저자는 도움이 토론과 문화원 퀴리의 동물 시설에 대한 피브로넥틴, 에릭 Theveneau에 이식편의 사진을 Adeline 돌리 감사합니다. CM은 지역 일 드 프랑스 (도멘 드 Interet 죄르 장대 줄기), 대학교 파리 수드 (아타 Temporaire 디부 Enseignement 엣 드 공들인), 그리고 직원은 국립 드 라 공들인의 박사 후 동료입니다. 이 작품은 대학교 파리 수드 (Attractivite 2011), 센터 내셔널 드 라 공들인 Scientifique (액션 Thematique 등 Incitative 쉬르 프로그램), 협회 부어 라 공들인 contre 르 암 그랜트 SFI20101201882, 리그 contre 르 암과 직원은 국립 드 라에 의해 투자되었다 공들인 (직원은 국립 드 라 공들인 프로그램 블랑).

실험은 과학적 목적 및 교체, 감소 및 정제 국제적으로 확립 된 원칙에 사용되는 동물의 보호에 관한 국가와 유럽 규정을 준수합니다. 1. 피브로넥틴 코팅 요리 (14)의 준비 <ol><li> 표준 플라스틱 페트리 접시 (Ø 40 X 11mm)로 1X 인산염 완충 생리 식염수 (PBS)에 희석하여 10 ㎎ / ㎖ 문화 등급 피브로넥틴의 피펫 500 ㎖. 1 시간 동안 37 ℃에서 배양한다. 참고 : 다음 영상에 유리 그릇 또는 유리 커버 슬립을 사용하는 경우, 100 ㎎ / ㎖의 fibronectin 솔루션을 사용합니다. </li><li> 1X PBS로 3 회 씻어. </li><li> 1X PBS/0.1 %의 소 혈청 알부민 (BSA)의 500 ㎖의 PBS를 교체합니다. 30 분 동안 37 ° C에서 알을 품다. </li><li> 1X PBS로 3 회 씻어. </li><li> 1X PBS와 함께 입력하고 (보통 24-48 시간 동안) 사용할 때까지 4 ° C에 보관하십시오. </li></ol> 2. NC 외식 해부 &lt;올&gt; <li> 준비 3 / 4 일반 양서류 중간 (NAM) (110 mM의 NaCl을 2 ㎜의 KCl, 1 ㎜ 중 Ca (NO 3) 2, 1 ㎜ 망초, 0.1 mM의 EDTA, 1 mM의 탄산 수소 나트륨, 0.2 배 PBS, 50 ㎎ / ㎖ ) 10,15을 겐타 마이신. 4 ℃에서 해부, 저장소에 대해 1 주일 이상 보관하지 마십시오 이전 NAM은 아가 로스 코팅 해부 접시 (아래)의 제조에 사용 할 수 있습니다. </li><li> 3 / 4 NAM 2 % 아가로 오스 10 ㎖로 코팅 해부 용 60mm 페트리 접시를 준비합니다. </li><li> 해부 도구를 수집 플라스틱 파스퇴르 피펫, 최고의 곤충 핀은 핀 홀더 (참고 자료 표 참조), (유리 파스퇴르 피펫 (16)의 끝 부분에 파라핀 눈썹 머리로 만든) &quot;눈썹 칼&quot;, 두 개의 미세에 장착 해부 집게, P1000 피펫 팁, 50 배율 8-40X와 양안 입체경, 광학 섬유 가이드 밝은 조명. 모든 해부 도구를 청결하게 유지해야한다, 각 실험 후, 증류수로 2 회 위스콘신 헹구어100 % 에탄올을 토륨 건조 할 수 있습니다. 멀리 먼지 저장합니다. </li><li> 거의 정상에 신선한 NAM과 해부 접시에 기입하십시오. </li><li> X를 얻 표준 절차 (10) 그들이 (Nieuwkoop와 페이버 발달 테이블 (17)에 따라) neurula 스테이지 (16)에 도달 할 때까지를 나이에 따라 배아를 laevis의. </li><li> 16 배아 플라스틱 파스퇴르 피펫을 사용하여 해부 접시에 무대를 놓습니다. </li><li> 양안 범위에서 이후의 모든 단계를 수행합니다. 포셉 두 쌍의 난황 막 제거합니다. 배아의 등 부분이 손상되지 않도록주의하십시오. </li><li> 태아가 해부를 시작하는 단계 17에 도달 할 때까지 기다립니다. 신경 문장이 명확하게 초기 단계는 대조적으로, 신경관 따로 설정되어 있기 때문에 스테이지 (17)가 적절하다. 단 17 일 후, 노스 캐롤라이나 마이그레이션 및 주변 중간 엽과 섞여서 시작합니다. </li><li> 유지하기 위해 배아에 대한 틈새 시장을 집게와 아가로 오스의 작은 구멍을 파해부 동안 그들. 하나의 배아 등의면을 위로 놓습니다. 참고 : 모델링 점토 대신 아가로 구멍을 파고 배아를 유지하는데 사용될 수있다. </li><li> 눈썹 칼과 곤충 핀 앞쪽에 신경 배의 측면 부분에 절개를합니다. 깊은 배아로도 이동하지 않도록주의 : 2 ~ 3 세포층 (! archenteron만큼 깊이에 도달하지 않음)을 극복하는 것을 목표로하고 있습니다. 전체적인 박리 과정에서 해부 조직이 X.시기 공기 - 액체 계면에 접촉 입력 할 수 없다 laevis의 배아 조직은 표면 장력에 의해 용해된다. </li><li> 신경의 추가 지느러미 부분의 제 2 평행 절개 동일한 장비를 사용하여 접어 만든다. </li><li> 필요한 경우, 90 °의 주위에 요리를 켭니다. 처음 두 절개의 후방 측에, 눈썹 칼과 곤충 핀 세 번째 수직 절개를합니다. NC는 측면, 외배엽의 색소 외부 층 아래 세포의 두꺼운 질량신경 튜브에. </li><li> 세 번째 절개의 가장자리에서 시작하여 기본 NC에서 색소 외배엽 층을 분리 눈썹 칼의 끝 부분을 사용합니다. </li><li> 세 번째 절개로 시작하는 NC 조직을 분리 눈썹 칼의면을 사용합니다. 기본 두부 중배엽 흰 동안 NC 조직이 아니라 회색이다. 조심스럽게 중배엽에서 NC를 구분합니다. </li><li> 광학 소포에서 전방으로 NC 조직을 분리하고, 이식편을 무료로이 수준에서 잘라. 현장 하이브리드 화에 의해 일부 외식에 snail2 식 (정식 NC 마커)를 검사하는 첫 번째 실험에 대한 제어로 추천합니다. </li></ol> 3. 피브로넥틴에 도금의 Explants <ol><li> 강제로 피브로넥틴 코팅 접시 수정 Danilchick 중간 (MDM, 염화나트륨 53 밀리미터, Na2CO 5 mm의 K 글루 콘 산염 4.5 mm의 나 글루 콘 산염 32 밀리미터, 망초 1 ㎜, 염화칼슘 1 ㎜, BSA 0.1 %로, 산도를 조정 1 M Bicine 8.3에 <sup&gt; 18). </li><li> P1000 피펫을 사용하여 피브로넥틴 - 코팅 접시에 외식을 전송합니다. 외식 공기 - 액체 인터페이스를 터치 할 수 있도록하지 마십시오. 먼저, 다음 몇 가지 액체를 다시 외식 후, 끝으로 몇 가지 액체를 피펫. 피브로넥틴 - 코팅 접시에 외식을 전송하는 동안 끝으로 모든 공기를 허용하지 않습니다. 끝이 피브로넥틴 코팅 접시의 액체 내부되면, 외식 천천히 중력에 의해 아래로 가자, 또는 매우 조심스럽게 밀어 넣습니다. 너무 빨리 외식를 배출하지 마십시오. </li><li> 눈썹 칼을 사용하여 접시에 10-30 외식을 놓습니다. 다음 영상에 적합하지 않은 접시의 측면을, 피. </li><li> 외식이 (대략 15 ~ 30 분 소요) 피브로넥틴에 부착 할 때까지 요리를 움직이지 않도록하십시오. 필요한 경우, 아주 조심스럽게 접시를 이동합니다. </li><li> (온도 차트는 17 참조 15 ~ 20 ° C의 사이) 냉장 인큐베이터에서 접시를 놓습니다. 15 ° C에 배치하면, 세포가 아칸소전자 일반적으로 효율적으로 마이그레이션 및 배양 6 시간 후에 산란 (이 타이밍 실험 사이에 차이가있을 수 있습니다). </li></ol> 4. 호스트 태아로의 Explants를 접목 <ol><li> 거친 집게를 사용하여 매우 작은 조각으로 미세 유리 커버 슬립을 잘라. 조각의 크기는 약 1.5 mm 2 있어야합니다. 집게를 사용하여 3 / 4 NAM 또는 PBS를 함유하는 페트리 접시로 매 담금. </li><li> 호스트 배아의 제조 : 난황 막 제거하고 NC 해부 동안 호스트 배아를 손상시키지 않도록 특별히주의) 3의 설명에 따라 3 / 4 NAM에 호스트 NC를 해부. 도너 배아 해부 동안 호스트 배아 부분적으로 치유하자 : 티슈 장력 절제의 크기를 증가시키는 경향이있다. </li><li> 프로토콜 3에 설명 된대로 다른 아가 로스 코팅 접시 또는 동일한 접시의 다른 부분에서 도너 배아로부터 NC 이식편을 해부. 의 설명에 따라주의하여 호스트를 포함하는 접시에 NC 이식을 전송프로토콜 3. </li><li> 즉시 집게를 사용하여 호스트 절제 영역에 NC 이식편을 배치합니다. 이식편 부분적으로 연결해야합니다. 장소에 이식을 유지하기 위해 이식 배아에 유리 커버 슬립의 조각을 넣어. 그것은 손상을 방지하기 위해 배아보다 큰 유리 조각을 선택합니다. 배아는 평평 조금있을 것입니다. </li><li> 적어도 15 ~ 20 분 동안 기다린 후 부드럽게 커버 슬립을 제거합니다. 배아가 다른 10 분 동안 복구 할 수 조심스럽게 플라스틱 파스퇴르 피펫을 사용하여, 3 / 4 NAM 가득 깨끗한 접시에 피펫. </li></ol>

<ol>
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S., Luo, T., Xu, Y., Sargent, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin-X+is+required+for+cranial+neural+crest+cell+migration+in+Xenopus+laevis.">Myosin-X is required for cranial neural crest cell migration in Xenopus laevis.</a> Dev. Dyn. 238 (10), 2522-2529 (2009).</li><li>Lee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461 (7262), 402-406 (2009).</li><li>Mica, Y., Lee, G., Chambers, S. M., Tomishima, M. 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Dyn. 237 (11), 3404-3409 (2008).</li><li>Carmona-Fontaine, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+inhibition+of+locomotion+in+vivo+controls+neural+crest+directional+migration.">Contact inhibition of locomotion in vivo controls neural crest directional migration.</a> Nature. 456 (7224), 957-961 (2008).</li><li>Guiral, E. C., Faas, L., Pownall, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+migration+requires+the+activity+of+the+extracellular+sulphatases+XtSulf1+and+XtSulf2.">Neural crest migration requires the activity of the extracellular sulphatases XtSulf1 and XtSulf2.</a> Dev. Biol. 341 (2), 375-388 (2010).</li><li>Cousin, H., Abbruzzese, G., Kerdavid, E., Gaultier, A., Alfandari, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+the+Cytoplasmic+Domain+of+ADAM13+to+the+Nucleus+Is+Essential+for+Calpain8-a+Expression+and+Cranial+Neural+Crest+Cell+Migration.">Translocation of the Cytoplasmic Domain of ADAM13 to the Nucleus Is Essential for Calpain8-a Expression and Cranial Neural Crest Cell Migration.</a> Dev. Cell. 20 (2), 256-263 (2011).</li><li>Borchers, A., David, R., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+cadherin-11+restrains+cranial+neural+crest+migration+and+influences+neural+crest+specification.">Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.</a> Development. 128 (16), 3049-3060 (2001).</li><li>Borchers, A., Epperlein, H. -. H., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+to+study+migratory+behavior+of+cranial+neural+crest+cells+in+Xenopus.">An assay system to study migratory behavior of cranial neural crest cells in Xenopus.</a> Dev. Genes Evol. 210 (4), 217-222 (2000).</li><li>Kerney, R., Gross, J. B., Hanken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx2+is+essential+for+larval+hyobranchial+cartilage+formation+in+Xenopus+laevis.">Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis.</a> Dev. Dyn. 236 (6), 1650-1662 (2007).</li><li>Yan, C. Y. I., Skourides, P., Chang, C., Samba Brivanlou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Xenopus+hnRNP+expressed+in+neural+and+neural+crest+tissues.">a Xenopus hnRNP expressed in neural and neural crest tissues.</a> Dev. Dyn. 238 (1), 204-209 (2009).</li><li>Acloque, H., Adams, M. S., Fishwick, K., Bronner-Fraser, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions:+the+importance+of+changing+cell+state+in+development+and+disease.">Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.</a> J. Clin. Invest. 119 (6), 1438-1449 (2009).</li></ol>

저자가 공개하는 게 없다.

이 프로토콜은 X를 laevis의 태아에 premigratory 두개골 NC를 이식편하는 쉬운 방법을 설명합니다. 이러한 실험에 사용 된 배아는 견고하고 잘 치료해야합니다. 건강에 해로운 배아의 배치를 폐기하십시오. 또한, 그리고 나중에 17에 신경 능선을 절제하기 위해, (12 18 ~ 20 ° C)에서 다양한 온도에서 배아를 성장. 단 17 일 후, 신경 문장은 두개의 중배엽과 혼합 될 수 있으며 완전히 제거 할 수 없습?...

신경 능선 (NC)는 척추 동물의 배아에서 neurulation의 끝 부분에 신경 튜브에서 나온다 과도 배아 세포 집단이다. NC 사양을 제어하는​​ 신호 유전 이벤트는 초기 낭 배기로 시작합니다. NC는 주변 등의 조직으로부터의 신호에 의해 신경과 비 신경 외배엽의 경계에 지정됩니다. neurulation의 끝에서, NC 세포는 상피 - 투 - 중간 엽 전이 (EMT)를 거쳐 안내 신호를 주변에 응답하여 틀에 박힌 경로 다음 배아에서 광범위하게 마이그레이션 할 수 있습니다. 그들의 최종 목적지에 도달하면, 그들은 파생 상품, 예를 들면 신경 세포, 아교, 뼈, 연골, 그리고 색소 세포 1-5의 광대 한 배열로 분화. 때문에 많은 종류의 세포와 배아 조직, NC 세포 개발의 모든 단계에서의 결함, 유도에서 최종 분화에 기여으로, neurocristopathies 6이라는 선천성 증후군의 원인이 될 수 있습니다. E사양, EMT, 이주 및 분화 - - 다른 단계에서 개발하고 NC의 xperimental 조작 neurocristopathies에 대한 우리의 이해를 개선하고 잠재적 인 치료 전략의 디자인을 허용합니다. 는 Xenopus laevis의 배아는 NC의 개발을 연구하는 선택의 모델입니다. 배아의 큰 숫자는 쉽게 구할 수 있고, 외부 수정 개발의 첫 번째 단계에 액세스 할 수 있습니다. 많은 도구를 실험적으로 X를 조작 할 수 있습니다 배아 발달을 laevis의. 이득의 기능과 최저 유전자는 초기 blastulas의 개별 세포를 미세 주입에 의해 수행하기 쉽다. 배아 조직을 체외 재 연결 7-11 또는 백 접목 분석 (12, 13)에 절단 할 수 있습니다. 이 프로토콜에서는, 우리는 X에서 premigratory 두개골 NC를 해부하는 방법에 대해 설명합니다 이주 전에 후반 neurulas을 laevis의. 이러한 외식은 피브로넥틴 도금 파이프에 배양 할 수있다의 이동과 분화를 연구하는 D 판은 체외 실험 조건에서 제어. NC 외식은 생체 내에서 자신의 이동과 분화를 연구하는 정상 또는 조작 호스트 배아에 이식 할 수 있습니다.

<table><tbody><tr><td>Fibronectin from bovine plasma</td><td>Sigma</td><td>F4759-1MG</td><td></td></tr><tr><td>Bovine Serum Albumin, Fraction V</td><td>Euromedex</td><td>04-100-811-C</td><td>Lower quality grade may&amp;nbsp; be suitable for this application</td></tr><tr><td>Stainless Steel Insect Pins, Size 000</td><td>FST</td><td>26001</td><td></td></tr><tr><td>Dumont #5 forceps 0.05 mm x 0.02 mm</td><td>FST</td><td>11252-20</td><td></td></tr></tbody></table>

dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

신경 능선 (NC)이 neurulation의 끝에서 상피 - 투 - 중간 엽 전이 (EMT)를 거쳐 임시 지느러미 신경 튜브의 세포 집단이다, 다양한 기관으로 광범위하게 마이그레이션 및 파생 상품의 많은 종류의 (신경 교세포로 분화 연골과 뼈, 색소 및 내분비 세포). 이 프로토콜에서는, 우리는 생체 내 및 시험 관내에서 NC의 개발을 연구하기 위하여는 Xenopus laevis의 태아에서 premigratory 두개골 NC를 해부하는 방법에 대해 설명합니다. 개구리 모델은 초기 개발을 연구하는 많은 장점을 제공하고, 풍부한 일괄 배아 생체 이득과 기능 전략의 손실 전에 기증자 및 / 또는 호스트 배아에서 NC의 해부 유전자 발현의 조작을 허용, 신속하게 개발, 사용할 수 있습니다. NC 이식편은 피브로넥틴에 도금 및 시험 관내 연구를 위해 사용될 수있다. 이들은 무 혈청 한정 배지에서 몇 일 동안 배양 될 수있다. 우리는 또한 NC 외식을 다시 이식하는 방법에 대해 설명합니다생체 내에서 NC의 이동과 분화를 연구하는 호스트 배아에.

피브로넥틴에 도금 할 때, 신경 능선 외식 신속하게 (15 ~ 30 분)을 첨부하여 이식편은 2 시간 (그림 1A)에서 플랫 퍼집니다. 3-6 시간의 세포가 흩어 시작하면. 15 ° C에서 24 시간, 많은 세포가 떨어져 이식편 (그림 1B 및 1C)에서 이주하기 시작했다. 노른자는 위상차 (그림 1B)에서 세포가 매우 밝은 있습니다. 세포 돌기 (filopodes 및 lamellipodes)은 액틴 세포...

Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

이 프로토콜은는 Xenopus laevis의의 neurulas에서 premigratory 두개골 신경 능선 (NC)를 해부하는 방법에 대해 설명합니다. 이러한 외식은 피브로넥틴 및 체외 배양에 도금, 또는 다시 호스트 배아에 이식 할 수 있습니다. 이 기술은 NC 상피 - 투 - 중간 엽 이행, 이주 및 분화의 메커니즘을 연구 할 수 있습니다.

의 해부<em&gt;는 Xenopus laevis의</em&gt; 신경 크레스트<em&gt; 시험관</em&gt; 이식편 문화 또는<em&gt; 생체</em&gt; 이식

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

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Biology

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

13.2K 조회수.  Centre Universitaire.  이 프로토콜은는 Xenopus laevis의의 neurulas에서 premigratory 두개골 신경 능선 (NC)를 해부하는 방법에 대해 설명합니다. 이러한 외식은 피브로넥틴 및 체외 배양에 도금, 또는 다시 호스트 배아에 이식 할 수 있습니다. 이 기술은 NC 상피 - 투 - 중간 엽 이행, 이주 및 분화의 메커니즘을 연구 할 수 있습니다. 

비디오: Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

줄기 세포처럼<em&gt; Xenopus의</em&gt; 배아의 Explants는 초기 신경 발달의 특징을 연구하는<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

연구

발생학

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

에서 신경 Explant의 문화<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

신경과학

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

의 해부, 문화, 및 분석<em&gt; Xenopus laevis</em&gt; 배아 망막 조직

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

예 자궁 Electroporation 및 전체 반구의 Explants : 초기 두피 개발 연구 간단한 실험 방법

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

신경 문장 세포 박리 및 이동의 연구를 위한 마우스에서 1 차적인 두개골 신경 문장 세포의 해부, 문화 및 분석

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

제노푸스 라에비스의 신경 전구체 특성화를 위한 형광 칼슘 이미징 및 후속 시토 혼성화

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

병아리 두개골 신경 볏 세포 배양의 준비 및 형태학적 분석

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

두개골 Mesenchyme의 셀룰러 행동을 평가하기위한 Explant 분석

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

생물학

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

의 얼굴 이식<em&gt;는 Xenopus laevis의</em&gt; 태아

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Explant Culture of Neural Tissue

The intricate structure of the vertebrate nervous system arises from a complex series of events involving cell differentiation, cell migration, and changes in cell morphology. Studying these processes is essential to our understanding of nervous system function as well as our ability to diagnose and treat disorders that result from abnormal development. However, neural tissues are relatively inaccessible for experimental manipulations, especially in embryonic mammals. As a result, many scientists take advantage of explant culture in order to study neurodevelopmental processes in an &#8220;organotypic&#8221; environment, meaning that the tissue is removed from the organism but its complex cellular architecture is maintained. Generally, explant cultures are created by careful dissection of neural tissue that is then submerged in carefully designed growth media and cultured in vitro.
This video will first provide a brief overview of neural explant culture, including its advantages over other in vitro methods and important considerations for maintaining healthy tissue. Next, a general protocol will be provided for setting up an explant culture from embryonic mouse brain, outlining the isolation of embryos from the mother and dissection of the brain. The presentation also includes an overview of slice culture, in which thin sections of nervous system tissue are generated for improved visual access to the developing cells. Lastly, a few applications of these techniques will be provided to demonstrate how they can be used to answer important questions in the neurodevelopmental field.

신경 조직 절편배양

The intricate structure of the vertebrate nervous system arises from a complex series of events involving cell differentiation, cell migration, and ...

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의 해부, 문화, 및 분석