Name: visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish
Uploaded: 2016-12-21T13:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

S843 : LI1과의 Tg (EGFP kdrl) : 우리는 Tg가 (GFP wt1b)를 증여에 대한 교수 크리스토프 Englert 교수 디디에 Stainier 감사합니다 균주 각각과의 Tg (kdrl을 : mCherry) 제공하는 대만 Zebrafish의 핵심 시설 C15- 카르을. 이 연구는 102-, 과학 기술 (96-2628-B-029-002-MY3, 101-2313-B-029-001, 102-2628-B-029-002-MY3 대만의 정부에서 보조금에 의해 지원되었다 2321-B-400-018).

윤리 정책 : 제브라 피쉬의 모든 실험 절차는 동해 대학의 기관 동물 케어 및 사용위원회에 의해 승인되었습니다 (IRB 승인 NO 101-12합니다.) 및 승인 된 지침에 따라 수행 하였다. 3β-HSD 효소 활성 염색 1. 재고 솔루션 <ol><li> 트랜스 dehydroandrosterone [디메틸 술폭 사이드 (DMSO)에 10 ㎎ / ㎖]를 준비한다. </li><li> β - 니코틴 아미드 아데닌 디 뉴클레오티드 수화물 (0.1 M 인산염 완충액에서 1.2 ㎎ / ㎖, pH를 7.2)을 준비한다. </li><li> 니코틴 준비 (비타민 B3,을 50mg / ㎖의 H 2 O). </li><li> 4- 니트로 블루 테트라 졸륨 (50 ㎎ / ㎖ 70 % 디메틸 포름 아미드)를 준비한다. </li><li> -20 ° C에서의 미세 원심 분리 튜브에 나누어 저장하는 모든 이러한 구성 요소. 참고 : 우리의 경험, 반복 동결 모든 상기 용액의 분취 량의 해동에서 3β-HSD 활동 염색의 강도에 영향을주지 않습니다. </li></ol> 2. W홀 마운트 3β-HSD 활동 염색 참고 : 3β-HSD 효소 활성이 빠르면 28시간 게시물 수정 (HPF)와 같은 경우 배양 hsd3b1 2,7의 mRNA 발현의 발병과 일관성이 28.5 ° C,에서 제브라 피쉬 배아에서 감지 할 수 있습니다. 이 연구에서 전체 마운트 3β-HSD 활성 염색 프로토콜은 전술 한 방법 (8)에 기초하고 최대 7 일 후 시비 9 현상 제브라 성공적인 것으로 측정되었다. interrenal steroidogenic 조직의 혈관 미세 환경을 분석, 3β-HSD 활동 염색을 전체 마운트하는 것은 이러한 Tg는 같은 내피 세포 고유의 형광을 발현하는 형질 전환 라인 (kdrl : EGFP)에 적용해야합니다은 : C15- 카르 (15) (14)과의 Tg (mCherry kdrl를) S843 . 제브라 신장 개발을 고려 interrenal steroidogenic 조직을 분석, 3β-HSD 활성 염색 될 수 애플리LI1 물고기 (16), 사구체 및 pronephric 세관의 형성이 묘사되어 Tg가 (GFP wt1b)에 에디션. <ol><li> 색소 형성을 억제하기 위해, 12, 24 HPF 사이의 단계에서 시작, 달걀 물에 0.03 %의 페닐 티오 요소 (Phenylthiourea)에서 (탈 이온수에 0.06 ㎎ / ℓ 암초 소금)을 배양하여 개발 제브라 피쉬를 취급합니다. </li><li> 밤새 실온 (RT) 또는 (B) 2 % PFAT에서 1 시간 동안 0.1 % 트윈 20 (PFAT)으로 보충 된 인산염 완충 식염수 (PBS)에서 (A) 4 % 파라 포름 알데히드 (PFA)에 dechorionated 배아 또는 유충 해결 4 °의 C 또는 실온에서 4 시간 뒤에 4 ° C에서 2 시간에 대해. 주의 : PFA는 흡입 및 피부 접촉에 의해 독성. 그것은 피부, 점막, 눈, 상부 호흡기에 파괴. </li><li> PBS는 RT에서 0.1 % 트윈 20 (PBST)가 보충 10 분간 배아 4X 씻는다. 주 : 세탁 솔루션을 변경하면서 샘플을 건조하지에 중요합니다. </li><li> 갓 역을 준비반응 전에 ining 용액 (표 1). 준비, 소용돌이, 분리 튜브에서 원심 분리 부 A 및 B. 참고 : 바로 하나의 튜브에 모든 구성 요소를 추가하면 심한 침전을 일으킬 것입니다. </li><li> 부분 (B)의 전체 용액 A 부분의 전체 용액을 첨가 염색 용액을 제조 잘 혼합하고 즉시 고정 세정 배아를 포함하는 각 미세 원심 분리 튜브에 1 ㎖에 배포한다. </li><li> 회 전자 또는 실온에서 진탕 부드러운위한 락 플랫폼 중 하나에 빛과 장소에서 그들을 보호하기 위해 알루미늄 호일로의 microfuge 튜브를 감싸. </li><li> 경험적으로 현미경을 통해 발색 신호를 모니터링함으로써, 반응 시간을 결정한다. 시간이 지남에 따라 개발한다 약간의 침전; 그러나, 이들은 반응 과정을 방해하지 않을 것이다. 참고 : 배아가 4 시간 동안 25 ° C에서 28 HPF, 염색 고정을 위해 interrenal 조직에서 명확한 신호를 생성한다. </li><li> PBST에서 10 분 동안 4 배를 세척하여 반응을 정지. 나는F 더 이상 면역 조직 화학 (IHC) 분석이 필요하지 않습니다, 이후 수정 실온에서 60 분 동안 4 % PFAT와 스테인드 배아를. 그렇지 않으면 더 사용할 때까지 4 ° C에서 세척 된 배아를 저장합니다. </li><li> 전체 마운트 현미경 들어, PBS에 50 % 글리세롤로, 염색 후 고정, 세척 배아를 취소 슬라이드에이 위를 향하도록 몸 윗면로 방향을, 밝은 필드 조명 아래 직립 현미경을 이용하여 관찰한다. 참고 : 권장 interrenal 조직 형태의 고해상도 이미지의 3β-HSD 활동 염색 및 deyolked 배아의 분석을 평면 마운트 확인하십시오. interrenal steroidogenic 조직이 바로 난황 위에 위치로, interrenal 조직의 복부 평면 마운트 분석은 interrenal 형태 (17)의 직접적이고 명확한 관찰 할 수 있습니다. </li></ol> 3β-HSD 활동 묻은 태아의 3 IHC 분석 주 : steroi의 혈관 미세 환경 분석을위한dogenic 조직은 형질 전환 내피 특정 형광 기자와 3β-HSD 활동 묻은 배아는 vibratome 절편 이후 IHC 분석 12, 18, 20, 22를 횡단하기 위해 실시한다. <ol><li> 후 고정 <ol><li> 4 ° C에서 1 시간 동안 1 % 트리톤 X-100 보충 2 % PFA와 3β-HSD 활동 염색 및 세척 배아를 포스트 - 수정 1 % 트리톤 X-100을 포함하는 PBS에서 10 분 동안 배를 씻어 (PBSTx) RT에서. </li></ol></li><li> 퍼가기 <ol><li> 4 %가 95 ° C, 4-8 ℃에서의 microfuge 튜브로 나누어지는 및 매장에서 PBS에서 아가로 오스를 용해하여 아가 로스 저 융점 준비합니다. </li><li> 10 분 동안 70 ° C에서 아가로 오스 4 % 저 융점의 나누어지는 용융 한 후 47 ° C에서 나누어지는을 유지합니다. 각각의 배아를 포함 용융 4 % 금형 역할을하는 1.5 ml의 미세 원심 분리 튜브의 분리 된 모자 아가로 오스 저 융점, 고체까지 냉각 할 수 있습니다. </li></ol></li><li> Vibratome 단면 <ol><li> 건조 페이지에 종이 테이프의 조각 스틱vibratome의 latform. </li><li> 면도날에 의해 금형에서 경화 아가로 오스 블록을 제거합니다. 플랫폼에 종이 테이프에 순간 접착제를 적용하고 위를 향 샘플의 앞쪽면, 종이 테이프에 아가로 오스 블록을 수정합니다. 각각 약 4mm 상부 및 하부면 양쪽에 8mm로, 사다리꼴 프리즘 형상 아가 블록 낸다. </li><li> 스포이드를 사용하여 0.5 % 트리톤 X-100 (0.5 % PBSTx) 보충 차가운 PBS로 샘플을 함유 아가로 오스 블록을 씻어. </li><li> vibratome의 사용 설명서에 따라 100 μm의 섹션으로 아가로 오스 블록을 슬라이스. 계속해서 건조를 방지하는 아가 블록을 헹군다. 각 슬라이스 절단되기 때문에, 26 G 주사기 바늘을 이용하여 vibratome에서 제거하고, 차가운 0.5 % PBSTx (500 μL / 웰)를 포함하는 스폿 플레이트에 옮긴다. </li><li> 해부 현미경 하에서 검사하여 3β-HSD 활성 양성 조직 절편을 수집하고, PI에 의한 부분 전송추운 0.5 % PBSTx (150 μL / 웰)를 포함하는 96 웰 플레이트에 끝 차단 (개방에서 약 1mm 내경)와 pette 팁. 조직 샘플은 통상적으로이 공정 후의 아가 블록으로부터 해리. </li></ol></li><li> Permeabilization 및 세척 <ol><li> PBS에서 10 분, 1 % 소 혈청 알부민 / 1 % DMSO / 0.1 % 트리톤 X-100 (PBDTx)로 보충을위한 슬라이스 RT에서 0.5 % PBSTx 15 분간 PBS에서 10 분, 4 배 6 배 씻는다. </li></ol></li><li> 블로킹 <ol><li> RT에서 1 시간 PBDTx 2 % 소 태아 혈청 (FCS)을위한 슬라이스 부화. </li></ol></li><li> 차 항체 반응 <ol><li> 차 항체를 함유하는 PBDTx 1.5 일 동안 인큐베이션 슬라이스 (예를 들어 토끼 다 클론 항 - 인간 피브로넥틴 및 마우스 항 - 인간 국소 부착 키나아제) 4 ℃에서. </li></ol></li><li> 세탁 <ol><li> 조각은 실온에서 PBDTx에서 10 분 동안 6 배 씻으십시오. </li></ol></li><li> 블로킹 <ol><li> PB에 2 % FCS에서 1 시간 동안 조각을 품어RT에서 DTX. </li></ol></li><li> 차 항체 반응 <ol><li> 4 ° C에서 차 항체 (형광 결합 염소 항 - 토끼 또는 항 - 마우스 IgG)를 포함 PBDTx 1.5 일 동안 조각을 품어. </li></ol></li><li> 세탁 <ol><li> 조각은 실온에서 PBST에서 10 분 동안 10 RT에서 PBDTx에서 분 및 4 배에 대한 6 배 씻으십시오. , 공 초점 현미경 분석을 위해 PBS에 50 % 글리세롤로 샘플을 취소 : 참고. </li></ol></li></ol> 4. 공 촛점 분석 <ol><li> 전체 마운트 배아 및 10X / 0.5 20X / 0.75 대물 렌즈가 장착 된 공 초점 현미경으로 단면을 관찰한다. </li><li> 다음 형광 및 3β-HSD 염색을 동시에 검출 들어, 멀티 모드를 설정 488nm의 아르곤 레이저 및 GFP의 검출 505-530 nm의 대역 통과 필터; 543 nm의 헬륨 - 네온 레이저 mCherry 감응 560-615 nm의 대역 통과 필터; 및 3β-HSD의 얼룩의 검출을위한 광 채널 전송된다.핀홀 크기는 1 Airey 부이고, 해상도가 1024 X 1024 픽셀이다. </li><li> 3 차원으로 투영 (6 μm의 간격)의 Z 스택을 조립하고 이용함으로써, 돌기와 시야 병합 내장 촛점 소프트웨어. </li></ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Proulx, K., Lu, A., Sumanas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+vasculature+in+zebrafish+forms+by+angioblast+cluster-derived+angiogenesis.">Cranial vasculature in zebrafish forms by angioblast cluster-derived angiogenesis.</a> Dev Biol. 348, 34-46 (2010).</li><li>Perner, B., Englert, C., Bollig, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wilms+tumor+genes+wt1a+and+wt1b+control+different+steps+during+formation+of+the+zebrafish+pronephros.">The Wilms tumor genes wt1a and wt1b control different steps during formation of the zebrafish pronephros.</a> Dev Biol. 309, 87-96 (2007).</li><li>Chou, C. W., Chiu, C. H., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+mediates+correct+positioning+of+the+interrenal+organ+in+zebrafish.">Fibronectin mediates correct positioning of the interrenal organ in zebrafish.</a> Dev Dyn. 242, 432-443 (2013).</li><li>Chiu, C. H., Chou, C. W., Takada, S., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+fibronectin+signaling+requirements+of+the+zebrafish+interrenal+vessel.">Development and fibronectin signaling requirements of the zebrafish interrenal vessel.</a> PLoS One. 7, e43040 (2012).</li></ol>

The authors have no competing financial interests to declare.

3β-HSD 활동의 신호 강도는 반응의 과정을 통해 증가. 3β-HSD 활동의 명확한 신호는 이후 28 HPF에서 단계에 대한 반응 4 시간 후에 발견되었다. 그러나, 반응 시간은 분석의 목적에 따라 경험적 결정이 필요하다. 염색 밤새 처리 요구의 경우, 약간 엷은 배경 샘플을 개발하는 경향이있다. 이 문제는 HSD-3β 활성 염색 전에 고정 지속 시간 (예를 들어, RT에서 4 % PFAT에서 4 시간)의 증가에 의해 극복 ?...

부신은 시상 하부 - 뇌하수체 - 부신 축의 중요한 구성 요소, 스테로이드를 분비하고 스테로이드 항상성과 스트레스에 대한 신체 반응을 조정한다. 부신은 카테콜아민을 합성 ㄱ 영역 별 방식으로 스테로이드를 분비 바깥 쪽 피질, 및 내부 수질를 포함한다. 경골 어류의 interrenal 선은 포유 동물의 부신의 상대이며, 부신 피질과 수질, 각각 1-3의 기능적 등가물이다 steroidogenic interrenal과 크롬 친화 세포로 구성되어있다. 제브라 피쉬 모델을 사용하여 수행 연구는 모두 steroidogenic 및 크롬 친화 세포의 계보가 매우 포유류 1, 2에서와 유사한 분자 및 세포 메커니즘에 의해 형성되는 것으로보고있다. 따라서, 제브라 피쉬는 유전 질환, 신경 내분비 조절하고, 시상 하부 - 뇌하수체 - 부신 (interrenal) 축 시스템 생물학 연구를위한 잠재적으로 강력한 모델이다. 4,5에서 17α-hydroxypregnelolone에서 프레 그네 놀론, 17α-hydroxyprogesterone에서 프로게스테론의 변환 및 안드로 스텐 디온을 촉매. 3β-HSD 호르몬 스테로이드, 즉 황체 호르몬, 글루코 코르티코이드, 무기질 코르티코이드, 안드로겐과 에스트로겐의 모든 클래스를 생합성을 위해 필수적이다. 두 사람의 3β-HSD는 HSD3B1을 동질 효소와 HSD3B2는 차동 (6) 표현된다. HSD3B2가 신피질 생식선에서 발현되는 반면 HSD3B1는 태반 및 말초 조직에서 발현된다. 인간 HSD3B1 및 HSD3B2는 interrenal 조직과 성인 생식선에서 표현되는 제브라 피쉬 hsd3b1, 공동 orthologs이다; 제브라 피쉬 hsd3b2 누구의 성적 기관 형성 칠하기 전에 사라질 모체 표현 유전자이다. 제브라 피쉬의 전체 마운트 3β-HSD 효소 활성 분석의 프로토콜, 레비의 방법을 수정하여 개발 된밀라노 등의 알에 의해 설명 s의. 여덟 경골 종 (8)의 동결 절편에. 때문에 조직 투과성 현상 지브라 피쉬의 광학 투명성, 3β-HSD 조직 화학 성공적 고정 제브라 배아와 유충에 사용할 수있는 전체 마운트 구체적 차별화 interrenal 조직을 서술. 이 민감하고 신속한 분석은 interrenal dysmorphogenesis의 다른 유형을 보여주는 다양한 돌연변이 morphants 적용되었다. interrenal 3β-HSD 활성은 interrenal 조직 명세가 Ff1b 전사 인자의 특정 녹다운을 중단하고 interrenal 분화가 Ff1b의 coregulator Prox1 9,10-의 최저 의해 영향으로 감소 된 배아 부재. 특히, 3β-HSD 활동과 같은 하나 아이드 핀 머리 심한 초기 결손 돌연변이 체에서 검출 될 수 있고, 여기서 H-3β, 곁눈질SD 조직 화학은 interrenal 세포 이동 (11)를 어떻게 영향을 받는지 묘사. interrenal 조직의 분화에도 혈액과 혈관의 완전한 부재에 손상되지 않습니다. 따라서, 내피 세포 유래 신호를 형성하는 방법을 개발 interrenal 기관은 12, 13를 측정 할 수있다. 전반적으로,이 조직 화학적 분석은 성공적으로 제브라 피쉬 모델의 사양, 차별화, 그리고 steroidogenic 세포의 이동을 연구에 사용되었다. 따라서, 효율적이고 부신 및 interrenal 장기 장애를 타겟팅하는 유전자 또는 화학 스크린에 대한 신뢰할 수있는 도구를해야한다.

<table><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></td></tr></tbody></table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

pronephric 신장 사구체 및 초기 혈관과 steroidogenic interrenal 조직 codevelops에서, 3β-HSD 효소 활성 분석은 이중 형질 전환의 Tg (wt1b : GFP)에서 수행 된 방법을 결정하려면 LI1, Tg는 (kdrl : mCherry) C15- 카르 배아를 34 HPF에서 (그림 1). 이 단계에서, 3β-HSD 활동을 양성 steroidogenic 조직을 바로 중간 선에 위치하고 있으며 일부 steroidogenic 세포가 중?...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

제브라 피쉬의 interrenal 선은 포유 동물의 부신의 teleostean 대응이다. 효소 활성 분석, 개발 제브라 피쉬에서 차별화 된 steroidogenic 세포를 감지,이 프로토콜은 3-β-하이드 록시 탈수소 효소 (3β-HSD Δ 5-4 이성화 효소)를 수행하는 방법을 소개합니다.

Interrenal Steroidogenic 조직 및 제브라 피쉬의 혈관 미세 시각화

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.9K 조회수.  Tunghai University.  제브라 피쉬의 interrenal 선은 포유 동물의 부신의 teleostean 대응이다. 효소 활성 분석, 개발 제브라 피쉬에서 차별화 된 steroidogenic 세포를 감지,이 프로토콜은 3-β-하이드 록시 탈수소 효소 (3β-HSD Δ 5-4 이성화 효소)를 수행하는 방법을 소개합니다. 

비디오: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

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

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

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

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

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

연구

발생학

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

전체 마운트 Zebrafish의 태아의 섬모 기관에서 면역 염색 포스 - 항원 결정기

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

화학

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 ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

제브라피시 종양 이종이식에서 숙주 혈관조형성 반응의 생체 내 이미징 및 정량화

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

암 연구학

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

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

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

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

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

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

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

성인 Zebrafish 부상 모델 뼈 조직 재생에 Prednisolone의 효과 연구

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

브라이트필드 현미경을 사용하여 애벌레 제브라피시의 간 크기 정량화

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

성인 Zebrafish의 신장의 해부

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

생물학

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

물고기는 눈을 통해 당뇨병을 공부: 기정, 시각화, 및 성인 tg(fli:EGFP) Zebrafish 망막 맥 관 구조 분석

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

의학

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

전체 마운트하여 제브라 피쉬 배아 시편 스테인드 관찰 및 분석을위한 평면 마운트 준비<em&gt; 현장에서</em&gt; 하이브리드

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...