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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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(NO3)2 Calcium nitrate hydrate, 99.996%&#160;</td>
			<td>Aldrich</td>
			<td>202967-50g</td>
			<td>To prepare 30x Danieau&#39;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>Fluka Analytical</td>
			<td>A5040-100G</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>35mm petri dish, 14mm microwell, No. 0 coverglass</td>
		</tr>
		<tr>
			<td>Glass needles</td>
			<td>World Precision Instruments Inc.&#160;</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&#39;s</td>
		</tr>
		<tr>
			<td>High vacuum grease</td>
			<td>Dow Corning&#160;</td>
			<td>DCC000001242 150g</td>
			<td>Silicon dioxide grease</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>Heraeus</td>
			<td>To maintain zebrafish embryos at 28.5&#8304; C</td>
		</tr>
		<tr>
			<td>KCl 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S7643-5kg</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>MgSO4.7H2O &#160;&#160;Magnesium sulfate heptahydrate 98+% A.C.S reagent</td>
			<td>Sigma-Aldrich</td>
			<td>230291-500g</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>Microinjector&#160;</td>
			<td>Eppendorf</td>
			<td></td>
			<td>FemtoJet</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>0.5-20uL</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 &#62;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1kg</td>
			<td>To prepare 30x Danieau&#39;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&#160;</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&#160;</td>
			<td>821472</td>
			<td>92 x 16mm&#160;</td>
		</tr>
		<tr>
			<td>Plastic molds&#160;</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&#160;</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&#160;</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 
			Stir vigorously at room temperature&#160; 
			Store at -20 oC in 1 ml aliquots 
			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 
			Add 2 ml 1M Tris base (pH9) 
			Adjust to pH 7&#160; 
			Store at -20 oC in 1 ml aliquots 
			Use at 1x working solution</td>
		</tr>
		<tr>
			<td>Danieau&#39;s Solution (30x Stock)</td>
			<td></td>
			<td></td>
			<td>1740 mM&#160; NaCl&#160; 
			&#60;21 mM&#160;&#160;&#160;&#160;&#160; KCl&#160; 
			12 mM&#160; &#160;&#160;&#160;&#160;MgSO4.7H2O&#160; 
			18 mM&#160;&#160;&#160;&#160;&#160; Ca (NO3)2 
			150 mM&#160;&#160;&#160; HEPES buffer&#160; 
			Distilled water upto 1 L&#160; 
			Store at 4 oC&#160; 
			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

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

광 변환 한 후 형광 붕괴를 사용하여 생활 Zebrafish의 태아의 단백질 안정성을 측정 (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

연구

발생학

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Zebrafish의 배아에 주입 마이크로 비드

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

에반스 블루 염료와 Zebrafish의 애벌레 골격 근육 무결성 분석

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Photoconvertible PSmOrange 시스템을 사용하여 GFP 유전자 변형 Zebrafish의 세포를 추적

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

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 ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

살아있는 개구리와 Zebrafish 태아의 저산소증 유도

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

분석, 동적, 안정과 초기 Microtubules Zebrafish 태아에 있는 Immunolabeling의 사용

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

AFM와 Microrheology Zebrafish 태아 노른자위 셀에

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

생활 그대로 Zebrafish에 단일 셀 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Zebrafish 태아에 있는 세포 Photoconversion 통해 다음 Endocardial 조직 움직임

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...