAL4-UAS 시스템에 상당한 개선의 맥락에서 사용되는 경우 (26, 27)을 관찰한다. 리포터 유전자는 GAL4 결합 상류 활성화 서열 (UAS)의 하류에 클로닝하는 동안이 분형 발현 시스템에서, 세포 유형 특이 적 프로모터 요소는 GAL4 전사 활성의 발현을 구동한다. 적절한 GAL4 드라이버와 UAS 리포터 조합하여, 발현은 다른 프로모터 뒤에 특이 적 발현 패턴이 요구 될 때마다 리포터 유전자를 복제 할 필요를 회피, 특정 세포 유형에 한정된다. 또한, 다수의 UAS 리포터 유전자의 발현 할 수있다하나의 GAL4 활성화에 의해 구동. GAL4-UAS 시스템은 따라서 세포 내 라벨에 대한 다양하고 유연한 유전 적 접근 방식을 제공합니다. 넓은 필드와 공 초점 현미경은 대부분 실험실의 일꾼입니다. 와이드 필드 시스템은 일반적으로 광원으로서 아크 램프를 사용하여 광 통로의 단부에 배치 된 카메라로 민감한 발광을 검출한다. 아웃 오브 포커스 빛이 두꺼운 샘플에 초점 정보를 가린다로이 영상 양상은 일반적으로 얇은 샘플로 제한됩니다. 공 초점 현미경은 초점 (즉, &quot;광학 절편&quot;) (28)에서 발생한 것들 위에 초점면에서 발생한 신호를 선호하는 내장되어 있다는 점에서 넓은 필드 시스템 다르다. 광학 절편을 달성하기 위해 핀홀은 점 광원에 대​​한 공액 위치에 배출 경로에 배치된다. 레이저가 광원으로 사용되며, 신호는 광전자 증 배관 튜브 (PMT가)로 검출된다. 실제로, 레이저빔을 시료 위에 포인트 별 각 스폿 (화소)에서의 형광 방출 PMT를 검출되어 와이프된다. 여기 이미지 모두 현미경 양식의 직접적인 비교를 제공하는 넓은 필드와 공 초점 현미경을 모두 사용하여 제브라 피쉬 배아 생활에 매우 동일한 세포 내 구조. 이러한 비교를 제공하는 기본 목적은 손의 특정 질문에 가장 적합한 현미경 기술 선택을위한 가이드 라인을 제공하는 것입니다. 우리는 미토콘드리아와 중심체의 GAL4-UAS 기반 유전 라벨을 보여 여기에 설명 된 방법을 사용. 이러한 소기관 각 영상 기법의 적합성을 입증 넓은 필드와 공 촛점 현미경을 사용하여 신경계와 근육 세포가 다른 세포 유형에 이미징된다. 여기에 설명 된 방법을 용이하게 살아있는 배아 제브라 다른 세포 내 소기관 및 구조를 조사하도록 구성 할 수있다.

<table><tbody><tr><td>Agarose (2-hydroxyethylagarose)</td><td>Sigma-Aldrich</td><td>A4018-10G</td><td>Low-gelling temperature Type VII</td></tr><tr><td>Block heater</td><td>Eppendorf</td><td></td><td>Thermomixer compact</td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt; Calcium nitrate hydrate, 99.996%&amp;nbsp;</td><td>Aldrich</td><td>202967-50g</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>CCD camera</td><td>Qimaging</td><td>Retiga Exi Fast 1394</td><td></td></tr><tr><td>Ceramic Coated Dumont #5 Forceps</td><td>Dumont - Fine Science Tools</td><td>11252-50</td><td>#5 Forceps</td></tr><tr><td>Confocal laser-scanning microscope</td><td>Olympus</td><td>FV1000 Fluoview</td><td></td></tr><tr><td>Culture dish heater</td><td>Warner Instrument Corporation</td><td>DH-35</td><td>Heating ring</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfate salt</td><td>PharmaQ</td><td>Tricaine PharmaQ-25g</td><td>Tricaine (anesthetic)</td></tr><tr><td>Fluorescence dissecting microscope</td><td>Leica</td><td>M205 FA</td><td></td></tr><tr><td>GeneClean kit</td><td>MP Biomedicals</td><td>111001200</td><td></td></tr><tr><td>Glass Bottom Culture Dishes</td><td>MatTek Corporation</td><td>P35G-0-14-C</td><td>35 mm petri dish, 14 mm microwell, No. 0 coverglass</td></tr><tr><td>Glass needles</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>TW100F-4</td><td>For microinjections</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-250g</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>High vacuum grease</td><td>Dow Corning&amp;nbsp;</td><td>DCC000001242 150g</td><td>Silicon dioxide grease</td></tr><tr><td>KCl 99%</td><td>Sigma-Aldrich</td><td>S7643-5kg</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O &amp;nbsp;&amp;nbsp;Magnesium sulfate heptahydrate 98+% A.C.S reagent</td><td>Sigma-Aldrich</td><td>230291-500g</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>Microinjector&amp;nbsp;</td><td>Eppendorf</td><td></td><td>FemtoJet</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>930001007</td><td>0.5-20 ul</td></tr><tr><td>Micromanipulator</td><td>Maerzhaeuser Wetzlar</td><td>MM33 Rechts/00-42-101-0000/M3301R</td><td></td></tr><tr><td>Micropipette holder</td><td>Intracel</td><td>P/N 50-00XX-130-1</td><td></td></tr><tr><td>mMESSAGE mMACHINE SP6 Transcription Kit</td><td>Ambion</td><td>AM1340</td><td>To transcribe PCS-Transposase</td></tr><tr><td>NaCl BioXtra &gt;99.5%</td><td>Sigma-Aldrich</td><td>P9541-1kg</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>Nanophotometer</td><td></td><td></td><td>To measure DNA/RNA concentration</td></tr><tr><td>Needle puller</td><td>Sutter Instrument</td><td>P-1000</td><td>Flaming/Brown</td></tr><tr><td>NIR Apo 40x/0.80W&amp;nbsp;</td><td>Nikon</td><td></td><td>Water-dipping-cone objective</td></tr><tr><td>N-Phenylthiourea Grade I, approx. 98%</td><td>Sigma</td><td>P7629-10G</td><td>PTU (prevents pigmentation)</td></tr><tr><td>Petri dishes</td><td>Sarstedt AG&amp;nbsp;</td><td>821472</td><td>92 x 16mm&amp;nbsp;</td></tr><tr><td>Plastic molds&amp;nbsp;</td><td>Adaptive Science Tools</td><td>TU-1</td><td>For microinjections</td></tr><tr><td>Plexiglas cover-with a hole</td><td>Custom-made</td><td></td><td>The hole in the Plexiglas cover should be 3 mm larger than the diameter of the water-dipping-cone objective</td></tr><tr><td>Tea-strainer (Plastic)</td><td></td><td></td><td>To collect zebrafish eggs</td></tr><tr><td>Temperature controller</td><td>Warner Instrument Corporation</td><td>TC-344B</td><td>Dual Automatic Temperature Controller</td></tr><tr><td>Transfer pipettes</td><td>Sarstedt AG&amp;nbsp;</td><td>86.1171</td><td>3.5mL plastic transfer pipettes</td></tr><tr><td>UMPlanFI 100x/1.00W</td><td>Olympus</td><td></td><td>Water-dipping-cone objective</td></tr><tr><td>UMPlanFLN 20x/0.50W&amp;nbsp;</td><td>Olympus</td><td></td><td>Water-dipping-cone objective</td></tr><tr><td>Widefield microscope</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>PTU (50x Stock)</td><td></td><td></td><td>Dissolve 76 mg PTU in 50 ml distilled water&lt;br/&gt; Stir vigorously at room temperature&amp;nbsp;&lt;br/&gt; Store at -20 &lt;sup&gt;o&lt;/sup&gt;C in 1 ml aliquots&lt;br/&gt; Use at 1x working solution</td></tr><tr><td>Tricaine (20x Stock)</td><td></td><td></td><td>Dissolve 200 mg Tricaine in 48 ml distilled water&lt;br/&gt; Add 2 ml 1M Tris base (pH9)&lt;br/&gt; Adjust to pH 7&amp;nbsp;&lt;br/&gt; Store at -20 &lt;sup&gt;o&lt;/sup&gt;C in 1 ml aliquots&lt;br/&gt; Use at 1x working solution</td></tr><tr><td>Danieau&#039;s Solution (30x Stock)</td><td></td><td></td><td>1,740 mM&amp;nbsp; NaCl&amp;nbsp;&lt;br/&gt; 21 mM&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; KCl&amp;nbsp;&lt;br/&gt; 12 mM&amp;nbsp; &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;&lt;br/&gt; 18 mM&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Ca (NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 150 mM&amp;nbsp;&amp;nbsp;&amp;nbsp; HEPES buffer&amp;nbsp;&lt;br/&gt; Distilled water upto 1 L&amp;nbsp;&lt;br/&gt; Store at 4 &lt;sup&gt;o&lt;/sup&gt;C&amp;nbsp;&lt;br/&gt; Use at 0.3x working solution</td></tr></tbody></table>

imaging subcellular structures in the living zebrafish embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

여기에 넓은 필드와 이미지 미토콘드리아와 제브라 피쉬 배아 생활의 중심체에 공 초점 현미경의 사용을 직접 비교하고 대조된다. 피검 소기관 동성이있는 셀들의 위치 및 특정 세포 내 사건의 고유 주파수에 따라 일반적으로 넓은 필드 또는 공 초점 현미경 하나가 더 좋은 선택이다. 우리는 배아의 표면 상에 위치 RB 뉴런 깊은 위치 망막 세포 소기관을 묘화. 자신의 두 ?...

Watch this Scientific Journal Video about Imaging Subcellular Structures in the Living Zebrafish Embryo at JoVE.com

Imaging Subcellular Structures in the Living Zebrafish Embryo

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

살아있는 Zebrafish의 배아에서 세포 이하 구조를 이미징

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

imaging-subcellular-structures-in-the-living-zebrafish-embryo

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

11.5K 조회수.  Technische Universität München. Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy. 

비디오: Imaging Subcellular Structures in the Living Zebrafish Embryo

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Zebrafish의 Hindbrain에 라벨 부착 및 이미징 셀

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

연구

신경과학

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

해부 현미경을 사용하여 시간 경과 영상과 Zebrafish의 신장 개발의 분석 광학 단면에 대한 장착

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

발생학

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

라이브 Zebrafish의 배아에서 유사 분열 부문 및 역학을 관찰

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Multiciliated 셀 전체 마운트 형광 제자리에서 교 잡 및 면역 형광의 결합 된 프로토콜을 통해 Zebrafish에 시각화

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

생물학

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

세포 운동성 및 Zebrafish 태아의 개별 뉴런과 신경 크레스트 세포의 굴지의 Cytoskeleton의 라이브 영상

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

<meta charset="utf8"></head><body>제브라피시 배아를 위한 개선된 Multiview Light Sheet Microscopy 프로토콜

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

<meta charset="utf8"></head><body>저자 스포트라이트: 고해상도 멀티뷰 광시트 현미경을 위한 제브라피시 배아 장착 전략 — 이미징 및 이미지 재구성 기법

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

이미징 및 배아 제브라 피쉬의 뇌 혈관 구조의 3 차원 재구성

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

살아있는 Zebrafish의 배아에서 세포 이하 구조를 이미징

요약

더 많은 비디오 탐색

이 비디오의 챕터

Zebrafish의 Hindbrain에 라벨 부착 및 이미징 셀

해부 현미경을 사용하여 시간 경과 영상과 Zebrafish의 신장 개발의 분석 광학 단면에 대한 장착

라이브 Zebrafish의 배아에서 유사 분열 부문 및 역학을 관찰

Multiciliated 셀 전체 마운트 형광 제자리에서 교 잡 및 면역 형광의 결합 된 프로토콜을 통해 Zebrafish에 시각화

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

세포 운동성 및 Zebrafish 태아의 개별 뉴런과 신경 크레스트 세포의 굴지의 Cytoskeleton의 라이브 영상

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

이미징 및 배아 제브라 피쉬의 뇌 혈관 구조의 3 차원 재구성