Name: in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences
Uploaded: 2017-03-22T15:00:00.000+00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

이 작품은 (PO 1440 / 1-1) LP하는 PRJ에 ARC DECRA (DE120101311)에 의해과 독일 연구 협회 (DFG)의 연구 보조금에 의해 지원되었다. 호주 재생 의학 연구소는 빅토리아 주 정부와 호주 연방 정부의 자금 지원됩니다. 우리는 케이 등의 알에 게시 여기에 기술 된 실험을 실시 박사 제레미 잉 치 케이를 인정합니다. 2016 년 우리는 교수 Higashijima에서 형질 전환 어류의 규정에 대한 감사 및 PROFS 감사합니다. H2B-RFP 및 H2A-GFP 구조를 생성하기위한 PCS2 + 플라스미드 박사 윌킨슨의 제공을위한 터너와 렆. 우리는 우리의 동물을 돌보는에 대한 FishCore 시설 직원 (모나 쉬 대학) 감사합니다.

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The authors have nothing to disclose.

효율적 특정 포스트 - 유사 분열 세포 유형 또는 패터닝 된 조직으로 분화하는 배아 또는 유도 된 다 능성 줄기 세포를 지시하는 것을 목표로 할 때 넓이가있는 이웃하는 셀은 중요한 세포의 운명 결정 인자의 발현의 타이밍에 영향을 이해하는 것이 필수적이다. 또한, 살아있는 동물의 개발 세포에서 이러한 분자 이벤트를 검사하면 추가로 관련 동적 생체 내에서 이러한 이벤트와 관련된 특...

<html> 생체 내 척추 동물의 중요한 발달 과정을 시각화하는 능력은 정상과 질병 조건을 공부 제브라 피쉬에게 키 모델을 만들기에 기여하고있다 (1 검토, 2). 특히, 신경 망막은 중추 신경계의 접근 가능한 부분이다. 망막 인해 고도로 조직 아직 비교적 간단한 구조 및 척추 동물 종 3에서 고도로 보존 된 신경 세포 유형에 쉽게 신경의 연구를 수행하기 위해 빌려 준다. 이러한 증식, 세포주기 출구, 비대칭 세포 분열, 운명 사양, 차별화 및 신경 회로의 형성과 같은 세포 행동의 역학은 3 일의 postfertilization에 의해 제브라 피쉬의 중심 망막에 완료 retinogenesis의 전체 과정에 걸쳐 올 수 있습니다 ( DPF) 4, 5,REF &quot;&gt; (6, 7). 또한, 전술 한 각 단계에서 상이한 유전자의 기능적 요구 병용 단 후 부검시에 평가 될 수있는 유전자 녹아웃 기술의 적용으로 인한 표현형 다른 척추 동물 모델에 비해 이점을 제공 제브라 망막 평가할 수있다 의 조직을 고정. 특히, 시각화 및 망막 리포터 유전자 형광 단백질의 발현을 모니터 할 수있는 트랜스 제닉 라인의 사용은 우리가 특정 신경 세포 유형 기원의 기초의 유전자 발현의 시간 해상도를 얻을 수있다. 인해 지브라 피쉬의 급속한 발전으로, 이러한 이벤트는 따라서 신경 세포 식별 획득 및 셀의 동작과 관련하여 유전자 발현의 시간 중요도에 깊은 통찰력을 제공하여 전체 개발 기간을 시각화 할 수있다. 마지막으로,이 방법은 유전자 기능의 두 가지 주요 형태로 통계 결과 이식을 통해 키메라의 생성과 제브라 효율적으로 결합 될 수있다. 우선, 그들이 비 표지 야생형 환경에서 개발하면서 특정 유전자를 넉다운시킨 도너 배아에서 이식 세포 검사, 세포 자율 방식으로 유전자 기능에 대한 관련 정보를 얻을 수있게 해준다. 따라서, 그들은 일반적으로 더 이상 유전자 -4,6-에서 기능성 단백질을 생성 할 수 없다 전구 세포의 발달 운명 결과의 시험을들 수있다.이 표현되는 전구 세포 내 망막 운명 결정 인자의 기능에 관한 중요한 통계 리드 8,9. 이 방법을 활용하여, 우리는 많은 운명의 결정 요인 (예를 들어, Vsx1, Atoh7, Ptf1a, Barhl2가) 세포의 역할을하는 것으로 나타났습니다-autonomously 특정 망막 신경 세포의 운명을 구동하는 단계; 유전자 발현의 부족은 주로 유전자 녹다운 세포가 다른 세포 운명 4, 6, 7, 10을 채용하여 생존 유지되도록 운명 스위치로 이끈다. 둘째, 이러한 키메라 실험은 서로 다른 유전 적 환경에서 개발할 때 형 선조 동작 방식 야생 평가하는데 사용될 수있다. 예를 들어, 일반적으로 조작 된 호스트 환경 (예를 들어, 유전자 녹아웃 / 최저) 대 WT 관심 (리포터 유전자)의 유전자를 발현 WT 세포의 발달을 비교하여, 유전자 발현 및 세포 운명 얻어진 효과를 평가할 수있다 . 호스트 환경의 특정 신경 세포 유형의 부재가 예를 들어, 소수 O로 분화 향해 바이어스 그들 셀 비 자율 방식으로 야생형 전구 동작에 영향을 미치는 것으로 밝혀졌다신경 세포 타입 4, 7, 11, 12을 r에 누락. 망막 신경 세포가 (13에서 검토) 특정 신경 세포 운명 결정하는 유전자의 순차적 초과 식 (운명의 유전자 발현)에 의해 보존 histogenic 위해 태어난 것을 감안할 때, 우리는 입증하기 위해 이러한 방법을 사용하는 방법 야생형 전구 세포의 운명 유전자 발현의 타이밍 이러한 전구 세포가 유도 된 비정상적인 세포 조성 망막 호스트 환경에서 개발할 때 영향을받습니다. 비교적 널리 사용되는 표준 및 기술의 조합은 망막 전구 세포를 8, 9 개발 운명 유전자 발현의 타이밍의 시험을 가능하게하는 방법을 여기에서는 증거 이러한 방법을 설명합니다. 이 프로토콜은 당의 용이성으로 시간 경과 영상을 결합하는 실험 방법을 설명합니다전 생체 개발 제브라 피쉬 배아에 이식을 형성하는 개별 mosaically에게 발달 retinogenesis의 전체 기간 동안 표지 세포를 수행합니다. 기능 유전자 조작을 수행함으로써 어느 호스트 배아 도너 배아, 또는 둘 모두에서 하나의 유전자 기능의 셀 자율성을 평가할 수있다. 이 방법은 여기에 설명 된 개별 구성 요소가 적합있는 다른 시스템에서 유사한 연구 질문에 널리 적용 할 수 있습니다.

<table><tbody><tr><td>Agarose</td><td>Bioline</td><td>BIO-41025</td><td>Agarose coated dishes can be prepared a few days prior, sealed with parafilm to prevent drying and stored at 4 &amp;#176;C</td></tr><tr><td>Agarose low melt</td><td>Sigma</td><td>A9414-100G</td><td>Can be prepared in larger volume, microwaved to liquify and then kept molten in a 40 &amp;#176;C water-bath. 1 mL aliquots can be prepared (one for each group of embryos mounted).</td></tr><tr><td>borosillicate glass capillary tube w/o filament</td><td>SDR Scientfic</td><td>30-0035</td><td>alternatives with similar diameter can be used&amp;nbsp;</td></tr><tr><td>borosillicate glass capillary tube with filament</td><td>SDR Scientfic</td><td>30-0038</td><td>alternatives with similar diameter can be used&amp;nbsp;</td></tr><tr><td>glass petri dishes (60 mm diameter)</td><td>Science Supply</td><td>1070506</td><td>any glass alternative of any size</td></tr><tr><td>Injection tube (2 m)</td><td>Eppendorf</td><td>524616004</td><td>This connects the needle holder to the syringe during transplantation</td></tr><tr><td>Microinjection mold (plastic mold with wedge-shaped protrusions)</td><td>Adaptive Science Tools</td><td>PT-1</td><td>alternatives with similar shape can be use</td></tr><tr><td>microneedle holder</td><td>Narishige</td><td>M-152</td><td>any alternative that fits the outside diameter of the injection needles can be used</td></tr><tr><td>microinjector</td><td>Narishige or Eppendorf Femtojet</td><td></td><td>Pneumatic injector using gas pressure</td></tr><tr><td>micromanipulator</td><td>Coherent Scientific</td><td>M330IR</td><td>similar alternatives can be used</td></tr><tr><td>microloader tip</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M5904-500ML</td><td></td></tr><tr><td>needle puller</td><td>Sutter Instruments</td><td>Model P-2000</td><td>Settings used for this puller are: H 430, Fil 4, Vel 50, Del 225, Pul 75. Any needle puller can be used if it results in appropriate tip dimension</td></tr><tr><td>Parafilm</td><td>Interpath</td><td>PM996</td><td>any paraffin film</td></tr><tr><td>Pasteur pipette plastic</td><td>Samco Scientific</td><td>202</td><td></td></tr><tr><td>Pasteur pipette glass</td><td>Hirschmann Laborgeraete</td><td>9260101</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>P5942-25MG</td><td>&amp;nbsp;Protease Type XIV</td></tr><tr><td>N-phenyl thiourea</td><td>Sigma</td><td>P7629-25G</td><td>PTU is toxic and can be prepared as a stock solution to avoid frequent exposure to the powder, consult MSDS before purchase/use.</td></tr><tr><td>Qiaquick gel extraction</td><td>Qiagen</td><td>28704</td><td>any alternatives to purify DNA can be used</td></tr><tr><td>Rneasy Mini kit</td><td>Qiagen</td><td>74104</td><td>any alternatives to purify RNA can be used</td></tr><tr><td>SP6 mMessage kit</td><td>Qiagen</td><td>AM1340</td><td>any alternatives to transcribe DNA using SP6</td></tr></tbody></table>

in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences

유전 적 및 기술적 장점은 제브라 척추 유전자 조작의 결과가 급속한 발달 기간 동안 생체 내에서 추적 될 수있는 키 모델 생물 만들었다. 다중 처리는 세포 증식, 유전자 발현, 세포 이동 및 형태 형성을 포함하여 연구 될 수있다. 중요한 것은, 이식을 통해 키메라의 생성을 용이하게 호스트 환경의 영향하에 모자이크 표시와 개별 세포의 추적을 가능하게 수행 될 수있다. 예를 들어, 개별 이식 공여자 세포 (모르 폴리 마이크로 인젝션을 통해 예를 들어) 호스트 배아 기능 유전자 조작 및 라이브 영상, 외부의 영향 (유 전적으로 변형 환경에 의해 제공되는) 셀 비 자립적 신호를 조합하여 평가 될 수있다. 여기서 우리는이 방식 세포 운명 determin의 타이밍에 대한 프록시로서 형광 유전자 발현의 개시를 비교하기 위해 사용되는 방법을 보여다른 유전 호스트 환경에서 ATION. 이 글에서 공여 세포를 표시 유전자 호스트 배아 최저 키메라 배아를 생성하는 데 사용되는 이식 기술을 설명하고, 제조 및 생체 저속 촛점에서 실행을위한 프로토콜을 일으킬 제브라 피쉬 배아 마이크로 인젝션을위한 프로토콜을 제공 여러 개의 배아의 영상. 이러한 유전자 발현의 개시에 따라 이벤트의 타이밍을 비교하면 특히 다단 촬상을 행하는 것은 매우 중요하다. 이것은 다수의 제어와 동시에 처리 실험 배아에서 데이터 수집이 필요합니다. 이러한 접근 방식은 쉽게 장기 나 영상을 살 액세스 선택의 조직에서 외부의 영향에 대한 연구를 위해 확장 될 수있다, 이식이 이루어 배아 운명의지도에 따라 쉽게 대상이 될 수 있음을 제공했다.

이 작품은 야생형 망막 전구 세포가 morphant 호스트 배아 내에서 개발할 때 유전자 발현 타이밍의 변화를 평가하기 위해 실험 프로토콜을 제시한다. 실험 호스트 배아 망막 7, 26의 중간 태어난 수평 및 무 축삭의 interneurons 부족 Ptf1a의 morphants이다. 이러한 표준 제어 모르 주사 하였다 호스트 배아를 제어하기 위해 비교 하였다. ...

Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

서로 다른 유전 적 배경에서 개별 WT 망막 전구 세포의 실시간 추적은 신경 동안 셀이 아닌 자율적 인 신호의 기여도의 평가를 할 수 있습니다. 여기서, 배아 이식을 통한 생체 저속 촛점 촬상 유전자 녹다운, 키메라 세대의 조합이 목적을 위해 사용 하였다.

키메라 Zebrafish의 태아의 개별 망막 전구 세포의 형질 전환 유전자 발현의 생체 내 이미징에서 세포 비 자립적 영향을 연구하기 위해

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

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Research

JoVE Journal

Developmental Biology

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

7.6K 조회수.  The University of Melbourne.  서로 다른 유전 적 배경에서 개별 WT 망막 전구 세포의 실시간 추적은 신경 동안 셀이 아닌 자율적 인 신호의 기여도의 평가를 할 수 있습니다. 여기서, 배아 이식을 통한 생체 저속 촛점 촬상 유전자 녹다운, 키메라 세대의 조합이 목적을 위해 사용 하였다. 

비디오: In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

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.

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 ...

연구

발생학

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

분석<em&gt; 생체</em&gt; 셀 마이그레이션 Zebrafish의 배아에서 세포 이식 및 시간 경과 이미징을 사용하여

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

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

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

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 ...

신경과학

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

다중 광자 현미경으로 라이브 셀 이미징을위한 성인 형질 전환 Zebrafish의 망막의 Explants의 문화

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

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 ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

조직 타겟 배아 Chimeras : Zebrafish Gastrula 세포 이식

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

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 ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

의 이식 GFP - 표현 키메라 Zebrafish의 태아의 망막과 두뇌 개발의 라이브 영상에 대한 Blastomeres을

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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 ...

<meta charset="utf8"></head><body>재생 및 개발 연구를 위한 강력한 제브라피시 세포 이식

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>저자 스포트라이트: 발달 및 재생 중 제브라피시 신경계의 세포 연결성 매핑

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

배아 및 유충 제브라피시에서의 고분해능 세포 이식

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...