이 작품은 퍼듀 대학에서 생물학의학과에서 시작 기금에 의해 지원됩니다.

1 부 : microdissection 전 준비 <ol><li> 솔루션 <ol class="lalpha"><li> E3 매체 4 (5 MM NaCl, 0.17 MM KCl, 0.33 MM CaCl 2, 0.33 MM MgSO 4). </li><li> 링어의 솔루션 1, 5 (116 MM NaCl, 2.9 KCl MM 1.8 MM CaCl 2, 5 MM HEPES, pH7.2), 필터 살균. </li><li> 5N NaOH. </li></ol></li><li> 텅스텐 바늘의 화학 에칭 <ol class="lalpha"><li> 점토하여 페트리 접시에 5N NaOH를 포함하는 작은 비커를 확보. </li><li> 그것은 NaOH 용액과 접촉할 수 있도록 비커의 측면에 종이 클립을 부착합니다. </li><li> 종이 클립과 텅스텐 와이어의 짧은 조각 (~ 2cm)의 권력을 긍정적인 전극에 DC 전원 공급 장치의 음극을 연결합니다. </li><li> 작은 점토 공과 텅스텐 와이어의 다른 쪽 끝을 싸서 NaOH 용액에이 끝부분을 찍어. </li><li> 약 3V로 전압을 증가시키고 좋은 에칭 속도가 접수 때까지 NaOH 용액에 철사를 찍어 내려. 좋은 품질의 바늘은 보통 10 분 이내에 에칭 수 있습니다. </li><li> 솔루션에 노출 텅스텐 와이어 점차 얇은 될 것입니다. 흙 공이 꺼져 방울 때, 여전히 긍정적인 전극에 연결된 전선의 다른 한쪽은 날카로운 바늘 모양을 것입니다. </li><li> 소금 예금을 제거하는 링어의 솔루션 또는 E3와 날카로운 바늘을 씻어. </li><li> 테이프로 나무 작은 주걱이나 쉽게 처리할 수있는 바늘 홀더에 바늘을 연결합니다. </li></ol></li><li> 문화 플레이트 <ol class="lalpha"><li> 팰컨 60 X 15mm의 폴리스티렌 문화 번호판은 망막 microdissection에 사용할 준비가 된 것입니다. </li><li> RPE 연결된 망막을 해부를위한 30 분 해부하기 전에 표면의 접착력을 없애 링어의 솔루션을 포함하는 문화 판에서 완전히이 배아를 부술. 곧 절개 전에 신선한 링어의 솔루션을 광범위하게 접시를 씻으십시오. </li></ol></li><li> 배아의 수집 및 준비 <ol class="lalpha"><li> 성인 zebrafish 4, 5 설명된대로 유지됩니다. </li><li> 구분선에 의해 번식 전에 밤에 pairwise 정적 사육 탱크에있는 부모 물고기를 분리합니다. </li><li> 객실 표시등이 다음날 아침에 켜져 후 분할기를 제거합니다. 부모가 매 30 10 분 간격으로 시간을 통과하도록 허용합니다. 각 교차로 후 생선을 분리. </li><li> 28 별도로 E3 매체의 각 교차로에서 수집한 배아를 유지 ° C. </li><li> 설립 기준 1, 6에 따라 10~12시간 포스트 수정 (HPF)와 restage 즉시 특정 시간 지점에서 해부하기 전 무대 배아. </li></ol></li></ol> 2 부 : 해부 - 뇌의 제거 및 안구 노출 1 <ul><li> 이 절에서 설명하는 절차는 망막 (부품 3A)와 RPE 연결된 망막 (파트 3B) 해부 일반적인 수 있습니다. </li><li> 모든 미세 해부는 더몬트 집게의 끝부분에 의해 행해지며 조직을 위치에 대한 더몬트 포셉 또 한 쌍의에 의해 지원됩니다. 화학적 - 새겨져 텅스텐 바늘은 미세한 조작 필요한 경우 사용할 수 있습니다. </li><li> 모든 미세 해부는 1X 목적 또는 동급을 갖춘 올림푸스 SZX16 현미경 ~ 5 배속 배율에서 수행됩니다. </li><li> 28 벤치 최고 인큐베이터에서 E3 중간에 배아를 유지 ° C 해부하는 동안 쉽게 배아 액세스에 대한 현미경 옆에. </li><li> 현미경 단계는 유전자 발현에 대한 온도 변동의 영향을 최소화하기 위해 온도 플레이트에 의해 28 ° C로 예열합니다. </li></ul><ol><li> 제 1 부 # 3에서 설명한대로 팰컨 60 X 15mm 문화 플레이트에 링어의 솔루션에 대한 특정 발달 단계에서 배아를 전송 준비. </li><li> 시체에서 빠르게 앞쪽에 트렁크의 일부로 머리를 잘라. </li><li> 지원 포셉과 문화 판에서이 조직의 뒷부분 끝을 핀. </li><li> 앞쪽에 이마에서 시작 등의 측면에 머리를 엽니다. </li><li> 눈의 중간 측면이 노출되고 더 조작에 대한 상향 직면되도록 머리를 제거합니다. </li></ol> 부품 3A : 망막 절개 1 <ol><li> 로 제 2에 설명되어있는 배아를 준비합니다. </li><li> 조심스럽게 망막에있는 작은 오프닝을 볼 때까지 포셉의 일각에 의해 위쪽으로 직면하고있는 안구 공의 중간 측면에 노출된 RPE를 브러시. </li><li> 빗질과 망막의 중간 측면은 거의 모든 노출 때까지 작업을 필링을 계속합니다. 긁힘 및 노출된 망막을 펀칭을 피한다. </li><li> 이제 아래 직면하고있는 망막, 접근의 측면 RPE를 제거하려면 해당과 문화 플레이트 표면에서 약 45 °의 각도로 칫솔. </li><li> C로 RPE의 분리 부분을 달아하여 오라 세라타 상대적으로 단단히 연결되어 RPE를 제거다음 ulture의 해부에 대한 판하고 망막을 부드럽게 리프트. </li><li> 잔류 RPE를 정리하기 위해 문화 접시의 바닥에있는 망막을 롤. <ol class="lalpha"><li> 우리는 배아가 깔린되면이 특정 팰컨 폴리스티렌 문화 플레이트가 약 20 분 RPE의 우선 준수를 가지고 것으로 나타났습니다. 이 속성은 RPE 잔존물의 제거를 위해 활용됩니다. 그러나, 망막 세포는 높은 배율에 따라 밀접하게 검사 수있는 낮은 정도로 표면에 막대기를 않습니다. 하나는 RPE 제거 및 망막 무결성의 완성도간에 균형을 공격한다. </li><li> 렌즈는 종종 문화 판을 준수하고 압연 과정에서 망막에서 분리합니다. 때때로, 그것은 렌즈 표면에 소용돌이 치는 동작과 함께 새겨져 텅스텐 바늘에 의해 렌즈를 분리하는 것이 필요합니다. </li></ol></li><li> 이러한 절차를 성공적으로 조직의 무결성을 손상시키지 않고 망막의 RPE를 제거할 수 있습니다. 이것은 좋은 전반적인 형태 (그림 1B와 B &#39;) 및 조직학 (그림 1B &quot;)로 표시되는 화살표 특히, photoreceptor 레이어와 RPE 사이에있는 세포외 기질의 보존 (그림 1B 매우 좋습니다.&quot;; 전체 배아 (그림 1A &quot;))에서 조직학 비교. </li></ol> 부품 3B : RPE 연결된 망막 절개 3 <ol><li> 로 제 2에 설명되어있는 배아를 준비합니다. </li><li> 보조 포셉하여 문화 판에 머리를 핀. 뒷부분 측면 측면에서 부드럽게 눈을 들어 앞쪽에쪽으로 롤. <ol class="lalpha"><li> RPE 계층의 외부에 추정 맥락막과 scleral 조직은 피부에 상대적으로 긴밀하게 연결되어 있으며, 주로 앞쪽에쪽으로 조심스럽게 눈을 압연에 의해 해제 벗겨 수 있습니다. </li></ol></li><li> 눈 측면 측면이 노출되고 안구가 계속 피부에 신경을 쓰는 경우 후 텅스텐 바늘로 렌즈를 제거합니다. </li><li> 이러한 절차를 성공적으로 망막을 가지고있는 전체의 RPE 계층을 보존할 수 있습니다. 추정 choroids과 scleral 조직은 크게 (그림 1C, C &#39;와 C&#39;) 제거할 수 있습니다. </li></ol> 4 부 : RNA 작업에 조직 샘플 컬렉션 <ol><li> 해부 표본으로 1 일 이전에 설명한 스트림 RNA 특성화를위한 RNase 무료 microfuge 튜브에 TRIzol 수집하실 수 있습니다. </li></ol> 대표 결과 <img src="/files/ftp_upload/2028/2028fig1.jpg" alt="그림 1" /> 그림 1. (A) 래터럴과 절개 전 52 HPF에서 zebrafish 애벌레 머리 (A &#39;) 등의 볼 수 있습니다. (A) &amp; (A &#39;). (B) 래터럴과 (B&#39;54 HPF에서 해부 망막의) 지느러미 참조하십시오. 망막의 표면 측면 모두에서 손상되지 않았어요에서 애벌레 머리 ( &quot;) 해당 histological 섹션 그리고 지느러미 전망. (B) &amp; (B &#39;)에서 해부하는 망막의 (B &quot;) 해당 histological 섹션을 참조하십시오. 망막과 망막 라미네이션의 구조는 손상되었다. 특히, photoreceptor 계층 및 RPE (A 사이의 세포외 기질은 &quot;(화살표, 화살표) 잘 해부 망막 B)에 보존되었다.&quot; (C) 래터럴 52 HPF에서 해부 RPE 연결된 망막의 (C &#39;) 중간 볼 수 있습니다. RPE 계층도를 해부 조직의 histological 섹션 (C &#39;)로 표시되었던, 손상 및 지속되었다. C&#39;의 하얀 부분은 시신경 (화살표)입니다. 조직학 들어, 조직 샘플은 4 %의 수집 및 고정되었습니다 3 설명된대로 paraformaldehyde. 삽입 이러한 샘플 sectioning 플라스틱이 수행되었다. 스케일 막대 = 50 μm의이.

<ol>
	<li>Leung, Y. F., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retina.">Gene expression profiling of zebrafish embryonic retina.</a> Zebrafish. 2, 269-283 (2005).</li><li>Leung, Y. F., Ma, P., Link, B. A., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factorial+microarray+analysis+of+zebrafish+retinal+development.">Factorial microarray analysis of zebrafish retinal development.</a> Proc Natl Acad Sci U S A. 105, 12909-12914 (2008).</li><li>Leung, Y. F., Ma, P., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retinal+pigment+epithelium+in+vivo.">Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo.</a> Invest Ophthalmol Vis Sci. 48, 881-890 (2007).</li><li>Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish : a practical approach. , (2002).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+book+:+a+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio).</a> , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Fadool, J. M., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+eye+genetics.">Zebrafish: a model system for the study of eye genetics.</a> Prog Retin Eye Res. 27, 89-110 (2008).</li></ol>

동물 실험은 퍼듀 애니멀 케어 및 사용위원회에 의해 명시된 지침과 규정에 따라 수행되었다.

zebrafish 아이 조직의 Microdissection 효과적으로 그대로 망막 및 RPE 연결된 망막을 얻을 수 있습니다. 이것은 실질적으로 특정 안구 조직 (예 : 망막 또는 RPE)에 관한 표현 연구를 지원합니다. 사실, 우리는 성공적으로 전체 망막 1 RPE 3 RNA 발현 프로파일을 얻기 위해이 절차를 이용합니다. 해당 프로필의 유틸리티는 강력 망막 차별 돌연변이에 불안정하게된다 는걸 알수 경로와 ...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cordless pestle motor</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-370</td>
<td>&nbsp;</td>
<tr>
<td>DC power supply</td>
<td>&nbsp;</td>
<td>Lascar</td>
<td>PSU130</td>
<td>Any DC supply would work. The specific voltage of a different machine will need further optimization.</td>
</tr>
<tr>
<td>Disposable pestle &amp; microtube, 1.5 mL (DNase, RNase and pyrogen-free)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-366</td>
<td>These are used for tissue collection in TRIzol for expression analysis.</td>
</tr>
<tr>
<td>Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>500341</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-00</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Falcon polystyrene culture plates, 60 X 15 mm</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>351007</td>
<td>These plates are used as dissection plates.</td>
</tr>
<tr>
<td>Olympus SZX16 Stereomicroscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZX16</td>
<td>Any stereomicroscope would work. We used Leica stereomicroscope in previous studies1-3 without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed. </td>
</tr>
<tr>
<td>Sharpening stone</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>29008-01</td>
<td>Use this to sharpen the tip of the forceps if necessary</td>
</tr>
<tr>
<td>Thermo plate</td>
<td>&nbsp;</td>
<td>Tokai Hit</td>
<td>MATS-U55SZX2B</td>
<td>This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28&deg;C during dissection in previous studies1-3 with good success.</td>
</tr>
<tr>
<td>Trizol, 100 mL</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>&nbsp;</td>
<tr>
<td>tungsten wire, 0.015 inch diameter</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TGW1510</td>
<td>&nbsp;</td>
<tr>
<td>Wooden Applicator</td>
<td>&nbsp;</td>
<td>Puritan</td>
<td>807</td>
<td>This is used for holding the chemically-etched tungsten needle.</td>
</tr>		</tbody>
		</table>
		</div>

microdissection of zebrafish embryonic eye tissues

Zebrafish는 때문에 급속한의 눈 개발에 연구에 인기있는 동물 모델입니다<em&gt; 예 utero</em&gt; 개발과 좋은 생산력. 3 일간 게시물 수정 (DPF)으로, 애벌레는 첫 번째 시각 응답을 표시합니다. 많은 유전자가 적절한 눈 개발을 제어 식별하지만, 우리는 근본적인 유전자 아키텍처의 완전한 이해를 멀리 아르되었습니다. 전체 게놈의 유전자 발현 프로 파일링은 눈 개발을위한 유전자 규제 네트워크를 명료하게하다하는 유용한 도구입니다. 그러나, zebrafish의 배아 눈의 작은 크기는 어려운 표현 분석을위한 온전하고 순수한 눈 조직을 얻을 수 있습니다. 렌즈의 직경이 적은 100 μm의 동안 예를 들어, 2 일 및 3의 눈 앞쪽에 - 후부 길이에만 약 200-300 μm의 수 있습니다. 또한, 망막을 기본 망막 안료 상피 (RPE)는 단지 하나의 레이어 상피이다. 유전자 발현 프로필은 전체 배아에서 얻을 수 있지만, 그들은 정확하게 이러한 조직의 표현을 대변하지 않습니다. 따라서 조직은 순수한 눈 개발의 성공적인 유전자 발현 프로 파일링에 대한 취득해야합니다. 이 문제를 해결하기 위해, 우리는 눈을 morphogenesis의 주요 단계를 커버 1-3 DPF의 부착 RPE와 함께 그대로 망막과 망막을 microdissect하는 접근 방식을 개발했습니다. 모든 절차는 표준 stereomicroscopes 이하 벌금 포셉 및 일반 실험실 공급을 할 수 있습니다. 망막 절개 들어, 단일 레이어 RPE 제거하고 작업 및 해부를위한 문화 판의 표면에 RPE 잔존물의 특혜 준수 닦고으로 해제 벗겨. RPE 완전히 망막과 보존 수 있도록 RPE 연결된 망막 절개 들어, 해부 접시에 RPE의 준수는 해부하기 전에 제거됩니다. 이 조직의주의 리프팅 작업은 효율적으로 추정 맥락막과 공막엔을 분리할 수 있습니다. 렌즈는 화학적으로 에칭 텅스텐 바늘로 두 경우 모두에서 제거할 수 있습니다. 즉, 우리의 접근 방식은 그대로 눈 조직을 얻을 수 있으며, 성공적으로 zebrafish 망막의 조직 구체적인 표현 프로필을 연구 활용되었습니다<sup&gt; 1, 2</sup&gt; 및 망막 색소 상피<sup&gt; 3</sup&gt;.

Watch this Scientific Journal Video about Microdissection of Zebrafish Embryonic Eye Tissues at JoVE.com

Microdissection of Zebrafish Embryonic Eye Tissues

이 문서는 망막 안료 상피 하나에서 세 일 postfertilization의 배아에게 부착과 않고 zebrafish의 망막 microdissect하는 방법을 설명합니다.

Zebrafish 배아 눈 조직의 Microdissection

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

microdissection-of-zebrafish-embryonic-eye-tissues

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JoVE Journal

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16.0K 조회수.  Purdue University. 이 문서는 망막 안료 상피 하나에서 세 일 postfertilization의 배아에게 부착과 않고 zebrafish의 망막 microdissect하는 방법을 설명합니다.

비디오: Microdissection of Zebrafish Embryonic Eye Tissues

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

포유 동물 조직의 레이저 캡처 Microdissection

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

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생물학

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

렌즈 Zebrafish의 이식과 눈 돌연변이의 분석에의 응용

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

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

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

조직 타겟 배아 Chimeras : Zebrafish Gastrula 세포 이식

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

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

유전자 발현 분석을위한 배아 Zebrafish 및 cDNA 합성에서 RNA 격리

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

블랙 위도우 스파이더 실크 생산 땀샘의 Microdissection

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

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.

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Zebrafish 배아 골격근의 레이저 쏜 부상

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...