We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

이 프로토콜은 독일과 베를린 주법의 동물 보호 지침을 다음과; 제브라 피쉬의 처리는 동물 보호 (LaGeSo, 베를린 - 브란 덴 부르그)에 대한 지방 자치 단체에 의해 모니터링 하였다. 1. 심장 추출을위한 Zebrafish의 배아를 얻기 <ol><li> 형질 전환 라인 9 twu34 마음 별 GFP 발현과 배아를 얻기 위해 : 같은 Tg는 (EGFP myl7)와 같은 크로스 심장 기자 제브라 피쉬. </li><li> 원하는 배아 단계 10, 11까지 28.5 ° C에서 계란 물 배아를 유지한다. 수집 된 배아 인구는 유전자 발현의 변화를 제한하는 두 유전자와 발달 단계에 의해 균일 있는지 확인하십시오. </li><li> 수동으로 배아를 Dechorionate. </li></ol> 2. 해부 Zebrafish의 배아 마음 참고 : 얼음의 모든 솔루션을 보관하십시오. 가능하면, 팀워크를 수행 한 사람이 EMB에서 마음을 해부한다ryos 다른 사람이 형광 실체 현미경 마음을 정렬한다. 이 작업은 몇 시간에서 배아의 수백의 처리를 할 수 있습니다. <ol><li> 1.5 ㎖의 원심 분리 관에 약 100 배아를 전송합니다. tricaine와 배아 (E3​​ 매체에 0.16 ㎎ / ㎖)을 마취. 배아가 명확하게 마취 일단 시험 (그들은 수영 튜브의 바닥에 침전물, 그러나 그들의 마음은 여전히​​ 치고있다하지 않음). </li><li> tricaine / E3 솔루션을 제거하고 1 ml의 L-15 / 10 % FBS 배지로 한 번 배아를 씻어 얼음에 유지한다. 참고 : L-15 / 10 % FBS 매체는 마음 RNAlater 또는 트리 졸에 전송 될 때까지 전체 해부 과정에서 살아 장기와 세포를 유지하는 것이 중요합니다. </li><li> 노른자가 완전히 중단 될 때까지 라운드 젤로드 팁과 위아래로 배아 및 피펫에 5-8 시간을 1 ml의 L-15 / 10 % FBS 매체를 추가합니다. 배아 드롭 현명한 용액의 표면에, 드롭 버스트 수 있도록 피펫배아는 부드럽게 중단된다. 참고 : 최대 피펫 아래로, 즉 더 피펫 팅이 후반 단계 (예를 들어 56 HPF에서)에 필요, 태아의 발달 단계에 따라하는 방법을 적극적으로 얼마나 자주 배아가있다. </li><li> 너무 부드럽게 피펫 경우 어떤 마음이 배아에 부착 남아있을 수 있기 때문에 해부 배아의 첫 번째 일괄 들어, 배아의 무결성과 실체 현미경을 해부 마음의 비율을 평가합니다. 이에 따라 절개의 다음 라운드 피펫의 방식을 조정합니다. 참고 : 플라스틱에 집착 마음을 최소화하기 위해 낮은 유지 피펫 팁과 마이크로 원심 튜브를 사용합니다. </li><li> 50 ㎖ 원심 분리 튜브에 배치 된 100 ㎛ 필터 상에 시료를 적용; 1ml의 L-15 / 10 % FBS 배지를 1.5 ml의 튜브 헹구어 낸 후 (일부 하트 튜브의 벽에 달라 붙을 수있는) 샘플의 손실을 최소화하기 위해 필터에 이것을 적용한다. 1 ml의 L과 필터를 2 회 세척-15 / 10 % FBS. 이 단계에서는 마음이 필터를 통과하고 50 ML 튜브에 수집됩니다. </li><li> 15 ml의 원심 분리 튜브에 배치 된 30 μm의 필터로 관류를 적용하고 1 ㎖의 L-15 / 10 % FBS 배지로 한번 필터를 헹군다. 이 초 여과 공정에서, 하트 필터에 보유되고, 작은 먼지는 세척된다. </li><li> 거꾸로 필터를 켜고 1 ml의 L-15 / 10 % FBS와 세 개의 연속 세척을 적용하여 1 % 아가로 오스 코팅 된 배양 접시에 필터 중 마음을 세척하십시오. </li><li> 수동 형광 실체 현미경 포셉 쌍 비 형광 배아 파편 (예 : 아이 렌즈)로부터 GFP 양성 하트 분리 접시의 중앙에이를 농축시킨다. 3 단계에 따라이를 수집합니다. 참고 : 배아 나이가 24 HPF 경우, 마음, 구타 조직이 아직도 생리 학적으로 정상을 나타내는해야한다. </li></ol> 3. Dissec에서 mRNA의 격리를테드 마음 <ol><li> (얼음) 0.75 ㎖의 RNAlater를 포함하는 1.5 ML 튜브에 가장 작은 부피의 마음 (예를 들어 10 μL)과 펫을 수집합니다. 더 마음 형광 실체 현미경을 보면서 피펫 팁에 남아 있는지 확인합니다. </li><li> 대안 적으로, 화학적 후드 형광 현미경의 바로 근처에서 사용할 경우, 화학 후드 트리 졸 (주)에 수집 된 하트 옮긴다. 이 피펫 팁과 또한 마음의 원심 분리 동안 고집 마음의 가능한 손실을 회피 (3.4, 3.5 및 3.7 단계). 트리 졸와 같은 튜브에 격리 몇 차례의 마음을 풀 3.8 단계로 바로 이동; 최종 부피는 트리 졸 원래 부피의 10 %를 초과하지 않아야한다. 참고 : 사용하기 전에 트리 졸 물질 안전 보건 자료 (MSDS)를 참조하십시오. 후드 아래 트리 졸 시약을 처리하고 개인 보호 장비를 권장 착용하십시오. 피부, 눈, 및 clothi과의 접촉을 피할 것NG. 모든 점화원을 제거 할 것. </li><li> RNA 분리의 효율이 수집 된 조직의 양의 향상으로, (얼음) RNAlater와 같은 튜브에 격리 몇 차례의 마음을 풀. 총 샘플 부피 RNAlater 원래 부피의 10 %를 초과하는 경우, (얼음) 저장 RNAlater 0.75 ㎖를 추가로 관을 사용한다. </li><li> 마음을 침전 4 ° C에서 20 분 동안 15,700 XG에서 샘플을 원심 분리기. </li><li> 형광 실체 현미경을 이용하여, 3 ㎖ L-15 / 10 % FBS 배지를 함유하는 1 % 아가로 코팅 된 배양 접시에 최대한 신중만큼 상등액을 수집하지만, 원심 분리 튜브 바닥에 침강 마음을 방해하지 않는다. 마음의 최대 15 % 인해 RNAlater의 높은 점도에 뜨는에 남아있을 수 있기 때문에 단계 3.7에 설명 된대로, 그들을 검색 할 수 있습니다. </li><li> 화학 후드에서 침전 마음이 들어있는 튜브에 0.5 ml의 트리 졸을 추가하고 얼음에 보관하십시오. </li><li> 형광 microsco에서PE, 희석 3 ㎖ L-15 / 10 % FBS와 다른 1 % 아가 로스 코팅 접시에 RNAlater / L-15 / 10 % FBS 용액을 함유하는 1 % 아가 로스 코팅 접시에서 (단계 3.5)에서 하트 전송할 RNAlater 아웃. 페트리 접시의 중앙에 마음을 수집하고 화학 후드 트리 졸에 침전 된 마음의 튜브에 작은 볼륨을 전송합니다. </li><li> 소용돌이는 트리 졸에 마음을 포함하는 1.5 ML 튜브는 마음에 혼란을 실온에서 5 분 동안 품어합니다. </li><li> 화학 후드에서 100 μl의 클로로포름 (주의)를 추가, 철저하게 혼합하고 (사용하기 전에 최대 속도로 원심 분리기 30 초)를 1.5 ml의로 미리 회전 PLG 튜브를 트리 졸 / 클로로포름 용액을 전송합니다. 실온에서 3 분 동안 배양한다. 참고 : 사용하기 전에 클로로포름 MSDS를 참조하십시오. 후드 클로로포름 시약을 처리하고 개인 보호 장비를 권장 착용하십시오. 피부, 눈, 및 의복에 접촉하지 않도록 할 것. 알칼리 금속과 멀리 혼합 위험성에서 보관하십시오. </li><li> CentrifuGE의 4 ° C에서 15 분 동안 15,700 XG에 샘플 및 새로운 1.5 ML 튜브로 수성 단계를 전송합니다. </li><li> 5 ~ 10 μg의 글리코겐과 이소프로판올 (-20 ° C에서) 예비 냉각 25​​0 μl를 추가; 잘 혼합 및 -20 ° C에서 밤새 품어. </li><li> 4 ° C에서 30 분 동안 15,700 XG에서 샘플을 원심 분리기 상층 액을 버린다. </li><li> 4 ° C에서 15 분 동안 15,700 XG에 1 ml의 75 % 에탄올, 원심 분리기와 펠렛을 세척하고 상층 액을 제거한다. </li><li> 그것은 (예를 들면 10 ~ 15 분 동안)의 백색이 될 때까지 상온에서 펠렛을 건조하여 에탄올을 증발하자. </li><li> 펠렛 20 μL RNA 분해 효소가없는 DDH 2 O를 추가하고 RNA를 용해 55 ° C에서 10 ~ 15 분 동안 배양; 다음 얼음에 보관하십시오. </li><li> 흡광도를 측정하여 RNA 농도와 순도를 평가합니다. 1 % 아가 로스 겔상에서 샘플을 실행하여 RNA 무결성을 평가. </li></ol>

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The authors have nothing to disclose.

이 프로토콜은 유전자 발현 용 제브라 피쉬 배아 심장 조직의 신속한 분석을 보충한다. RNA 정제 할수있는 충분한 작동하기 때문에, 하트 손실 프로토콜의 모든 단계에서 방지 할 경우에는 먼저, 시료의 수량이 크게 향상된다 : 심장 특이 RNA 샘플의 양과 질을 크게 몇 결정적인 단계에 따라 출발 물질. 둘째, 실험에 전적으로 의존하는 샘플의 순도는, 정렬 및 엄격하게 다른 조직을 제외한 동안 배양...

제브라 피쉬 (다니오 레 리오 (rerio))는 널리 때문에 작은 크기와 형광 단백질의 조직 특이 적 발현과 유전자 변형 기자 라인의 가용성과 결합의 빠르고 투명하고 자궁 외 배아 발달로 생체 내에서 기관 형성을 연구하기 위해 발달 생물학에서 사용된다. 이 작은 척추 동물은 특히 초기 제브라 피쉬 배아의 산소가 심장 박동, 혈액 흐름에 의존하지 않기 때문에 심장 개발을 연구하기에 적합하다; 이러한 기능은 심장 혈관 돌연변이 1,2 많은 수의 특성을 허용하고 제브라 피쉬는 이제 심장 질환 (3) 연구 널리 알려진 모델 생물이다. 배아 개발하는 동안 유전자 발현을 연구하기 위해, 성적 증명서는 일반적으로 전체 마운트 현장 하이브리드에 (WISH) 4 분석, RT-qPCR에 5, 마이크로 어레이 (6), 또는 차세대 시퀀싱 (RNA-SEQ) 7. 동안WISH 전체 배아 내의 유전자 발현의 시공간적 분석, 전사 레벨은 일반적으로 RT-qPCR에, 마이크로 어레이 또는 RNA-SEQ 접근법에 의해 평가되어 있습니다. 그러나 이러한 방법은 특정 유전자 발현 프로파일 링에 대한 조직 농축이 필요합니다. 제브라 피쉬 배아 심장이 전체 배아의 작은 부분을 나타 내기 때문에, 심장 개발하는 동안 사체 연구는 해부과 마음의 농축을위한 프로토콜이 필요합니다. 또한, 생리 학적으로 관련 데이터를 얻기 위해, 그것은 RNA 추출까지 완전히 기능 심장 조직을 유지하는 것이 중요하다. 여기서 우리는 신속하게 생리 학적으로 정상 분리하고 효율적으로 추가 분석을위한 고품질의 RNA 샘플을 얻기 위해 제브라 피쉬 배아의 수백 마음을 구타하기위한 프로토콜을 설명합니다. 본 발명의 방법은 화상 및 맥레이 2006 년 8 의해보고 된 프로토콜을 기반으로합니다. 심장 조직의 농축의 경우, 두 가지 방법은 형질 전환 심근 기자 행을 사용하여및 다른 조직에 비해 제브라 피쉬 배아 마음의 차동 접착 특성을 활용. 간단히, 좁은 피펫 팁을 통해 아래로 많은 배아를 피펫으로, 마음이 동시에 배아의 몸에서 해제되고 순차적으로 두 개의 빠른 여과 단계에서 배아 파편 분리; 형광 표지 된 마음은 수동으로 남아있는 파편에서 분류 및 추가 처리를 위해 수집됩니다.

<table><tbody><tr><td>Equipment for raising fish and collecting eggs&amp;nbsp;</td><td></td><td></td><td>see the Zebrafish Book&lt;sup&gt;11&lt;/sup&gt; for details</td></tr><tr><td>Fluorescence stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Refrigerated Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>UV-Spectrophotometer</td><td></td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; Thermo Scientific Nanodrop 2000</td></tr><tr><td>Nucleic acid electrophoresis chamber</td><td></td><td></td><td></td></tr><tr><td>Petri dishes 4 cm &amp;Oslash;, coated with 1% agarose in E3 medium</td><td></td><td></td><td></td></tr><tr><td>Micropipettes and tips (P20, P100, P1000)</td><td></td><td></td><td></td></tr><tr><td>1.5 ml centrifugation tubes</td><td></td><td></td><td></td></tr><tr><td>15 ml and 50 ml centrifugation tubes</td><td></td><td></td><td></td></tr><tr><td>Pair of Dumont #5 forceps</td><td></td><td></td><td></td></tr><tr><td>ExactaCruz&amp;trade; Round Gel Loading Tips in Sterile Rack, 1-200&amp;mu;l&amp;nbsp;</td><td>Santa Cruz</td><td>sc-201732</td><td></td></tr><tr><td>100 &amp;mu;m filter (BD Falcon 100 mm Cell Strainer)</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>30 &amp;mu;m filter (Pre-Separation Filters-30 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>Phase lock gel, heavy, 1.5 ml tubes&amp;nbsp;</td><td>Prime</td><td>2302810</td><td></td></tr><tr><td>Egg water medium</td><td></td><td></td><td>60 &amp;mu;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td></tr><tr><td>E3 medium&amp;nbsp;</td><td></td><td></td><td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Tricaine (3-amino benzoic acidethylester)</td><td>Sigma-Aldrich</td><td>A-5040</td><td>4 mg/ml Tricaine stock solution, pH 7</td></tr><tr><td>1% agarose (in E3 medium)</td><td></td><td></td><td></td></tr><tr><td>1% agarose gel (in TBE buffer)</td><td></td><td></td><td></td></tr><tr><td>Leibovitz&amp;acute;s L-15 medium&amp;nbsp;</td><td>Gibco</td><td>21083-027</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>RNAlater</td><td>Ambion</td><td>AM7020</td><td></td></tr><tr><td>Trizol</td><td>Ambion</td><td>1559606</td><td></td></tr><tr><td>Glycogen&amp;nbsp;</td><td>Invitrogen</td><td>10814-010</td><td>20 &amp;micro;g/&amp;micro;l in RNase-free water</td></tr><tr><td>chloroform</td><td></td><td></td><td></td></tr><tr><td>isopropanol</td><td></td><td></td><td></td></tr><tr><td>75% ethanol (in DEPC-ddH2O)</td><td></td><td></td><td></td></tr><tr><td>Nuclease-free water or sterilized DEPC treated ddH2O</td><td></td><td></td><td></td></tr><tr><td>Nucleic acid loading buffer</td><td></td><td></td><td></td></tr><tr><td>TBE (Tris/Borate/EDTA) buffer for electrophoresis</td><td></td><td></td><td></td></tr></tbody></table>

large-scale zebrafish embryonic heart dissection for transcriptional analysis

제브라 피쉬 배아 심장은 전체 배아의 작은 부분을 나타내는, 몇백 세포로 구성된다. 따라서, 글로벌 배아 사체에 의해 마스크되는 심장 사체를 방지하기 위해, 상기 분석 하트 충분한 수를 수집 할 필요가있다. 제브라 피쉬 심장 개발이 급속히 진행 더욱이, 심장 수집 및 RNA 추출 방법은 샘플의 균일 성을 확보하기 위해서는 신속한 있어야한다. 여기서 우리는 제브라 피쉬 배아에서 기능 / 꺾고 마음을 수집하기위한 빠른 매뉴얼 해부 프로토콜을 제시한다. 이것은 전 사체 분석에서는 이들 정량적 실시간 중합 효소 연쇄 반응 (RT-qPCR에)와 같은 심장 특이 유전자 발현 수준을 결정하기 위해 후속 심장 특이 RNA 추출을위한 필수 조건이다. 방법은 다른 조직에 비해 제브라 피쉬 배아 심장의 접착 성 차이에 기초한다; 이것은 빠른 PHY 허용유체 전단력 중단, 단계적 여과 및 형질 전환 형광 표지 된 마음의 설명서 모음의 조합에 의해 심장 외 조직에서 심장의 이외에도 클래식 분리.

독점적으로 심근. (그림 1)에서 twu34 형질 전환 라인 (9), 녹색 형광 단백질 (GFP)을 표현한다 : 여기서 우리는 제브라 피쉬의 Tg (GFP myl7)를 사용하여 대표적인 심장 해부 실험을 설명합니다. 우리는 해부 하트 그들이 심장 시료의 순도를 평가하기 위해 유도 된 배아로부터 모두 모았다. 간단히, 동형 접합의 Tg (myl7 : GFP) twu34 제브라 피쉬 (9)는 ...

Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

제브라 피쉬의 심장 개발하는 동안 심장 유전자 발현 프로파일을 분석하기 위해, 총 RNA는 고립 된 마음에서 추출해야합니다. 여기서 우리는 심장 별의 mRNA를 얻기 위해 제브라 피쉬 배아에서 빠른 설명서를 해부하여 기능 / 구타 마음을 수집하기위한 프로토콜을 제시한다.

전사 분석을위한 대규모 Zebrafish의 배아 심장의 해부

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

large-scale-zebrafish-embryonic-heart-dissection-for-transcriptional

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

14.3K 조회수.  Max Delbrück Center for Molecular Medicine.  제브라 피쉬의 심장 개발하는 동안 심장 유전자 발현 프로파일을 분석하기 위해, 총 RNA는 고립 된 마음에서 추출해야합니다. 여기서 우리는 심장 별의 mRNA를 얻기 위해 제브라 피쉬 배아에서 빠른 설명서를 해부하여 기능 / 구타 마음을 수집하기위한 프로토콜을 제시한다. 

비디오: Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

분리 및 Zebrafish의 배아에서 단일 세포의 특성

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

연구

발생학

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

애벌레 Zebrafish의 생식소 조직의 해부

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

심근 기계 변형의 변위 분석 (DIAMOND) 배아 제브라피시에 있는 심장 기능의 분할 이질성을 제시합니다

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

개발 Zebrafish 마음에 전단 응력을 조절의 효과의 4 차원 이미지를 캡처 빛 시트 형광 현미경 검사 법

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

생체공학

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

애벌레의 심장 해부, 소년과 성인 Zebrafish, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

생물학

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

해부 Zebrafish 배아 심장의 Immunostaining

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

얼룩말 물고기 배아에서 빠른 심장 역학의 빛 시트 현미경 검사

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9 매개 제브라피쉬 하트의 정확한 녹인 편집

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...