Name: understanding early organogenesis using a simplified in situ hybridization protocol in xenopus
Uploaded: 2015-01-12T12:00:00
Description: Watch this Scientific Journal Video about Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus at JoVE.com

The authors would like to acknowledge the CIHR for fellowship support of Steve Deimling and the Department of Paediatrics, University of Western Ontario for support of Steve Deimling, Rami Halabi and Stephanie Grover. This work was supported by the NSERC grant R2654A11 and an NSERC Discovery Accelerator Supplement

1. 배아 준비 <ol><li> 배아 문화, pH가 8.0 이전의 고정 8 2.5 % 시스테인을 사용하여 드 젤리 배아의 일환으로 정기적으로하지 않으면. 이 절대적으로 필요하지 않지만 수동으로 미세 집게를 사용하기 전에 고정에 시비 봉투를 제거하려면 유용합니다. <ol><li> 배아를 전송하는 데 유리 파스퇴르 피펫을 사용합니다. 피펫은 배아 그렇게 배아를 선택하기에 충분히 넓은 지점에서 유리 피펫을 절?...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+is+required+to+regulate+anterior-posterior+patterning+in+the+Xenopus+lateral+plate+mesoderm.">Fgf is required to regulate anterior-posterior patterning in the Xenopus lateral plate mesoderm.</a> Mech Dev. , (2011).</li><li>Fletcher, R. B., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+FGF+signaling+in+the+establishment+and+maintenance+of+mesodermal+gene+expression+in+Xenopus.">The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus.</a> Dev Dyn. 237, 1243-1254 (2008).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of Xenopus laevis : a laboratory manual. , (2000).</li><li>Park, E. C., Hayata, T., Cho, K. W., Han, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+cDNA+microarray+identification+of+genes+with+endodermal+organ+expression.">Xenopus cDNA microarray identification of genes with endodermal organ expression.</a> Dev Dyn. 236, 1633-1649 (2007).</li><li>Horb, M. E., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoderm+specification+and+differentiation+in+Xenopus+embryos.">Endoderm specification and differentiation in Xenopus embryos.</a> Dev Biol. 236, 330-343 (2001).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin) : a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Gebhardt, D. O., Nieuwkoop, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Lithium+on+the+Competence+of+the+Ectoderm+in+Ambystoma+Mexicanum.">The Influence of Lithium on the Competence of the Ectoderm in Ambystoma Mexicanum.</a> J Embryol Exp Morphol. 12, 317-331 (1964).</li><li>Nieuwkoop, P. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPOX2-peroxidase+expression+and+the+XLURP-1+promoter+reveal+the+site+of+embryonic+myeloid+cell+development+in+Xenopus.">XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus.</a> Mech Dev. 117, 173-186 (2002).</li><li>Drysdale, T. A., Tonissen, K. F., Patterson, K. D., Crawford, M. J., Krieg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+troponin+I+is+a+heart-specific+marker+in+the+Xenopus+embryo:+expression+during+abnormal+heart+morphogenesis.">Cardiac troponin I is a heart-specific marker in the Xenopus embryo: expression during abnormal heart morphogenesis.</a> Dev Biol. 165, 432-441 (1994).</li><li>Carroll, T., Wallingford, J., Seufert, D., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+pronephric+development.">Molecular regulation of pronephric development.</a> Curr Top Dev Biol. 44, 67-100 (1999).</li><li>Tonissen, K. F., Drysdale, T. A., Lints, T. J., Harvey, R. P., Krieg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XNkx-2.5,+a+Xenopus+gene+related+to+Nkx-2.5+and+tinman:+evidence+for+a+conserved+role+in+cardiac+development.">XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for a conserved role in cardiac development.</a> Dev Biol. 162, 325-328 (1994).</li><li>Davidson, L. A., Ezin, A. M., Keller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+wound+healing+by+apical+contraction+and+ingression+in+Xenopus+laevis.">Embryonic wound healing by apical contraction and ingression in Xenopus laevis.</a> Cell Motil Cytoskeleton. 53, 163-176 (2002).</li><li>Hurtado, R., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sensitivity+and+stability+in+two-color+in+situ+hybridization+by+means+of+a+novel+chromagenic+substrate+combination.">Enhanced sensitivity and stability in two-color in situ hybridization by means of a novel chromagenic substrate combination.</a> Dev Dyn. 235, 2811-2816 (2006).</li></ol>

특정 유전자의 발현 패턴을 시각화하는 계내 혼성화에 사용하는 능력은 제노 푸스 배아의 특정 기관 또는 세포 유형을 식별하기 위해 가장 일반적으로 사용되는 방법에 남아있다. 이는이 기술에 의해 제공되는 몇몇 이점이다. 유전자의 발현은 해당 셀 (18)의 명확한 경계하기 전에 심장 전구 세포에서 nkx2.5 식의 사례로 잘 분화의 조직 학적 서명하기 전에 특정 구조를 ...

The expression pattern of a specific gene is an important piece of information in determining the potential role for that gene in the development of a specific organ or cell type. Simply put, if it is not expressed at the right time and place it is unlikely to play a key role. In Xenopus, as in most early embryos, the most commonly used assay for detecting the expression of a gene is whole mount in situ hybridization using labeled antisense RNA probes. The use of antibody staining to assess expression of a gene in Xenopus is becoming more common as researchers discover antibodies, usually raised against mammalian proteins, that cross react to the Xenopus homologue or generate their own 1-3. However, the vast majority of studies on Xenopus organogenesis still utilize antisense RNA probes. When antibodies are used, each individual antibody often requires optimization for the primary antibody concentration or fixation protocols. In contrast, the protocol for in situ hybridizations is essentially invariant for different probes. The basic concept is relatively simple and an excellent standard protocol has been well established 4. Our protocol is a streamlined version of the original protocol 4 that still provides excellent detection of gene expression patterns in the early embryo. The embryos are fixed and then prepared for hybridization by changing solutions and temperatures such that it allows for high stringency binding of the labeled antisense RNA probe to its target mRNA. The unbound probe is washed away and the embryos are then prepared for binding of an antibody against the label on the RNA probes. Excess antibody is then washed away and an enzymatic color reaction is used to localize where the RNA probe is bound in the embryo. There are now a number of Xenopus transgenic lines that drive expression of fluorescent proteins in specific tissues and these are available at the Xenopus stock centers such as the National Xenopus Resource in Woods Hole. While very useful for many experiments that require examining organogenesis in living embryos, this option requires separate housing for the transgenic lines. 
In situ hybridization can clearly delineate where specific organs or cell types will form in the early embryo (Figure 1). The technique is remarkably sensitive given that one can detect gene expression in a small number of cells in a single embryo 5. However, in situ hybridization using the intensity of colorimetric staining is not considered quantifiable because the color reaction is not a linear one. Despite difficulty in quantifying staining intensity, changes in expression are often quite noticeable; particularly when the in situ hybridization shows quantifiable increases or decreases in the size of expression domains 6,7. 
The clear advantages of whole mount in situ hybridization make it a critical assay in the study of early development. However, it is a time consuming one that requires many steps over several days. This protocol is a simplified version of the standard protocol that eliminates several steps without reducing the quality of the in situ result. The simplification also eliminates sources of variability, making trouble shooting easier if an in situ hybridization is not optimal. Specifically, we have eliminated the use of proteinase K and RNAse treatments of the embryo, two steps that can depend on reagent quality and can also reduce signal intensity if overdone. The protocol also provides some degree of cost saving due to eliminating the use of several reagents. Finally, this protocol also provides some simple guidelines for improved capturing of images of in situ hybridization results. Although this protocol is optimized for work in Xenopus embryos, it is likely that at least some of the simplifications will be applicable to in situ hybridization work in other embryo systems.

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understanding early organogenesis using a simplified in situ hybridization protocol in xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

조직 특이 적 프로브의 사용은 특정 기관에 대한 개발의 상태에 관해서 뛰어난 정보를 제공 할 수있다. 다음 예에서, 배아 단계는 Nieuwkoop 및 파버 스테이징 테이블 (11)에 기초한다. 하나는 프로브 형태 분화 후 유전자 발현, 단계 28-30에서 심장 트로포 닌 I (예를 들어,도 1C), 차별화 된 장기의 존재 또는 크기를 사용하는 경우, 어느 단계 포스트 분화에서 평가 될 수있다. ...

Watch this Scientific Journal Video about Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus at JoVE.com

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

이해 간체를 사용하여 초기 형성기<em&gt; 현장에서</em에서&gt; 하이브리드 프로토콜<em&gt; 아프리카 발톱 개구리</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

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Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

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

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

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, ...

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Technique to Target Microinjection to the Developing Xenopus Kidney

개발에 미세 주입을 대상으로 기술<em&gt; Xenopus의</em&gt; 신장

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

멀티 타겟 원체 전체 마운트<em&gt; 현장에서</em&gt; 하이브리드에서 유전자 발현의 도메인을 비교<em&gt; 초파리</em&gt; 배아

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Multiciliated 셀 전체 마운트 형광 제자리에서 교 잡 및 면역 형광의 결합 된 프로토콜을 통해 Zebrafish에 시각화

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Prometaphase 팔 응집력과 분열의 상태를 모니터링 하려면 형광 제자리에서 교 잡 (물고기)를 사용 하 여 나 초파리 Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

2 단계 접근-그리고 늦은-초기 조류 모델에서 기관 형성의 탐구: Thymus 및 부 갑 상선 동맥 Organogenesis 패러다임

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

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

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 ...

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount Astyanax 배아에 대 한 제자리 교 잡

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

제브라피시의 인시투 혼성화 염색의 게놈 및 정량화

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...