The authors thank Drs. Hukriede and Tsang for discussions. M.K. was supported by an NIH training grant (T32EB001026). This work was supported in part by grants from the American Liver Foundation, the March of Dimes Foundation (5-FY12-39), and the NIH (DK101426) to D.S.

Zebrafish의 표준 절차에 따라 제기 자란; 실험은 피츠버그 기관 동물 관리 및 사용위원회의 대학에 의해 승인되었습니다. 배아 / 애벌레 1. 준비 <ol><li> 시간 제한 교배, 설정 성인 남성과 여성의 Tg (fabp10a : CFP-NTR) 수행 S931 hemizygous 또는 동형 접합체 물고기 O / N과 그들 사이에 디바이더를 배치합니다. 상대가 요구 될 때이 분할에게 다음 아침을 제거합니다. 미세 플라스틱 메쉬 스트레이너를 사용하여 물을 긴장에 의해 배아를 수집합니다. </li><li> 거꾸로 스트레이너를 켜고 스퀴즈 병에서 분배 계란 물 좋은 스트림과 메쉬 표면을 씻어. 계란 물 25 ㎖로 100mm 배양 접시에 100 배아를 전송합니다. 페트리 접시 당 최대 100 배아를 유지하지 않고 28 ℃에서을 올립니다. </li><li> 색소 침착을 억제하기 위해, 10 시간 포스트 수정 (HPF) 이전에 배양 접시에 페닐 티오 (PTU)를 추가, 전자를 제기80 HPF까지 mbryos / 애벌레. PTU의 최종 농도는 0.2 mm이다. 주의 : PTU 독성이다. 적절한 처리 지침에 따라 사용합니다. </li><li> 배양 접시에, 0.016 %의 최종 농도에서 3- 아미노 벤조산 에틸 에스터 (Tricaine)을 첨가하여 애벌레를 마취. 그런 다음, 표면 형광 현미경 종류 CFP 양성 유충을 사용. 비슷한 간 크기의 애벌레를 사용하여 작거나 큰 간을 가진 사람을 버린다. 주 : hemizygous 동형 접합체 유충 사이 간세포 박리의 정도에 차이가 없기 때문에 어떤 CFP 양성 유충 관계없이 강도를 사용할 수있다. 주의 : tricaine 독성이다. 적절한 처리 지침에 따라 사용합니다. </li><li> Tricaine을 포함하는 계란 물을 제거하고 0.2 mM의 PTU 보충 계란 물을 추가합니다. 28 ° C에서 배아 / 애벌레를 유지합니다. </li></ol> 2. MTZ 치료 <ol><li> 1 MTZ 분말 0.171 g을 용해하여 신선한 10 mM의 MTZ 솔루션을 준비00 ml의 달걀 물은 0.2 % DMSO 0.2 mM의 PTU 보충. 완전히 MTZ를 해산하기 위해 20 ~ 30 분 동안 신속하게 교반하면서 실온에서 솔루션을 섞는다. MTZ를 준비 할 때 MTZ을 용해하기 위해 및 유충에 MTZ 투과을 향상시키기 위해, DMSO를 추가합니다. 주의 : 장기간 노출 또는 MTZ의 증가 농도는 독성이다. 적절한 처리 지침에 따라 사용합니다. </li><li> 두 개의 서로 다른 100mm 페트리 접시 또는 멀티 웰 플레이트에 유충의 원하는 번호를 전송하여 제어 및 실험 그룹으로 유충을 분리합니다. 실험군의 경우, 10 mM의 MTZ 용액 유충을 유지; 대조군을 위해, 0.2 % DMSO를 함유하는 달걀 물에 보관. </li><li> 플레이트 또는 MTZ의 광 불 활성화를 막기 위해 알루미늄 호일로 MTZ 용액을 함유하는 페트리 접시를 커버하고, 28 ° C에서 배양한다. MTZ 치료 기간은 애벌레 단계 및 형질 전환 라인에 의존한다. </li><li> 비슷한 심한 간세포 절제를 관찰하려면 <em은&gt;의 Tg (fabp10a : CFP-NTR) 라인은 (36 시간의 기간 동안) 또는 5 DPF에서 (24 시간의 기간 동안) 중 3.5 일 이후 수정 (DPF)에서 MTZ 솔루션 유충을 취급합니다. </li></ol> 담즙 중심의 간 재생 3. 분석 <ol><li> 절제 기간의 끝에서, 판에서 MTZ 용액을 제거한다. 계란 물 25 ㎖를 추가하여 MTZ 처리 유충을 씻으십시오. 계란 물을 폐기하기 전에 유충을 씻어 판을 2 ~ 3 회 소용돌이 친다. 세 번 반복 한 후 다시 한 번 달걀 물에 애벌레를 체험. 유충 고정화, 0.008 %의 최종 농도에서 Tricaine의 낮은 농도를 추가 MTZ 처리 된 유충 심장 부종 및 유생 사망을 방지한다. </li><li> 간 기능 분석을 위해, CFP 필터 표면 형광 현미경 MTZ 처리 된 유충의 간 크기를 조사한다. 간 상대적인 크기에 기초하여 간세포 박리 수준을 평가 : 대 (비 절제 0-10 %의 유충), 중간 (부분적으로 절제하는 단계; 10-20 %), 및 완전히 절제 (매우 작은; 80~90%). </li><li> 비 절제된 또는 부분적으로 절제 간와 유충을 제거합니다. 만 심각한 간세포 절제 나타내는 작은 간으로 유충을 유지합니다. 재생 0 시간 시점 (R0H)에서 간 분석을 위해, MTZ의 세척 및 CFP 정렬 후 유충을 수확. 그렇지 않으면, 수확에 대한 준비가 될 때까지 0.2 mM의 PTU 보충 달걀 물에 정렬 된 유충을 유지합니다. </li><li> 1.5 ml의 microcentrifuge 관에 수확 유충을 전송하고 PEM 버퍼에 3 %의 포름 알데히드의 고정 솔루션 계란 물을 교체합니다. 회 전자의 O / N에서 4 ° C의에 튜브를 품어. </li><li> PBSDT 1 ㎖​​로 고정 솔루션을 교체하고 5 분 동안 회전에 애벌레가 들어있는 튜브를 회전. </li><li> 수동으로 해부 현미경 노른자와 가슴 지느러미를 제거, 100mm 페트리 접시 PBSDT를 포함하고 집게를 사용으로 고정 된 유충을 전송합니다. 1.5 ML 튜브에 20 해부 유충 TP까지 전송합니다. </li> &lt;리&gt; 차단 솔루션 1 ml의 PBSDT 솔루션을 교체하고 2 시간 동안 실온에서 회 전자에 튜브를 바위. </li><li> 일차 항체를 함유 용액을 차단 100 ㎕로 차단 솔루션 교체합니다. 천천히 회전의 O / N에서 4 ° C의에 튜브를 바위. </li><li> 10 분마다 5 회 차 항체 용액을 제거하고 PBSDT 씻어. 그런 다음 차 항체를 포함하고 2 시간 동안 실온에서 회 전자에 바위 차단 솔루션의 100 UL를 추가합니다. </li><li> 회 전자에 흔들어 실온에서 10 분마다에 대한 PBSDT 5 회 반복한다. </li><li> 현미경 슬라이드에 옆으로 유충의 방향과 설치 미디어의 한 방울을 추가합니다. 조심스럽게 커버 유리로 커버 및 손톱 광택제와 커버 글래스를 밀봉. 지금, 그것은 공 초점 현미경에 대한 준비가되어 있습니다. </li></ol>

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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

심각한하지만 온화한하지, 간세포 절제는 담즙의 중심 간 재생을 유도한다. CFP-NTR 트랜스 : 따라서 MTZ 처리 및 세척을 다음, 그것은 fabp10a에서 극한 CFP 형광에 의해 평가 될 수 MTZ 처리 된 유충의 간 크기를 조사하는 것이 중요하다. 절제된 비 (0~5%) 또는 부분 (10 내지 20 %의) 간 절제를 표시 MTZ 처리 애벌레의 적은 비율 때문에, 소트 및 매우 작은 간으로 유충을 분석하는 것이 필수적이?...

간은 고도로 회생 기관이다. 간 재생 중에 간세포를 재생, 간 실질 세포, 간세포 선재 (간세포 중심 간 재생) 또는 BECS (담도 중심 간 재생) -1,2-로부터 유도된다. 간 손상은 일반적으로 기존의 간세포의 증식을 유도한다; 간세포 증식가 손상된 경우에는, BECS 2-4 간세포에 기여할 수있다. 간 재생이 두 모드는 임상 적으로 중요하다. (인해 간 종양 또는 라이브 간 도너의 예), 인간 간 부분의 수술 제거하여 남은 간세포가 간에서 간 질량 손실을 복구하기 위해 증식. BECS 간 또는 전구 세포 (된 LPC)는 간세포의 재생에 기여하는 5,6- 나타나도록 반대로, 중증 간 질환 환자에서, 간세포 증식이 크게 손상된다. 설치류 2/3 부분 절제술 모델있는간세포가 잃어버린 간 질량을 복구 할 증식, 크게 간세포 중심의 간 재생 7,8의 현재 이해에 기여하고있다. 그러나, 재생 간세포가 주로 BECS에서 파생되는 유효한 설치류 모델이 없습니다. 여러 설치류 간 독소 모델 담즙 중심의 간 재생 2-4의 식별을 주도하지만, 생쥐에서 최근 혈통 추적 연구는 이러한 모델 9,10에서 간세포 재생에 BECS 최소한의 기여를 나타냅니다. 부분 간절 11-13 및 아세트 아미노펜 유도 간 손상을 포함 14,15 쥐 간 손상 모델의 일부는 제브라 피쉬인가 간 재생에 관여하는 유전자 또는 신규 경로의 식별을 주도하고있다. 그러나 간세포가 주도하지만, BEC-구동되지, 간 재생이 제브라 피쉬의 간 손상 모델에서 발생합니다. 따라서, 신규의 간 손상 모델되는 BECS 광범위 regenerat 기여간세포를 보내고하는 BEC 중심의 간 재생의 더 나은 이해를 위해 필요하다. 간세포 절제 모델의 전체적인 목적은 (1) BECS 광범위 재생 간세포에 기여하고, (2) BEC 중심 간 재생을 기본 분자 세포 메커니즘을 규명 된 간 손상 모델을 생성하기 위해 여기에 설명. 우리는 부상의 정도가 간 재생의 모드를 결정한다는 가정; 따라서, 우리는 담즙 중심의 간 재생이 심한 간세포 손상에 시작할 것이라고 예측했다. 이 가설을 테스트하기 위해, 우리는 유전자 변형 라인을 생성하여 제브라 피쉬의 간 손상 모델을 개발, Tg는 (fabp10a : CFP-NTR) S931, 즉 높은 간세포 특이 fabp10a에서 시안 형광 단백질 (CFP)와 융합 박테리아 Nitroreductase (NTR)를 표현 발기인. NTR은 세포 독성 약물에 비 독성 전구 약물, 메트로니다졸 (MTZ)를 변환하기 때문에, 단지 의도 절제한다 NTR-이 경우에는, 세포를 16-18을 발현, 간세포. MTZ 치료의 지속 시간을 조작함으로써, 간세포 박리의 정도를 제어 할 수있다. 이 모델을 사용하여, 우리는 최근 심한 간세포 손실시 BECS 광범위 또 다른 두 개의 독립적 인 연구에 의해 확인 하였다 (20, 21)의 재생 간세포 (19)을 야기한다는보고. 따라서, 상기 설치류 및 지브라 피쉬 간 손상 모델에 비해, 우리 간세포 절제 모델 BEC 중심 간 재생을 연구하는데 더욱 유리하다. 이 프로토콜은 제브라 간세포 절제 모델을 사용하여 간 재생 실험을 수행하기위한 절차를 설명한다. 이 모델은 담즙의 중심 간 재생을 기본 메커니즘을 결정하기위한 화학 화면을 억제 또는 간 재생을 보강 할 수 있습니다 작은 분자를 식별하기에 적합합니다.

<table><tbody><tr><td>3-amino benzoic acid ethyl ester (Tricaine) powder</td><td>Sigma-Aldrich</td><td>A5040</td><td>Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7.&amp;nbsp;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Metronidazole</td><td>Sigma-Aldrich</td><td>M1547</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8532</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>252549</td><td></td></tr><tr><td>Calcium sulfate dihydrate</td><td>Sigma-Aldrich</td><td>C3771</td><td></td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629</td><td></td></tr><tr><td>Mounting media (Vectashield)</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>100 x 15 mm petri dish&amp;nbsp;</td><td>Denville Scientific Inc.</td><td>M5300</td><td></td></tr><tr><td>clear nail polish</td><td>Fisher Scientific</td><td>50949071</td><td></td></tr><tr><td>fine forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>Dumont #5&amp;nbsp;</td></tr><tr><td>glass slide</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>glass coverslip</td><td>Fisher Scientific</td><td>12-540-A and 12-542-A</td><td>18 x 18 mm</td></tr><tr><td>zn-5 (mouse anti-Alcam)</td><td>ZIRC</td><td>zn-5</td><td>1:10 dilution</td></tr><tr><td>goat anti-Hnf4a</td><td>Santa Cruz</td><td>sc-6556 (C-19)</td><td>1:50 dilution</td></tr><tr><td>rat anti-RFP</td><td>Allele Biotechnology</td><td>ACT-CM-MRRFP10</td><td>1:300 dilution</td></tr><tr><td>AlexaFluor 647 AffiniPure Donkey Anti-goat IgG (H+L)</td><td>Jackson laboratories</td><td>705-605-147</td><td>1:500 dilution</td></tr><tr><td>AlexaFluor 647 AffiniPure Goat Anti-mouse IgG (H+L)</td><td>Invitrogen</td><td>A21240</td><td>1:500 dilution</td></tr><tr><td>Cy3 donkey anti-rat IgG</td><td>Jackson laboratories</td><td>712-165-150</td><td>1:500 dilution</td></tr><tr><td>dissecting microscope</td><td>Leica Microsystems</td><td>S6F</td><td></td></tr><tr><td>epifluorescence microscope</td><td>Leica Microsystems</td><td>M205FA</td><td></td></tr><tr><td>confocal microscope</td><td>Carl Zeiss</td><td>LSM700</td><td></td></tr><tr><td>egg water</td><td></td><td></td><td>0.3 g/L of sea salts &amp;lsquo;Instant Ocean&amp;rsquo; and 0.5 mM CaSO&lt;sub&gt;4 &lt;/sub&gt;in distilled water.&lt;br /&gt;
&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>10X PBS</td><td></td><td></td><td>25.6 g Na2HPO4&amp;middot;7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4.&amp;nbsp; Bring to 1 L with distilled water.&amp;nbsp; Adjust pH 7.&amp;nbsp; Autoclave for 20 min at 121&amp;deg;C.</td></tr><tr><td>1X PBSDT</td><td></td><td></td><td>0.2% Triton X-100 and 0.2% DMSO in 1 X PBS.</td></tr><tr><td>PEM buffer</td><td></td><td></td><td>30.2 g PIPES, 761 mg EGTA, 1 mM MgSO4.&amp;nbsp; Bring to 1 L with distilled water.&amp;nbsp; Adjust pH 7.</td></tr><tr><td>Blocking solution</td><td></td><td></td><td>Mix 1 ml of heat-inactivated horse serum with 9 ml of 1X PBSDT.</td></tr></tbody></table>

hepatocyte-specific ablation in zebrafish to study biliary-driven liver regeneration

간은 재생 할 수있는 좋은 능력을 가지고있다. hepatocyte- 또는 담즙 중심의 간 재생 : 간세포, 간 실질 세포는 두 가지 방법 중 하나를 다시 생성 할 수 있습니다. 담즙 중심의 재생에, 재생 간세포가 담도 상피 세포 (BECS)에서 파생 된 반면 간세포 중심의 간 재생에 재생 간세포는, 기존의 간세포에서 파생됩니다. 간세포 중심의 간 재생에 크게 간 재생의 현재 이해에 기여한 우수한 설치류 모델이 있습니다. 그러나, 이러한 설치류 모델은 담즙 중심의 간 재생에 존재하지 않습니다. 우리는 최근에 BECS 광범위 심한 간세포 손실시 간세포을 야기하는 제브라 피쉬 간 손상 모델에보고했다. 이 모델에서, 간세포는 특히 약물 유전 학적 방법으로 절제되어있다. 여기에서 우리는 간세포를 브레이션과 BEC 중심의 간 재생 과정을 분석하는 상세 방법을 제시한다.이것은 간세포 특이 절제 모델은 또한 담즙 중심 간 재생의 기본 분자 세포 메커니즘을 발견하는데 사용될 수있다. 더욱이, 이러한 방법은 보강 또는 간 재생을 억제 작은 분자를 식별하는 화학 스크린에 적용될 수있다.

3.5 DPF 5에 극적으로 감소 간 크기 (그림 1B)에서 36 시간 (A36h,이 절제를 의미)에 대한 MTZ 처리. CFP-NTR 및 fabp10a : MTZ의 세척 한 후, 간 재생 (R), 강한 fabp10a의 시작으로 간주을 DsRed 발현 30 시간 (그림 1B)에서 다시 나타났다. 간내 담즙 네트워크 BECS (22)의 막에 존재 ALCAM의 발현에 의해 평가로, 초기 붕괴 그러나 54 시간의 후 세척 (R54h) (그림 1C)에</strong...

Watch this Scientific Journal Video about Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration at JoVE.com

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

담즙 중심의 간 재생을 연구하는 Zebrafish의에서 간세포 특이 절제

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

hepatocyte-specific-ablation-zebrafish-to-study-biliary-driven-liver

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8.5K 조회수.  University of Pittsburgh. To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

비디오: Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

겐타 마이신 유도 부상으로 성인 Zebrafish의에서 신장 재생

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

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Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

코스닥 시스템의 사용은 분자 메커니즘 Zebrafish의 재생의 연구를

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Zebrafish의에서 에탄올에 의한 섬유 성 간 모델의 개발은 전구 세포 매개 간세포의 재생을 연구하는

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

수술 절제 분석 플라나리아에 아이 재생을 유학을위한

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Zebrafish에서의 급성 알코올성 간 손상의 조직 학적 분석

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

제브라피쉬에서 비알코올성 지방간 질환 관련 간세포암의 초기 진행 중 간 미세환경 분석

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

암 연구학

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 ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

마우스 간에서 세포 유형 별 유전자 발현 프로파일링

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

생물학

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

성인 얼룩말 피시의 부분 간 절제술

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

Targeted Liver Ablation: Inducing Hepatocyte Death with Metronidazole in Transgenic Zebrafish Larvae

Source: Choi T.Y., et al. <a href="https://www.jove.com/v/52785/" target="_blank">Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration</a>. J. Vis. Exp. (2015)
The article describes targeted liver ablation, a method for inducing hepatocyte death with metronidazole in transgenic zebrafish larvae. The sample protocol describes the process for generating, manipulating and assessing targeted liver ablation in zebrafish.

표적 간 절제술: 형질전환 제브라피시 유충에서 메트로니다졸을 사용한 간세포 사멸 유도

Source: Choi T.Y., et al. Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration. J. Vis. Exp. (2015)
The article describes ...