Name: grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression
Uploaded: 2016-01-17T14:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression at JoVE.com

This work was partly funded by a University of Nottingham Early Career award to DS. RM is funded by the Higher Committee for Education Development of Iraq.

윤리 문 : 이러한 모든 실험 동물 관리 및 노팅엄 대학의 윤리 지침을 따르십시오. 손톱에 대한 헤파린 비즈 1. 준비 <ol><li> 사용하기 전에 PBS에 철저 헤파린 구슬을 씻으십시오. 참고 : 비즈는 PBS의 슬러리로 4 ° C에 저장할 수 있습니다. <ol><li> PBS의 1 ml의 드롭에 20 μL 피펫으로 주식에서 그들을 제거하여 이식을 위해 구슬을 선택합니다. 그런 다음 3cm 페트리 접시에 PBS의 20 μL 드롭에 구슬을 전송하는 2 μL로 설정 마이크로 피펫을 사용합니다. P2 피펫 팁으로 선택한 구슬을 전송, 현미경을 해부 스테레오를 사용하여 크기에 따라 구슬을 선택하고, 그 약 100 μm의 직경은 이상적이다. </li></ol></li><li> 구슬에서 PBS를 제거합니다. 20 μL 피펫 및 미세 워치 집게와 모세관 작용에 의해 잔류 액체와 액체의 대부분을 제거합니다. 주 : 사실상 성장하므로 가능한 한 많이 제거하는 것이 중요이 비드에 첨가 될 때, R는 희석되지 않는다. </li><li> 비드에 직접 성장 인자 피펫. FGF18 위해 PBS에 0.​​1 % BSA 0.5 ㎎ / ㎖에서 재구성 재조합 FGF18의 0.5 μl를 추가합니다. 증발 액체를 방지하기 위해 접시의 가장자리 주위에 물 몇 방울을 놓습니다. </li><li> RT에서 1 시간 동안 인큐베이션 비즈. 파스퇴르 피펫과 접시에서 물 방울을 제거하고 이식에 대한 준비가 얼음에 배치합니다. </li><li> 를 시각화하기 위해 이식 전에 (투명 구슬) 전송을 2 % 페놀 레드에 4-5 구슬이 필요합니다. 배아에 전송하기 전에 PBS로 씻어. </li></ol> 손톱 확장을위한 AG 1-X2 비즈 2. 준비 <ol><li> 0.2 N 포름산 유도체 화 비드를 사용하는 종래. <ol><li> 이 사용을 1.5 ml의 microcentrifuge 관에 구슬을 전송하는 주걱을 수행합니다. 0.2 N 포름산의 1ml를 추가하고 1 시간 동안 진탕 플랫폼에 씻는다. </li><li> 그런 다음 P1000 피펫으로 포름산을 제거하고 1로 교체ml의 물. 1 시간 동안 세척하고 포름산 나머지 제거하기 위해 여섯 번 반복합니다. </li><li> P1000 피펫에 남아있는 액체를 제거하고 4 ℃에서 구슬을 저장합니다. 주 :이 단계에서 모든 잔여 수분을 제거 할 필요가 없다. </li></ol></li><li> 3cm 페트리 접시에 작은 주걱을 사용하여 주식에서 약 5 ~ 10 μL 볼륨의 구슬의 작은 펠렛을 제거하고 DMSO 1 ㎖를 추가 (또는 약물에 용해 용매 중). 참고 :이 적당한 크기의 사람들을 선택할 수있는 수백 개의 구슬을 제공해야한다. </li><li> 그런 다음 다른 3cm 페트리 접시에 용매의 20 μL 드롭에 약 100 μm의 직경 비즈를 전송하는 데 2​​ μL로 설정 P2 피펫을 사용합니다. </li><li> 20 μL 피펫과 2 μL 피펫과 잔류 용매와 용매 제거합니다. 약 20 μL를 적용 할로 교체하십시오. 주 : 일관성 (또는 이제까지 용매가 사용되는) DMSO에 100 μM의 농도로 약물을 용해 들어일부 작은 분자 억제제 -80 ° C에서 20 μL 및 저장소로 나누어지는이 불안정하고 반복 동결 융해를 방지하는 것이 가장 좋습니다. 필요한 경우 다음 구슬을 몸을 담근 전에 원하는 농도로 약물을 희석. 효과적인 농도는 각각의 약물에 대한 경험적으로 결정해야 할 수도 있습니다. </li><li> 1 시간 동안 품어. 이러한 분자의 대부분은 민감한 빛으로 빛으로부터 보호합니다. </li><li> 물에 용해 2 % 페놀 레드의 20 μL에 2 μL로 설정 P2 피펫으로 전송에게 4-5 구슬을 이식하기 전에. 구슬을 시각화하기 위해 스테레오 해부 현미경을 사용하여이 단계를 수행합니다. 고급 시계 제조 집게와 단일 비드 또는 와이어 루프를 제거하고 배아에 전송하기 전에 PBS로 씻어. 이 활동의​​ 손실이 발생할 수 있으므로 사용하기 전에 15 분 이상 2 % 페놀 빨간색 구슬을 두지 마십시오. </li></ol> 3. 텅스텐 바늘을 준비 <ol><li> 3-4cm 길이로 미세 텅스텐 와이어를 잘라. 의 끝 부분에 와이어의 한 조각을 삽입분젠 버너에 유리 파스퇴르 피펫과 녹아은 위치에 고정합니다. 그들이 사용하는 동안 손상된 경우 여분이 있다는 것을 확인하기 위해 10 바늘까지 준비합니다. </li><li> 토치 불꽃으로 와이어의 끝을 배치하고 (2 ~ 3 분 정도) 텅스텐 흰색으로 점등과 끝이 날카로운 될 때까지 기다립니다. 주 : 사용 중에 바늘은 세정 될 수 있으며, 필요에 따라 토치에 re-은 날카롭게. 주의 : 이러한 바늘은 또한 비드를 전송하는 루프를 만들기 위해 사용될 수있다. 부드럽게 벤치에 바늘을 터치하고 그 루프를 형성하도록 구부려 것이다. </li></ol> 구슬 이식 4. 준비 배아 <ol><li> 그들이 원하는 단계 (대부분의 사지 꽃 봉오리 조작 3-5 일)에 도달 할 때까지 무딘 끝이 위로 알을 품어. 쉘을 중단 한 후 껍질을 제거하고 배아를 노출하는 집게를 사용하는 무딘 끝에 무딘 집게 탭을 사용. 달걀 껍질 막 않도록 확인도 제거됩니다. 참고 : PBS의 1-2 ml를 지원하기 위해 파스퇴르 피펫으로 추가 할 수 있습니다이 멤브레인은 노른자을 고수합니다. 부드럽게, 노른자의 표면에 PBS를 피펫 노른자를 손상하지 않도록주의하면서 집게로 막을 잡고 살짝 떨어져 계란에서 잡아 당깁니다. </li><li> 10 ML의 주사기와 계란 19 G 바늘에서 알부민 5 ㎖까지 제거합니다. 배아 생존율을 감소시킬 수있다 큰 볼륨의 제거와 같은 계란을 밀봉하는데 사용되는 테이프와 접촉하지 않는 배아를 보장하기 위해 필요한만큼만 받아. </li><li> 배아는 15 (100) U 페니실린을 포함하는 PBS ml의 0.1 mg의 스트렙토 마이신 / ml의 예술가 인도 잉크의 5-6 방울을 추가 시각화합니다. 0.5을 주입 ml로 직접 1 ML의 주사기와 배아와 25 G 바늘 아래. </li><li> 탈수하지 않는 배아를 보장하기 위해 난자에 1 % 소 태아 혈청과 100 U 페니실린과 0.1 mg의 스트렙토 마이신 / ml의 PBS의 2-3 ML을 추가합니다. 날카롭게 시계 제작자의 집게를 사용하여 난황 막을 제거하고 개발 사지 싹 통해 양막을 엽니 다. 배아가 건조하지 않도록하고필요한 경우, 더 PBS / FCS를 추가합니다. 참고 : 사지 싹이 명확 배아의 측면에 쌍의 outgrowths로 볼 수있다. 이 단계에서 배아는 오른쪽이 일반적으로 가장 위에 켜집니다. 결과는 오른쪽 팔다리에 구슬을 접목과 반대측 컨트롤과 같은 왼쪽 사람을 사용하는 일반적으로 쉽습니다. </li><li> 비드가 이식 될 사지 꽃 봉오리에 절개를 만들기 위해 날카롭게 텅스텐 와이어를 사용합니다. 젊은 단계에서이 어렵지만 가능한 사지 꽃 봉오리를 통해 모든 방법을 절단하지 마십시오. 참고 : 구형 배아 (HH 스테이지 22 이상)에서는 그 래프팅뿐만 사지 자체가 작은 봉오리 같이이 더 어렵 이하 단계에서 사지 싹의 특정 영역을 선택할 수있다. 그것은 사지 봉오리 그래서, 특히 젊은 단계에서, 비드는 근위 사지에 남아 이식 비드지나 성장할 것을 기억하는 것이 중요하다. </li><li> 어느 극세 WA를 이용한 성장 인자 또는 약물 중 하나에서 비드 데리tchmakers 집게 또는 텅스텐 바늘로 만든 루프. 비드가 DMSO 또는 페놀 레드 하나에 침지 된 경우, 배아에 적용하기 전에 PBS로 헹군다. 집게 / 와이어 루프와 배아에 구슬을 전송합니다. 그런 다음 절개로 비드를 삽입 날카롭게 텅스텐 와이어를 사용합니다. </li><li> 배아가 수화 유지되도록 보장 계란에 PBS / FCS의 1-2 ML을 추가하지만이 배아를 손상 비드가 빠질 될 수 있습니다로 배아 자체에 직접 적용하지 마십시오. 테이프로 계란을 밀봉하고 인큐베이터로 돌아갑니다. </li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signals+provide+a+timing+mechanism+for+the+FGF-retinoid+differentiation+switch+during+vertebrate+body+axis+extension.">Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension.</a> Development. 134, 2125-2135 (2007).</li><li>Logan, M., Simon, H. G., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+T-box+and+homeobox+transcription+factors+suggests+roles+in+controlling+chick+limb-type+identity.">Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity.</a> Development. 125, 2825-2835 (1998).</li><li>Borycki, A. G., Mendham, L., Emerson, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+somite+patterning+by+Sonic+hedgehog+and+its+downstream+signal+response+genes.">Control of somite patterning by Sonic hedgehog and its downstream signal response genes.</a> Development. 125, 777-790 (1998).</li><li>Sweetman, D., 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=FGF-4+signaling+is+involved+in+mir-206+expression+in+developing+somites+of+chicken+embryos.">FGF-4 signaling is involved in mir-206 expression in developing somites of chicken embryos.</a> Developmental Dynamics. 235, 2185-2191 (2006).</li><li>Chapman, S. C., Collignon, J., Schoenwolf, G. C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+chick+whole-embryo+culture+using+a+filter+paper+carrier.">Improved method for chick whole-embryo culture using a filter paper carrier.</a> Developmental Dynamics. 220, 284-289 (2001).</li><li>Sweetman, D., Wagstaff, L., Cooper, O., Weijer, C., M&#252;nsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+migration+of+paraxial+and+lateral+plate+mesoderm+cells+emerging+from+the+late+primitive+streak+is+controlled+by+different+Wnt+signals.">The migration of paraxial and lateral plate mesoderm cells emerging from the late primitive streak is controlled by different Wnt signals.</a> BMC Developmental Biology. 8, 63 (2008).</li><li>Scott, B. B., Lois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tissue-specific+transgenic+birds+with+lentiviral+vectors.">Generation of tissue-specific transgenic birds with lentiviral vectors.</a> Proceedings of the National Academy of Sciences of the U.S.A. 102, 16443-16447 (2005).</li><li>Seidl, A. H., 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=Transgenic+quail+as+a+model+for+research+in+the+avian+nervous+system:+a+comparative+study+of+the+auditory+brainstem.">Transgenic quail as a model for research in the avian nervous system: a comparative study of the auditory brainstem.</a> Journal of Comparative Neurology. 521, 5-23 (2013).</li></ol>

The authors have nothing to disclose.

오보에서 조직 개발에 직접 적용 비드 이식편의 사용은 그들이 적용되는 발달 단계 및 노출 기간에 걸쳐 탁월한 제어를주는 개발 중에 성장 인자 신호 전달의 역할을 해부 강력한 도구이다. 분자의 각 유형에 대한 비드의 선택은 중요하다. 그것은 각각 실험적 이들 비드에 억제제의 효과를 시험 할 필요가 있지만, 여기에 설명 된 이러한 억제제 및 레티노 산 작은 소수성 분?...

조류 배아는 몇 년 1 개발의 연구를위한 강력한 도구를 제공하고 있습니다. 가장 유용한 특징 중 하나는 상대적으로 쉽게 조작 할 수 있다는 것이다. 외부 개발은 세포의 운명 2,3- 연구를위한 고전 메추라기 병아리 키메라 시스템과 같은 예를 포함하여 다양한 micromanipulations를 배아 액세스하는 달걀을 열고 수행하는 것을 가능하게 개발 4,5- 동안 특정 조직에서 과발현을위한 레트로 바이러스의 주입 및 이식편 문화는 발달 신호 소스 (6)를 식별합니다. 최근에 GFP를 발현하는 형질 전환 닭 줄에서 이식과 레이블이없는 호스트 사이에 발생하는 키메라 고전 이식 및 유전자 변형의 조합이 개발 7,8에 중요한 통찰력을 제공 할 수있는 것으로 나타났습니다. 조류 배아 조작 될 수있는 용이성을 사지 공부 그것을 우수한 모델을 만들었다개발 9. 생체 현상 사지 특정 성장 인자의 적용은 사지 10,11 패터닝을 변경하고이 프로세스 (12)에 대한 통찰력을 제공하고 요소를 식별하는 수단이되고있다. 이 접근법은 또한 근육 발달을 조절하는 인자를 연구하는 데 사용되었으며, 이러한 Wnts 13, 14, BMPs에 HGF (15) 같은 다양한 신호들에 대한 역할을 밝혀냈다. 최근 이러한 기술은 사지 싹 근육 조직의 유전자 발현을 제어하는 신호를 조사하기 위해 사용되었으며, FGF18 및 레티노 산 사이의 상호 작용이 MyoD 식 (16)의 타이밍을 제어 할 수 있음을 보여 주었다. 비드 상에 로딩하고 정의 발달 단계에서 특이 조직에 직접 그래프트 될 수 성장 인자 및 작은 분자의 조합을 이용하여 개발 중에 거의 모든 시간과 지역을 개입 할 수있는 기회를 제공한다. 이것은 INVE하는데 사용 된체절 패터닝 17, 18, ​​신경 사양 (19), 신경 크레스트 마이그레이션 (20)과 축 연장 (21)를 포함하여 많은 프로세스를 stigate. 여기에서 우리는 닭 사지 개발에 중 성장 인자 또는 억제제에 배어 구슬을 접목하는 방법을 설명합니다. 이것은 계내 혼성화에 근육과 특정 유전자의 발현을 분석하여 이들 신호 근육 생성에 미치는 영향을 결정하기 위해 사용되어왔다. 우리는 레티노 산 또는 특정 신호 전달 경로의 소분자 저해제로서 작은 소수성 분자에 대한 성장 인자, 또는 AG 1-X2 구슬에 사용되는 헤파린 침지 비드를 사용하여 이식 조직을 설명한다. 그러나 다른 구슬도 FGFs (22)과 쉬 (23)을 모두 전달하는 데 사용 된 어떤 사용할 수 있습니다.

<table><tbody><tr><td>Heparin acrylic beads</td><td>Sigma</td><td>H5263</td><td>The acrylic-heparin beads used have been discontinued. However a replacement product is available, Heparin agarose beads, cat no H6508. These are transparent so harder to work with but can be stained with phenol red in the same way as AG 1-X2 beads,</td></tr><tr><td>AG 1-X2 beads</td><td>Bio-Rad</td><td>140-1231</td><td></td></tr><tr><td>Affi Gel blue beads</td><td>Bio-Rad</td><td>153-7301; 153-7302</td><td>These beads have been used with a range of growth factors including Shh and FGFs and can be used to replace heparin beads</td></tr><tr><td>FGF18</td><td>Peprotech</td><td>100-28</td><td>Resuspend in PBS with 0/1% BSA, prepare single use aliquots of 0.5-1ul and store at -80&amp;deg;C. Batches and suppliers can vary so different concentrations should be tested to determine an effective dose.</td></tr><tr><td>U0126</td><td>Cell Signalling</td><td>9903</td><td>Make to 20mM stock in DMSO and store in single use aliquots at -80&amp;deg;C. Protect from light.</td></tr><tr><td>BMS-493</td><td>Tocris Biosciences</td><td>3509</td><td>Resuspend in DMSO and store in single use aliquots at -80&amp;deg;C. Protect from light.</td></tr><tr><td>Black Indian ink</td><td>Windsor and Newton</td><td>5012572003384 (30 ml)</td><td>While alternatives to this product are available care should be taken as some inks are toxic to embryos</td></tr><tr><td>Tungsten wire, 0.1 mm dia. 99.95%</td><td>Alfa Aesar</td><td>10404</td><td></td></tr><tr><td>Penicillin / streptomycin</td><td>Sigma</td><td>P0781</td><td>Dilute 100X in PBS/ink and PBS/FCS</td></tr></tbody></table>

grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression

닭 배아를 사용하여 직접 성장 인자 또는 유전자 발현 및 신호 전달 경로의 활성화에 대한 신호 경로의 어느 특정 억제제의 효과를 테스트 할 수있다. 이 기술은 특정 시간에 대한 정확한 정의 발달 단계에서 신호 전달 분자의 전달을 허용한다. 이 배아 수확 및 유전자 발현 원위치 혼성화, 또는 면역 염색으로 관찰 신호 전달 경로의 활성화에 의해, 예를 들어, 검사 될 수 후. U0126, MEK 억제제에 적신이 FGF18에 배어 비디오 헤파린 구슬 또는 AG 1-X2 구슬, OVO에서 사지 꽃 봉오리에 이식된다. 이 FGF18가 U0126에 의해 억제된다 MyoD 및 ERK의 인산화 및 내인성 및 FGF18 유도 모두 MyoD 발현의 발현을 유도 것을 보여줍니다. 레티노 산 길항제에 배어 구슬은 FGF18에 의해 조기 MyoD 유도를 강화시킬 수 있습니다. 이러한 접근 방식은 우리가 될 수 있습니다다른 성장 인자 및 억제제와의 광범위한 혼성 쉽게 배아에서 다른 조직으로 구성된다.

HH 단계에서 21 MyoD는 염색이 개발 somites (그림 1A)의 myotome에서 볼 수 있지만, 사지 근육 아세포 개발에 표시되지 않습니다. 그림 1B는 FGF18 구슬 이식 다음 MyoD 6 시간 동안 현장 하이브리드에 보여줍니다. 사지 반대측에는 발현은 없지만 MyoD 비드 부근 아세포에서 유도된다. U0126에 배어 구슬, MEK, MyoD (그림 1C)의 블록 FGF18 유도의 특정 억제제를 공동 이식. ?...

Watch this Scientific Journal Video about Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression at JoVE.com

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

유전자 발현에 영향을 미치는 신호 전달 진학을 확인하기 위해 닭 배아 사지 개발에 구슬의 접목

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

grafting-beads-into-developing-chicken-embryo-limbs-to-identify

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.8K 조회수.  University of Nottingham. By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

비디오: Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

최적화<em&gt; 전 비켜</em&gt; 개발의 고급 단계에 병아리 배아의 배양

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

연구

발생학

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

병아리GFP 신경관 이식 및 카르보시아닌 염료 DiI 주입을 사용하여 닭 배아 내신경염 세포 및 혈관의 이중 라벨링

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Zebrafish의 배아에 주입 마이크로 비드

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

초기 치킨 태아의 성장 팩터 - 코팅 비즈 배치

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

생물학

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

HH 스테이지 10 치킨 태아의 비켜 Electroporations에서

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

후보 인자 젖 구슬과 결합하여 불 침투성 장벽의 응용 프로그램은 병아리에서 유도 신호를 공부하기

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

칙의 배아에서 신경 유도에 대한 분석

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

병아리 예 비켜 문화와 예 비켜 CAM 어세이 : 그게 정말로 작동하는 방법

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Recombinant limbs are a powerful experimental model that allows for studying the process of cell differentiation and the generation of patterns under the influence of embryonic signals. This protocol presents a detailed method for generating recombinant limbs with chicken limb-mesodermal cells, adaptable to other cell types obtained from different organisms.

치킨 재조합 사지 분석은 모르포겐, 패터닝 및 세포 분화의 초기 단계를 이해하기 위한

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

Soaked-Bead Assay를 사용한 닭 배아 발생 동안의 세포 분화, Morphogenesis, 및 패터닝 분석

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...