这项工作是由R01 - DE018234和R01 - DE019638。

1。准备供体组织<ol><li>准备文化传媒，增强玻璃引脚，增强钨针。 </li><li>从壳收集胚胎，在冰冷的PBS洗。 <ol class="lalpha"><li>使用一个10毫升的注射器和18号针头从蛋壳指出删除1.0毫升的白蛋白。 </li><li>制作的外壳用剪刀点上方的一个小孔，然后切一个圆形开口揭露胚胎</li></ol></li><li>删除头和地点到DMEM培养液（无血清，室温） </li><li>额鼻过程剖析从胚胎（或其他捐助者的网站，如上颌或下颌过程） </li><li> 2.4U /毫升的PBS dispase精华为20分钟冰</li><li> 1％BSA停止消化封面用DMEM组织</li><li>削尖的钨针，使用单独的外胚层，间质细胞和神经外胚层</li><li>移植组织的DMEM 1％BSA的冰商店，直到主机是准备用于移植</li></ol> 2。准备主机<ol><li>揭露胚胎<ol class="lalpha"><li>使用一个10毫升的注射器和18号针头从蛋壳指出删除1.0毫升的白蛋白。 </li><li>制作的外壳用剪刀点上方的小孔。放置一块孔的磁带，然后切一个圆形开口揭露胚胎。 </li></ol></li><li>使用2双钳，把握羊膜，轻轻地撕了一个洞。 </li><li>转动头部，右眼上放置一对镊子和施加压力。按住头部稳定，使用钨针取出主机外胚层容纳嫁接。 </li><li>将移植到主机使用的玻璃吸管。 </li><li>嫁接，以取代删除外胚层的位置。玻璃针插入针嫁接嫁接组织的每个角落。为了使玻璃引脚，拉microcapillary管到细点超过酒精火焰。 </li><li>在孔放置磁带紧密，并返回适当的长度进行分析的时间，胚胎的孵化器。 </li><li>另见：9 </li></ol> 3。代表性的成果： 为了评估嵌合体胚胎应当收集和处理主机和捐助组织的分布进行分析。鹌鹑细胞，使用QcPN抗体免疫组织化学检测（中描述：3）就业，石蜡切片原位杂交检测小鼠细胞，用于6。应只限于供体细胞的上皮细胞，和捐助方的间叶组织不应该有证据（图1）。孤立的供体细胞在间质的存在表明污染的神经嵴细胞的存在，这将混淆的任何形态的解释，由于面部发展的许多方面（如10），这些细胞的影响。一旦确信污染的间质细胞移植技术是免费的，可用于进一步的形态学或分子的结果措施评估嵌合体。对于我们而言，我们发现对应的上颚被移植的组织（图2），并在嫁接外胚层间叶组织中的分子变化引起的（图3）重复异位软骨和骨骼的存在5,6。 <img src="/files/ftp_upload/2557/2557fig1.jpg" alt="图1" /> 图1。嵌合体的评估（一）反QcPN染色是用来检测鹌鹑鹌鹑，鸡嵌合体细胞。有没有证据证明在间质细胞染色。 （二） 在原位杂交技术用来检测鼠标小鸡嵌合体表达正弦B2成绩单。表达仅限于外胚层组成的嫁接。 <img src="/files/ftp_upload/2557/2557fig2.jpg" alt="图2" /> 图2：三色染色显示上颚骨骼（一）鹌鹑鸡嵌合体的重复，和（B）鼠标小鸡嵌合体。 <img src="/files/ftp_upload/2557/2557fig3.jpg" alt="图3" /> 图3。外胚层regultes间质细胞的基因表达。 （a）正常BMP7的表达在小鸡胚胎的间质（ 放射性原位杂交积极的信号，在Adobe Photoshop中的伪彩色红色）。 （二）BMP7的表达，是诱发相邻的间质细胞在嵌合胚胎移植（箭头）。

<ol>
	<li>Noden, D. 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+the+Neural+Crest+in+Patterning+of+Avian+Cranial+Skeletal,+Connective,+and+Muscle+Tissues.">The Role of the Neural Crest in Patterning of Avian Cranial Skeletal, Connective, and Muscle Tissues.</a> Developmental Biology. 96, 144-144 (1983).</li><li>Bronner-Fraser, M., Stern, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Mesodermal+Tissues+on+Avian+Neural+Crest+Cell+Migration.">Effects of Mesodermal Tissues on Avian Neural Crest Cell Migration.</a> Developmental Biology. 143, 213-213 (1991).</li><li>Schneider, R. A. <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+can+form+cartilages+normally+derived+from+mesoderm+during+development+of+the+avian+head+skeleton.">Neural crest can form cartilages normally derived from mesoderm during development of the avian head skeleton.</a> Developmental Biology. 208, 441-441 (1999).</li><li>Couly, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+Hox-negative+cephalic+neural+crest+cells+and+the+foregut+endoderm+in+patterning+the+facial+skeleton+in+the+vertebrate+head.">Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head.</a> Development. 129, 1061-1061 (2002).</li><li>Evans, D. J., Noden, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+relations+between+avian+craniofacial+neural+crest+and+paraxial+mesoderm+cells.">Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells.</a> Dev Dyn. , (2006).</li><li>Hu, D., Marcucio, R., Helms, J. 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+zone+of+frontonasal+ectoderm+regulates+patterning+and+growth+in+the+face.">A zone of frontonasal ectoderm regulates patterning and growth in the face.</a> Development. 130, 1749-1749 (2003).</li><li>Hu, D., Marcucio, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+organization+of+the+frontonasal+ectodermal+zone+in+birds+and+mammals.">Unique organization of the frontonasal ectodermal zone in birds and mammals.</a> Dev Biol. 325, 200-200 (2009).</li><li>Le Li&#232;vre, C. S., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+derivatives+of+the+neural+crest:+analysis+of+chimaeric+quail+and+chick+embryos.">Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos.</a> Journal of Embryology and Experimental Morphology. 34, 125-125 (1975).</li><li>Bollag, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+repetitive+mouse+B2+element+to+identify+transplanted+mouse+cells+in+mouse-chick+chimeras.">Use of a repetitive mouse B2 element to identify transplanted mouse cells in mouse-chick chimeras.</a> Exp Cell Res. 248, 75-75 (1999).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Eames, B. F., Schneider, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quail-duck+chimeras+reveal+spatiotemporal+plasticity+in+molecular+and+histogenic+programs+of+cranial+feather+development.">Quail-duck chimeras reveal spatiotemporal plasticity in molecular and histogenic programs of cranial feather development.</a> Development. 132, 1499-1499 (2005).</li><li>Odent, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Sonic+hedgehog+(SHH+)+gene+during+early+human+development+and+phenotypic+expression+of+new+mutations+causing+holoprosencephaly.">Expression of the Sonic hedgehog (SHH ) gene during early human development and phenotypic expression of new mutations causing holoprosencephaly.</a> Hum Mol Genet. 8, 1683-1683 (1999).</li><li>Szabo-Rogers, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+skeletogenic+patterning+roles+for+the+olfactory+pit.">Novel skeletogenic patterning roles for the olfactory pit.</a> Development. 136, 219-219 (2009).</li></ol>

使用这种移植方法，使我们能够确定外胚层包含信令信息，调节背腹极性和proximodistal扩展上颌。鹌鹑或鼠标外胚层，并在这在许多物种6,11组织中的分子信号的保护成果时使用的相似性，表明这是一个高度保守的信号脊椎动物之间的中心。此外，其他研究者使用了类似的技术来测试头外胚层表面的不同区域的信号特性，并发现存在多种信号， 有助于面对12的形态发生在外胚层中心?...

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<td>1x PBS</td>
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<td>Microcapillary tube </td>
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<td>Razor blade</td>
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<td>Fine Science Tools</td>
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assessing signaling properties of ectodermal epithelia during craniofacial development

实验胚胎学家了解细胞的命运，在发展和组织的相互作用，调节脊椎动物的图案和形态发生（ 例如，1，2，3，4）作用，帮助禽流胚胎无障碍。在这里，我们说明了一种方法，利用此辅助测试期间面部发展的信令和外胚层组织的图案属性。在这些实验中，我们创建了鹌鹑鸡5或鼠标小鸡6嵌合体移植头的表面外胚层，涉及到同一地区或鸡胚异位地区从鹌鹑或鼠标的上颚。作为供体组织移植到雏鸡的鹌鹑是一个目前在鹌鹑，但不是小鸡细胞的核仁标记的优势，从而使调查来区分主机和捐助组织7。同样，一个重复的元素是在小鼠基因组中是无所不在表示，这使我们能够区分主机和捐助组织在鼠标小鸡嵌合体8。使用鼠标作为供体组织的外胚层，将大大扩展我们对这些组织间的相互作用的理解，因为这将使我们能够测试外胚层的信号来自各种突变胚胎的属性。

Watch this Scientific Journal Video about Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development at JoVE.com

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

本文介绍了组织移植技术的目的是测试头的表面外胚层在颅面部发育中的信令和图案属性。

评估外胚层上皮细胞在颅面发育的信号属性

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

assessing-signaling-properties-ectodermal-epithelia-during

Research

JoVE Journal

Biology

9.4K 次观看.  University of California San Francisco. 本文介绍了组织移植技术的目的是测试头的表面外胚层在颅面部发育中的信令和图案属性。

视频: Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

科研

生物学

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

评估具体物种的贡献颅面开发利用鹌鹑，鸭嵌合体

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...