We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

1. 높은 처리량 모르 폴리 노 디자인 전략 <ol><li> 관심의 유전자 (들) (예를 들어 후보 유전자 접근 방식, 시스 - 규제 분석, RNAseq 및 / 또는 단백체 차동 화면)를 확인합니다. </li><li> 자주 업데이트되는 웹 사이트에서 게놈, transcriptomic 및 유전자 발현 데이터를 사용 (예 : SpBase http://www.echinobase.org (20)와 S. purpuratus 게놈 검색에 http : ///urchin.nidcr.nih.gov/blast/index .html)을 시공간 발현 프로파일이 문제의 발달 메커니즘과 겹치는 것을 확인합니다. 어떤 발현 데이터가 없다면, qPCR에 프라이머를 생성 및 / 또는 인 시츄 프로브는 안티센스 유전자 발현 패턴을 평가. </li><li> 시공간 발현 패턴을 결정한 후, 게놈 웹 사이트로부터의 DNA 서열을 얻었다. <ol><li> 번역 차단 모르 폴리 노 올리고 뉴클레오티드를 생성하는 오 시퀀스를 획득F 5 &#39;비 - 번역 영역 (5&#39;UTR) 직접 상류에서 SpBase 또는 S. purpuratus 게놈 검색 웹 사이트에서 표현 된 시퀀스 태그 (EST) 데이터베이스에서 개시 코돈. 이 정보가 관심의 유전자를 사용할 수없는 경우, 다음 5 &#39;RACE를 수행합니다. </li><li> , 스플 라이스 차단 모르 폴리 노를 설계 엑손과 SpBase 또는 S. purpuratus 게놈 검색 도구의 발판 BLASTN 도구를 사용하여 인트론을 포함하여 게놈 시퀀스를 검색합니다. </li></ol></li><li> 목적하는 25 염기 쌍 모르 시퀀스를 설계하기 위해, 올리고 뉴클레오티드 설계 사이트 http://oligodesign.gene-tools.com를 사용한다. <ol><li> 병진 차단 모르 들어 병진 표적 서열의 소스로 &#39;3&#39;(5)로부터의 mRNA 서열을 사용한다. 5 &#39;UTR 서열 (전사 시작 사이트의 상류 약 70 뉴클레오티드) 플러스 마크 위스콘신 코돈 시작으로 (시작 사이트의 다운 스트림) 코딩 영역의 25 기지를 포함번째 괄호 (ATG). </li><li> 스플 라이스 차단 모르 들면, 인트론 - 엑손 또는 인트론 - 엑손 경계 시퀀스를 선택하고, 경계 영역 50 염기 (엑손 서열 25 염기 및 인트론 서열 25 염기)를 포함한다. 순서가 고유한지 확인 모르 폴리 노 서열과 게놈을 스캔합니다. </li></ol></li></ol> 모르 폴리 노 올리고 뉴클레오티드 2. 미세 주입 <ol><li> 300 nmol의 모르 폴리 노의 바이알에 클레아없는 물 100 μL를 첨가하여 폴리 노 올리고 뉴클레오티드의 3 mM의 스톡 용액을 준비한다. 참고 : 디 에틸 피로 카보네이트는 모르 폴리 노를 손상시킬 수 있기 때문에 재 현탁액에 대한 DEPC 처리 된 물을 사용하지 마십시오. </li><li> 최고 속도로 30 초에 대한 재고 올리고 뉴클레오티드 용액이 들어있는 유리 병 아래 첫 번째 재구성 스핀 - 5 ~ 10 분, 짧게 소용돌이 65 ° C에서 (14,000 16,000 XG), 간단히 소용돌이, 열, 룸에서 모르 폴리 노의 재고를 유지 적어도 1 시간 동안 온도. 모르 폴리 노 원액<html> 들 +4 ℃로 -20 ° C에서 저장됩니다. </li><li> 원하는 농도 모르 폴리 노 올리고 뉴클레오타이드를 포함하는 주사 솔루션을 준비합니다. 이 솔루션은 일반적으로 20 % 글리세롤 또는 캐리어 125 밀리미터의 KCl 15 % (형광 염료 덱스 트란 10,000 MW 2.5 밀리그램 / μL 원액) FITC-덱스 트란이 포함되어 있습니다. FITC 덱스 트란 등의 형광 덱스 트란 접합체 일상적 표면 형광 현미경으로 주입 된 배아를 식별하는 데 사용된다. -20 ℃에서 보관 주사액. </li><li> 5 분 - 적어도 2 열 블록 또는 수욕에서 65 ° C에서 모르 용액을 가열한다. </li><li> 적어도 10 분 - (16,000 XG 14,000) 최고 속도로 1 분간 원심 분리를위한 - (16,000 XG 14,000), 소용돌이 짧게 모르 폴리 노의 최고 속도에서 30 초 동안 솔루션을 스핀. </li><li> 모르 폴리 노 용액 주입 바늘을 넣습니다. 자세한 미세 주입 프로토콜 Stepicheva과 노래 2014 년 21 환호와 Ettensohn 2004를 참조하십시오&quot;&gt; 22. </li></ol> S. purpuratus 태아에서 24 시간 후 수정 (HPF)에서 3. 고정하고 제자리 프로토콜에서 참고 :이 프로토콜은 아레나스 - 메나 등에서 수정됩니다. 2000 년 23 세티 등. 2014 년 24. <ol><li> 정착 <ol><li> , 배아를 포함하는 웰 (아래 참조) 정착의 몇 방울을 추가 피펫으로 부드럽게 혼합하고, 그들이 정착 할 수 있습니다. 정착액 솔루션을 제거하고 고정액의 두 개의 추가 180 μL 세척과 배아를 혼합한다. 참고 : 철저하게 부드러운 피펫 위아래로 몇 번으로 세척하는 동안 배아를 혼합하는 것이 중요하다. 그렇지 않으면 신호 대 잡음비를 낮출 수있다. </li><li> pH를 7.0 10mM의 MOPS로 구성된 4 % 전자 현미경 등급 파라 포름 알데히드 실온에서 50 분 시간 1에 대한 두 번째 정착액 세척 (RT)에 수정 배아를 남겨, 0.1 % 트윈 20, 인공 seawateR (ASW). 최상의 결과를 위해 각 시간이 솔루션은 신선합니다. 또한, 편의성과 실용성 배아 96 웰 플레이트에 고정되어있다. <ol><li> 20 ML의 정착액 사용 5 mL의 16 % 파라 포름 알데히드, 15 mL의 ASW, 200 μL 1 M MOPS pH를 7.0, 20 μL 트윈 20. </li></ol></li><li> MOPS 180 μL에 실온에서 5 회 반복한다 0.1 M MOPS pH를 7.0, 0.5 M의 NaCl 및 트윈 20 적어도 5 분 동안 또는 배아 때까지이 우물의 바닥에 떨어 0.1 %로 구성된 버퍼를 씻는다. 다시, 충분히 부드럽게 피펫 팅하여 여러 번 아래 세척 완충액으로 배아를 혼합하는 것이 중요하다. (4)에 저장된 경우 MOPS 세척 완충액을 2 일 동안 사용될 수있다 기음. <ol><li> 40 mL를 들어 MOPS 버퍼 사용 4 mL의 1 M MOPS pH를 7.0, 4 ㎖의 5 M NaCl을 32 mL의 DH 2 O, 40 μL 트윈 20을 씻는다. </li><li> 고정 배아 2 일 동안 4 ℃로 저장 될 수있다. 주 : 이상 고정 된 배아를 저장하는 경우, 박테리아 성장을 방지하기 위해 버퍼 워시 MOPS에 0.2 % 아 지드 화 나트륨을 추가한다.</li></ol></li></ol></li><li> 사전 하이브리드 <ol><li> MOPS는 버퍼 워시 기음과 2의 180 μL를 추가, 하이브리드 버퍼에 버퍼 부드러운 피펫 몇 배 용액에 배아를 혼합하고 실온에서 적어도 20 분 동안 품어 씻어 MOPS의 1의 비율. 혼성화 완충액 70 % 포름 아미드, 0.1 M MOPS pH를 7.0, 0.5 M NaCl을 구성, 1 ㎎ / ㎖의 BSA, 0.1 % 트윈 -20. <ol><li> 40 mL의 하이브리드 버퍼 사용 4 mL의 1 M MOPS pH를 7.0, 4 ㎖의 5 M NaCl을, 4 mL의 DH 2 O, 0.04 g의 BSA, 40 μL 트윈 20. , 텍싱에 의해 잘 섞는다 다시 28 mL의 포름 아미드, 및 소용돌이를 추가합니다. </li></ol></li><li> MOPS 세척 및 하이브리드 버퍼의 1의 비율과 1의 180 μL를 추가 : 2를 제거, 하이브리드 버퍼에 버퍼 용액에 배아를 혼합하고 실온에서 적어도 20 분 동안 품어 씻어 MOPS 2 비율. </li><li> 하이브리드 버퍼 150 μL - 프로브 하이브리드 부드럽게 (100)에 배아를 혼합하기 전에. 적어도 1, 50 ° C에서 배아 부화시간. 참고 : 하룻밤 배아를 배양하는 것은 허용됩니다. 배양 전에 증발을 방지하기 위해 접착 시트 웰을 밀봉. </li></ol></li><li> 이종 교잡 <ol><li> 프로브 솔루션을 만들 부드럽게 한 다음, 하이브리드 버퍼에 소용돌이를 0.3 프로브의 NG / μL 500 μg의 / mL의 효모의 tRNA - 별도의 튜브에 0.1를 추가합니다. 효모의 tRNA는 안티센스 프로브의 비특이적 결합을 감소 시키도록 프로브 용액에 첨가된다. 이 50 ° C의 솔루션 및 흡인 하이브리드 버퍼를 예열. </li><li> 프로브 용액 100 μL에 미리 혼성화 된 배아 믹스 접착 시트로 밀봉하고, 상기 프로브에 따라 2 내지 7 일 동안 50 ° C에서 혼성화합니다 (foxq2 프로브 2 일 동안 배양 될 수있다). 참고 : 배양 시간은 프로브를 따라 달라질 수 있습니다. 낮은 수준으로 발현 일부 유전자는 7 일 동안 배양까지 필요로한다. 96- 웰 플레이트는 ADHES 경우 증발에 대하여 보험 등 습도 상자에 배치 될 수있다필자 시트가 제대로 밀봉되지 않습니다. </li><li> 갓 만든 MOPS 180 μL와 50 ° C에서 5 회 반복 3 시간 총 버퍼를 씻는다. 이어서, MOPS의 μL 실온에서 세척 완충액 (180)으로 3 회 (15 분) 세척 하였다. 세척에 배아 부드럽게 여러 번 피펫 팅에 의해 때마다 버퍼 혼합해야합니다. </li></ol></li><li> 항체 배양 <ol><li> MOPS는 버퍼 워시 대기음 10 % 정상 양 혈청 5 밀리그램으로 구성된 버퍼를 차단의 180 μL에 배아를 혼합 / 버퍼 하룻밤 실온에서 적어도 45 분 4 ° C에 대해 품어 씻어 MOPS에서 ㎖의 BSA. </li><li> 버퍼를 차단 제거한 다음 1 포함하는 버퍼 차단으로 배아를 혼합 : 버퍼를 차단에 희석 알칼리 포스 파타 아제 접합 된 항 - 디그 옥시 제닌 항체의 1,500 희석. 증발을 방지하기위한 밀봉 판에 RT에서 밤새 인큐베이션. 참고 : 하룻밤 이상에서 항체를 두지 마십시오. </li><li> 배아 6 번 씻으 (5 분 또는 배아가 떨어질 때까지) MOPS에 실온에서 버퍼를 씻는다. 배아 수4 ℃에서 하룻밤 저장 될 수있다. </li></ol></li><li> 현장 개발 <ol><li> 0.1 M 트리스의 pH 9.5, 100 mM의 염화나트륨, 50 mM의의 MgCl 2, 1 ㎜의 levamisole로 구성된 갓의 pH 9.5 완충액으로 실온에서 배아 3 회 (10 분) 세척하고, 0.1 % 트윈 20. <ol><li> 20 ㎖의 pH 9.5 버퍼 사용 2 mL의 1 M 트리스 산도 9.5, 400 μL 5M NaCl을 1 mL의 1 M의 MgCl 2, 20 μL 트윈 20, 16.6 mL의 DH 2 O, 및 0.0048 g의의 levamisole. </li></ol></li><li> 빛으로부터 보호 염색 버퍼에 37 ° C에서 배아를 품어. 염색 완충액 10 %의 디메틸 포름 아미드로 구성 4.5 μL / ㎖ 4- 니트로 블루 테트라 졸륨 클로라이드 (NBT) 및 갓의 pH 9.5 완충액 3.5 μL / ㎖의 5- 브로 모 -4- 클로로 -3- 인돌 일 포스페이트 (BCIP) . <ol><li> 염색 버퍼의 한 ML 100 μL 디메틸 포름 아미드, 4.5 μL NBT, 3.5 μL의 BCIP를 사용합니다. </li></ol></li><li> MOPS에 3 ~ 5 회 세척하여 알칼리 포스 파타 아제 반응을 중지 버퍼를 씻는다. 반응 위스콘신foxq2 프로브는 일반적으로 1 시간 30 분 소요 번째. 일부 프로브는 RT 4 °에 C 중 하나에서 하룻밤 배양을해야 할 수도 있습니다. </li><li> 70 % MOPS 세척 30 % 글리세롤의 솔루션으로 배아를 섞는다. 글리세롤 현미경 필요한 굴절률을 제공한다. 배아 몇 주 동안이 용액에 저장 될 수있다. 증발을 방지하기 위해 플라스틱 파라핀으로 판을 밀봉합니다. </li></ol></li><li> 슬라이드 준비 및 이미지 캡처 <ol><li> 슬라이드 위에 스트립 사이의 작은 틈으로 삼각형에 양면 테이프의 세 개의 작은 스트립을 배치하여 슬라이드를 준비합니다. </li><li> 삼각형의 중심에 70 % MOPS 세척하고 30 %의 글리세롤 용액에서 배아를 전송하고, 커버 슬립으로 덮고. </li><li> 커버 슬립의 가장자리 주위 매니큐어의 층을 도포하여 커버 슬립을 밀봉. </li><li> 20 배의 대물 렌즈와 연결된 카메라 복합 광학 현미경을 사용하여 이미지를 캡쳐. </li></ol></li></ol>

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The authors have nothing to disclose.

여기에 제시된 방법은 .. 많은 연구소가 해부 일찍 성게 개발 과정에서 유사한 분석을 사용하는 기본적인 발달 메커니즘을 지배하는 신호 전달 경로와 GRNs을 이해하는 척추 동물보다 작은 게놈과 형태 학적 복잡성 배아를 사용하는 능력을 보여주는 예입니다 다른 세포 운명 사양 이벤트에 관련된 신호 전달 경로 (예 노치 헤지 호그, TGF-β의와 FGF 신호) 27, <sup class="...

유전자 조절 네트워크 (GRNs) 신호 전달 경로는 성인 동물 신체 계획을 작성하는 데 사용되는 배아 발달 동안 유전자의 시공간적 식을 설정. 세포 간 신호 전달 경로는 세포가 통신하는 수단을 제공하는 이러한 규제 네트워크의 필수적인 구성 요소이다. 이러한 세포의 상호 작용을 수립하고 배아 1, 2 중에 다양한 지역과 중 규제 및 분화 유전자의 발현을 수정. 세포 외 분비 조절제 (리간드 길항제) 수용체 공동 수용체 간의 상호 작용은 신호 전달 경로의 활동을 제어한다. 세포 내 분자의 구색은 세포의 변형 된 유전자 발현, 분할 및 / 또는 형상의 결과로 이러한 입력을 transduces. 주요 경로에있는 세포 및 세포 수준에서 사용되는 키 분자의 대부분은 있지만공지 그것은 개개 신호 경로의 복잡성으로 인해 많은 부분에서 불완전한 지식이다. 또한, 다른 신호 경로는 종종 세포, 세포 내에서 양 또는 음 중 서로 상호 작용하고 전사 레벨 3, 4, 5, 6. 중요한 신호 전달 경로의 중심 구성 요소는 고도로 모든 후생 동물 종에서 보존하고, 특히 관련성 phyla의 발 미생물과 비교하면 현저하게 중요한 신호 전달 경로의 대부분은 종종 많은 종에서 유사한 발달 기능을 수행하는 7, 8, 9, 10, 11. 개발하는 동안 신호의 연구는 모든 생물의 발굴 작업이며,이척추 동물에서 큰 가능한 리간드 및 수용체 / 공동 변조기 상호 작용의 수, 세포 내 전달 분자뿐만 아니라, 거기에 1) : 가장 후구 동물 모델 (척추 동물, 무척추 척색, 반삭 동물 및 극피)의 신호 전달 경로를 연구하는 몇 가지 중요한 문제는 때문에 유전체 (12, 13), (14)의 복잡도에 서로 다른 신호 경로들 사이의 상호 작용 가능성; 2) 척추 동물의 복잡한 형태와 형태 형성의 움직임은 종종 더 어렵게 및 신호 전달 경로 사이에서 상호 작용을 해석 할 수 있도록; 3) 대부분의 비 극피 동물 무척추 동물의 후구 동물 모델 종의 분석은 일부 피막이 종 (15, 16)를 제외하고 임신 횟수의 짧은 창으로 제한됩니다. 그만큼성게 배아 상술 제한 몇 가지며 생체 내 신호 전달 경로의 상세한 분석을 수행하기 위해 많은 독특한 특성을 제공한다. 이들은 다음을 포함한다 : 1) 성게 게놈의 상대적 단순성 크게 가능한 리간드, 수용체 / 공동 수용체 및 세포 내 전달 분자의 수를 감소시킨다 (17)과의 상호 작용; 2) 세균 층 및 주요 배아 축 사양 및 패턴을 제어하는 GRNs 잘 신호 (18), (19) 수신 셀 / 지역의 규제 문맥의 이해를 돕고, 성게 배아 년에 설립된다 배아가 그 형태를 분석하기 쉽다 단층 상피 구성 때 3) 다수의 신호 전달 경로는 초기 및 절단 낭배 단계 사이 연구 될 수있다 4) 분자를 포함쉽게 조작하는 성게의 신호 전달 경로에서 D; 5) 많은 성게는 10 ~ 11 개월 년 (예 : Strongylocentrotus의 purpuratus 및 Lytechinus의 variegatus)에 대한 임신하는됩니다. 여기서 우리는 체계적이고 효율적으로 지정하고 성게 배아의 패턴 영역이 여러 무척추 동물 모델 시스템은 복잡한 분자 메커니즘의 연구에 제공하는 이점을 설명하기 위해 신호 전달 경로의 구성 요소를 특성화 할 수있는 방법을 제시한다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Translational-blocking morpholino and/or splice-blocking morpholino</td>
			<td>Gene Tools LLC</td>
			<td>Customized</td>
			<td>More information at www.gene-tools.com</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC (dextran fluorescein isothiocyanate)</td>
			<td>Invitrogen, Life Technologies</td>
			<td>D1821</td>
			<td>Make 25mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M1254-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>23336-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma Aldrich</td>
			<td>47671-1L-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase-conjugated anti-digoxigenin antibody</td>
			<td>Roche</td>
			<td>11 093 274 910</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride (levamisole)</td>
			<td>Sigma Aldrich</td>
			<td>L9756-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base UltraPure</td>
			<td>Research Products Internationall Corp</td>
			<td>56-40-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11 383 221 001</td>
			<td></td>
		</tr>
		<tr>
			<td>4 Nitro blue tetrazolium chloride (NBT)</td>
			<td>Roche</td>
			<td>11 383 213 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Formamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C1016-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

성게 배아에서 우리는 3 가지의 Wnt 신호 지점 (이 Wnt / β-catenin이,이 Wnt / JNK와의 Wnt / PKC) 4, 25 상호 작용은 전후방 (AP) 패턴에 적용의 Wnt 신호 전달 네트워크를 형성하는 것으로 나타났습니다. 이러한 시그널링 이벤트의 가장 중요한 결과 중 하나는 초기 넓게 표현 전방 neuroectoderm (ANE) GRN가 낭배의 시작 (S.의 purpuratus HPF 24)?...

Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

이 비디오 기사는 체계적이고 효율적으로 많은 무척추 동물 배아에서 복잡한 신호 전달 경로 및 규제 네트워크의 구성 요소를 특성화하는 데 사용할 수있는 생체 방법론에 직접적인을 자세히 설명합니다.

단순함의 힘 : 같은 성게 배아<em&gt; 생체</em&gt; 복잡한 세포 간 신호 전달 네트워크의 상호 작용 유학을위한 발달 모델

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the signaling pathways and the gene regulatory networks that they activate in an in vivo developmental context.Demonstrating this procedure will be Marina Martinez-Bartolom

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The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Unit 1

Principles of Cell Signaling

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7.8K 조회수.  Mississippi State University. The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the si...

비디오: The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

모델 콩과 식물에서 초기 배아 발달의 규정 유학을위한 접합체 및 체세포 배아를 얻기위한 프로토콜<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

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Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

흉선의 특성 사용하여 마우스 배아에서 전구 세포를 정착<em&gt; 생체</em&gt;와<em&gt; 체외</em&gt; 분석 실험

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

실시 항공의 패터닝 동안 Wnt 신호를 공부

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

작은 별 말미의 반구 Physa에서 전체 폴립 재생을 유도<em&gt; Nematostella의 vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

약물 치료와<em&gt; 생체</em&gt;은 메다카 물고기 골다공증 모델에서 조골 파골 세포 상호 작용의 영상

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

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

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

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

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

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

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

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Mouse Secondary Palate Fusion의 라이브 이미징

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

노드 및 스캐닝 전자 현미경 검사 법, 면역 형광 산 전체를 사용 하 여 Gastrulating 마우스 배아에서 Notochordal 접시를 시각화

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

자외선-C (UV-C) 손상에 응하여 1 차 마우스 안구 표면 세포/줄기 세포에 있는 산화 손상의 평가

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

전체 얼룩말 배아의 연장된 시간 경과 공초점 현미경 검사법을 위한 레이어형 장착 방법

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...