이 작품은 theNIH grantNS042626에 의해 지원되었다. 우리는 커트 손 및 이미징과 함께 도움을 UCSF 니콘 이미징 센터를 주셔서 감사합니다.

1. Zebrafish 태아의 준비 <ol><li> 이틀 electroporation 전에, 여성에서 남성으로 별도의 디바이더를 사용하여 야생 유형 물고기의 교배 새장을 설정합니다. </li><li> electroporation 전 어느 날, 전날의 새장을 짝짓기에 디바이더를 가져옵니다. </li><li> 배아를 수집하고 28.5에서 0.003 %의 phenylthiourea (PTU)를 포함하는 30 ML 배아 중간 ° C. 50 수정된 배아를 품어 </li><li> electroporation의 날, dechorionate 수동으로 배아는 22hpf의 발달 단계에 도달 벌금 포셉 (Inox 5 더몬트 전자, 스위스)와 배아. 다음 PTU 0.003 %를 포함 10ml electroporation 대타의 3 (180 MM NaCl, 5mM KCl, 1.8 MM CaCl2, 5 MM Hepes의 산도 7.2)로 배아를 전송합니다. </li><li> 링어이 배아를 마취 위하여 10ml의 electroporation을 420 μl tricaine 주식 (4mg/ml)를 추가합니다. </li></ol> 2. Electroporation 준비 <ol><li> 유리 사출 니들은 플라밍 - 브라운 P897 풀러 (셔터 인 스트 루먼트, 노바토, CA, 미국)에서 모세 혈관 (필라멘트와 1.2 mm OD, 0.9 mm ID가)에서 가져온되었습니다. 바늘 끝이 그림 1D와 같이 날카로운 끝을 얻으려면 포셉와 부러했다. 팁의 직경 10 μm의 주위해야하며 테이퍼 각도는 30도 근처에있을 것입니다. </li><li> 0.8 %을 준비하고 electroporation 대​​타의 솔루션에 1퍼센트 낮은 용융 점 아가로 오스 (셸턴 과학 주식 회사 카탈로그 # IB70050). 2 ML의 microcentrifuge 튜브에서 나누어지는 아가로 오스 솔루션입니다. ~ 37 ° C.에서 열 블록에 aliquots 보관 </li><li> Gal4/UAS 시스템 electroporated 태아의 신경 전구 세포의 유전자 발현을 유발하는 데 사용됩니다. 두 plasmids이 사용됩니다 : Gal4FF는 유비 쿼터스 모터의 EF1α에 의해 구동되는 플라스미드 EF1α - GFF - PT2KXIG, 그리고 플라스미드 UAS - E1B - EGFP - PT2KXIG합니다. </li><li> EF1α - GFF - PT2KXIG 및 GenEluteTM HP 플라스미드 Midiprep 키트 (시그마)를 사용하여 UAS - E1B - EGFP - PT2KXIG 플라스미드 DNA를 준비합니다. </li><li> plasmids은 다음 10 MM 트리스 · Club 호텔에서 1-2 μg / μl의 최종 농도 (PH 8.5)에 아세트산 나트륨 / 에탄올 침전에 의해 집중되고있다. </li><li> 두 plasmids를 섞어 각 플라스미드 500 NG / μl의 최종 농도 nuclease 무료 H2O로 희석. 솔루션에 페놀 레드 (10분의 1 총 볼륨을 희석)의 추가는 전달의 시각화를 선호합니다. 얼음의 DNA 솔루션을 유지. </li><li> 오른쪽 2 μl DNA 솔루션과 electroporation, backload 주사 바늘 이전 2 선배님은 사출 볼륨을 조정합니다. </li></ol> 3. Electroporation <ol><li> stero의 해부 현미경 (50x 최대 배율로 자이스 혈구 Stemi 2000), 공기 압력 주입기 (Narishige IM 300 microinjector) 두 micromanipulators (WPI M3301R, 세계 정밀 계측기, 사라소타, 플로리다, 미국), 그리고 전기 자극기 (SD9 그림 1A와 같이 사각형 펄스 자극기, 그라스 Tlefactor, 웨스트 워릭,로드 아일랜드, 미국)이 설정됩니다. </li><li> 1퍼센트 낮은 용융 점 아가로 오스의 배아, 잠그다이 배아를 마운트하려면 다음과 같이하십시오. 그런 다음 즉시 유리 피펫으로 아가로 오스의 배아를 제거합니다. </li><li> 아가로 오스의 개별 방울에 거꾸로 플라스틱 페트리 접시의 뚜껑에있는 배아를 놓습니다. 아가로 오스 너무 forebrain은 전극과 microinjection 니들 (그림 1B) 모두에 액세스할 수 굳은 전에 등의 접속 위치로 광섬유 프로브와 함께 부드럽게 배아를 동쪽을 향하게하다. 배아는 개인 아가로 오스 방울에 마운트해야합니다. </li><li> 왼쪽 micromanipulator로 전극을 조작. 그림 1B와 같이 아가로 오스에 전극을 삽입합니다. 사용자 플래티넘 이리듐 병렬 바이폴라 전극 직경 및 간격 500 μm의 125 μm의 간격은 electroporation (FHC, 카탈로그 # PBSA0575)에 사용됩니다. </li><li> 붉은 유체의 흐르는 및 뇌실의 팽창 관찰 될 수 있듯이, forebrain의 뇌실에 microinjection 바늘을 삽입하고 4nl DNA 믹스를 주입. </li><li> 바로 하나의 28.5 V 펄스 지속적인 1 밀리초는 수동으로 적용되었다. 다음 극성을 반대로 하나 이상 28.5 V 펄스 지속 1 밀리초를 적용합니다. 이것은 forebrain 신경 전구 세포의 양자 모자이크 라벨집니다. </li><li> 10 배아의 electroporation 후 배아를 공개 microsurgical 나이프로 배아를 커버하고 아가로 오스 껍질에 5ml 배아 매체를 추가하십시오. </li><li> 28.5에서 0.003 % PTU와 부화를 포함한 30 ML 배아 매체의 신선한 요리 ° C.에 electroporated 배아를 전송 </li></ol> 4. 장착 및 시간 경과 라이브 영상 <ol><li> epifluorescent 해부 현미경으로 볼 경우에는 electroporation 후 4시간 주변, EGFP 신호가 관찰됩니다. </li><li> 다섯 시간 electroporation 후 높은 모자이크하지만 forebrain 지역에서 강한 EGFP 표현으로 배아를 선택합니다. </li><li> 0.003 %의 PTU를 포함 10ml 배아 매체로 선택한 배아를 전송합니다. </li><li> 10ml 배아 매체 t에 420 μl tricaine 주식 (4mg/ml)를 추가O는 배아를 마취. </li><li> 배아를 탑재하기 위해 0.8 % 낮은 용융 점 아가로 오스 1 배아 젖어. 그런 다음 즉시 유리 피펫으로 아가로 오스의 배아를 제거하고 35mm 유리 바닥 문화 요리의 중심지 (MatTek 법인, 파트 # P35GC - 1.5-10 - C에 배아를 장소 <a href="http://www.glass-bottom-dishes.com/">www.glass - 하단 dishes.com</a> ) . </li><li> 아가로 오스는 굳은 전에 등의 접속 위치로 광섬유 프로브와 함께 부드럽게 배아를 동쪽을 향하게하다. </li><li> 그림 1C (우리가 오른쪽 목표와 니콘 C1 스펙트럼 공촛점 현미경을 사용하여)에 같이 공촛점 현미경의 온도 조절 무대에서 제대로 요리를 놓습니다. 28.5로 온도를 조절 ° C. </li><li> 3ml 28.5 추가 ° C 배아를 커버하는 PTU 0.003 %를 포함하는 preheated 배아 중간. 배아는 이제 이미지 준비가되었습니다. </li><li> 물이 마른 목표를 사용하여 고정 간격으로 시간이 경과 라이브 영상을 수행합니다. </li></ol> 5. 대표 결과 그림 2에 표시된 22 HPF에서 EF1α - GFF + UAS - E1B - EGFP의 electroporated 배의 시간이 경과 공촛점 라이브 영상의 프레임을 선택하고 있습니다. <img src="/files/ftp_upload/2594/2594fig1.jpg" alt="그림 1" /> 그림 1. 장치 셋업. (A) electroporation 설비의 조립 : 그라스 SD9 광장 펄스 자극기 (I), 왼쪽 micromanipulator (2), 전극 (3), 자이스 혈구가 현미경을 해부 (IV), 사출 바늘 (V)가, 오른쪽 micromanipulator (VI)과 Narishige IM 300 microinjector (VII). (B) 전극은 (i), forebrain 뇌실 (II) 및 사출 바늘 (3)의 상대 위치. 붉은 색의 DNA 솔루션 forebrain의 뇌실로 주입되어 있습니다. (C) 니콘 C1 공촛점 현미경에 살고 이미지의 조립 : 온도 컨트롤러 (I), 객관적인 렌 (2)와 유리 바닥 문화 요리 (3) (D에서 낮은 용해 지점 아가로 오스에 포함된 electroporated 배아 디핑 물 의 DNA microinjection에 뽑아 및 절단 바늘의) 이미지. <img src="/files/ftp_upload/2594/2594fig2.jpg" alt="그림 2" /> 그림 2. 22 HPF에서 EF1α - GFF + UAS - E1B - EGFP와 electroporated 배아의 18 시간 시간이 경과 공촛점 라이브 이미지의 선택된 프레임. 뇌실의 표면은 각각의 이미지 프레임의 하단 측면에 있습니다. 스케일 바는 10 μm의를 나타냅니다.

<ol>
	<li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stem+cells.">The development of neural stem cells.</a> Nature. 414, 112-117 (2001).</li><li>G#246;tz, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+neurogenesis.">The cell biology of neurogenesis.</a> Nat. Rev. Mol. Cell Biol. 6, 777-788 (2005).</li><li>Cerda, G. A., Thomas, J. E., Allende, M. L., Karlstrom, R. O., Palma, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+DNA,+RNA,+and+morpholinos+into+zebrafish+embryos.">Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.</a> Methods. 39, 207-211 (2006).</li><li>Hendricks, M., Jesuthasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+methods+for+in+vivo,+whole+mount+and+primary+culture+analysis+of+zebrafish+brain+development.">Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development.</a> Neural. Dev. 2, 6-6 (2007).</li><li>Tawk, M., Bianco, I. H., Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+electroporation+in+zebrafish+embryos+and+larvae.">Focal electroporation in zebrafish embryos and larvae.</a> Methods Mol. Biol. 546, 145-151 (2009).</li><li>Tawk, M., Tuil, D., Torrente, Y., Vriz, S., Paulin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+adult+fish:+a+new+tool+to+study+fin+regeneration.">High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.</a> Genesis. 32, 27-31 (2002).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 비디오에서는, 우리는 개발 zebrafish의 forebrain에서 clonal 수준에서 신경 전구 세포의 시간 경과 라이브 영상에 대한 방법을 보여줍니다. 우리는 유전자와 찬란 라벨 개별 신경 전구 세포에 테스트 기존의 electroporation 프로토콜에게 4-6을 수정했습니다. 단 몇 세포가 띄엄띄엄 electroporation의 상대적인 낮은 전압으로 표시 이후 clonally 관련 자손 세포로 구성된 혈통의 나무가 설정할 수 있습?...

time-lapse live imaging of clonally related neural progenitor cells in the developing zebrafish forebrain

분할, 마이 그 레이션 및 신경 전구 세포의 분화의 정확한 패턴을 적절한 두뇌 개발과 기능 1,2에 중요한 있습니다. 생체내 환경에서 복잡한에서 신경 전구 세포의 동작을 이해하기 위해서는 두뇌에 손상 신경 전구 세포의 시간 경과 라이브 영상이 매우 필요합니다. 이 비디오에서는, 우리는 생체내에 forebrain 신경 전구 세포의 발전을 시각화하는 zebrafish 배아의 고유한 기능을 이용. 우리는 유전자 및 띄엄띄엄 라벨 개별 신경 전구 세포에 electroporation을 사용합니다. 간단히, DNA가 형광 표식에 대한 코딩 구조 22 시간 게시물 수정 (HPF) zebrafish 배아의 전기 펄스가 즉시 배달되었고,의 forebrain의 뇌실로 주입했다. 여섯 시간 후, electroporated zebrafish의 배아는 유리 바닥 문화 요리 낮은 용융 점 아가로 오스와 함께 장착했다. 찬란 표시 신경 전구 세포는 다음 물 수영 객관적인 렌즈를 사용하여 공촛점 현미경 고정 간격 36시간 위해 몇 군데 있었다. 현재의 방법은 forebrain 신경 전구 세포의 생체내 개발에 대한 통찰력을 얻을 수있는 방법을 제공하고 zebrafish 배아의 중추 신경 시스템의 다른 부분에 적용할 수 있습니다.

Watch this Scientific Journal Video about Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain at JoVE.com

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

현재 비디오는 개발 zebrafish의 forebrain에서 개별 신경 전구 세포에 대한 라이브 영상을 수행할 수 electroporation과 공촛점 현미경의 조합을 활용하는 방법을 보여줍니다.<em&gt; 생체내에서</emclonal 수준 forebrain 신경 전구 세포의 개발&gt; 분석이 방법으로 얻을 수 있습니다.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

time-lapse-live-imaging-clonally-related-neural-progenitor-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.2K 조회수.  University of California San Francisco. 현재 비디오는 개발 zebrafish의 forebrain에서 개별 신경 전구 세포에 대한 라이브 영상을 수행할 수 electroporation과 공촛점 현미경의 조합을 활용하는 방법을 보여줍니다. 생체내에서 분석이 방법으로 얻을 수 있습니다.

비디오: Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

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

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

실시간으로 개발을 시각화 하기 위해 다중 광자 시간 경과 영상: 마이그레이션 Zebrafish 태아 신경 크레스트 셀의 시각화

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

발생학

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 ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

무지개와 타임랩스 공초점 이미징을 사용하여 살아있는 제브라피시의 뇌 발달 시각화

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

트렌스 제닉 Zebrafish의 여러 가지 빛깔의 시간 저속 영상 : 타겟의 연결을 세포 절제에 의해 활성화 떠올리 망막 줄기 세포

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

영상 모니터 세포 생물학과 여러 신경 인구의 계보 진행을 추적 하는 단일 셀 다음 라이브

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D 공 초점 현미경을 사용하여 제브라 피쉬의 두개 안면 형태 형성 분석

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

제브라피쉬 광학컵 모르포발생4차원 이미징

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

두개골과 피부 제거에 의해 깨어 얼룩말 물고기 애벌레와 청소년에서 완전히 활성 뇌 조직의 생체 내 이미징

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...