Name: analysis of transforming growth factor &#223; family cleavage products secreted into the blastocoele of xenopus laevis embryos
Uploaded: 2021-07-21T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Transforming Growth Factor ÃŸ Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos at JoVE.com

我们感谢玛丽·桑切斯对动物的出色照顾。作者的研究得到了国家卫生研究院国家儿童健康与人类发展研究所（NIH/NICHD）的资助，该研究所提供R01HD067473-08和R21 HD102668-01，国家糖尿病和消化和肾脏疾病研究所（NIDDK/NIH）授予R01DK128068-01。

所述的所有程序均由犹他大学机构动物护理和使用委员会 （IACUC） 批准。青蛙被安置在IACUC批准的设施中。雄性青蛙通过切开心脏心室，沉浸在三叶草中而安乐死。雌性青蛙在产卵激素诱导后，在实验室中最多安置24小时，以便收集卵子，然后返回护理设施。
1.收集 X. 莱维斯测试
<ol><li>麻醉性成熟的男性在0.2%特里卡因（表1）30-40分钟，直到动物没有反应?...

<ol>
	<li>Harrison, C. A., Al-Musawi, S. L., Walton, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prodomains+regulate+the+synthesis,+extracellular+localisation+and+activity+of+TGF-beta+superfamily+ligands.">Prodomains regulate the synthesis, extracellular localisation and activity of TGF-beta superfamily ligands.</a> Growth Factors. 29 (5), 174-186 (2011).</li><li>Gray, A. M., Mason, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+activin+A+and+transforming+growth+factor--beta+1+pro-regions+in+homodimer+assembly.">Requirement for activin A and transforming growth factor--beta 1 pro-regions in homodimer assembly.</a> Science. 247 (4948), 1328-1330 (1990).</li><li>Constam, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+TGFbeta+and+related+signals+by+precursor+processing.">Regulation of TGFbeta and related signals by precursor processing.</a> Seminars in Cell and Developmental Biology. 32, 85-97 (2014).</li><li>Wang, X., Fischer, G., Hyvonen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+activation+of+pro-activin+A.">Structure and activation of pro-activin A.</a> Nature Communications. 7, 12052 (2016).</li><li>Zhao, B., Xu, S., Dong, X., Lu, C., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prodomain-growth+factor+swapping+in+the+structure+of+pro-TGF-beta1.">Prodomain-growth factor swapping in the structure of pro-TGF-beta1.</a> Journal of Biological Chemistry. 293 (5), 1579-1589 (2018).</li><li>Cui, Y., 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=The+activity+and+signaling+range+of+mature+BMP-4+is+regulated+by+sequential+cleavage+at+two+sites+within+the+prodomain+of+the+precursor.">The activity and signaling range of mature BMP-4 is regulated by sequential cleavage at two sites within the prodomain of the precursor.</a> Genes &amp; Development. 15 (21), 2797-2802 (2001).</li><li>Goldman, D. 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=Mutation+of+an+upstream+cleavage+site+in+the+BMP4+prodomain+leads+to+tissue-specific+loss+of+activity.">Mutation of an upstream cleavage site in the BMP4 prodomain leads to tissue-specific loss of activity.</a> Development. 133 (10), 1933-1942 (2006).</li><li>Le Good, J. A., 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=Nodal+stability+determines+signaling+range.">Nodal stability determines signaling range.</a> Current Biology. 15 (1), 31-36 (2005).</li><li>Tilak, A., 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=Simultaneous+rather+than+ordered+cleavage+of+two+sites+within+the+BMP4+prodomain+leads+to+loss+of+ligand+in+mice.">Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.</a> Development. 141 (15), 3062-3071 (2014).</li><li>Neugebauer, J. M., 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=The+prodomain+of+BMP4+is+necessary+and+sufficient+to+generate+stable+BMP4/7+heterodimers+with+enhanced+bioactivity+in+vivo.">The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo.</a> Proceedings of the National Academy of Science U S A. 112 (18), 2307-2316 (2015).</li><li>Robertson, I. B., Rifkin, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+Bioavailability+of+TGF-beta+and+TGF-beta-Related+Proteins.">Regulation of the Bioavailability of TGF-beta and TGF-beta-Related Proteins.</a> Cold Spring Harbor Perspectives in Biology. 8 (6), 355-372 (2016).</li><li>Zhou, F., 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=GDF6-CD99+Signaling+Regulates+Src+and+Ewing+Sarcoma+Growth.">GDF6-CD99 Signaling Regulates Src and Ewing Sarcoma Growth.</a> Cell Reports. 33, 108332 (2020).</li><li>Nelsen, S. M., Christian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+cleavage+of+BMP4+by+furin,+PC6,+and+PC7.">Site-specific cleavage of BMP4 by furin, PC6, and PC7.</a> Journal of Biological Chemistry. 284 (40), 27157-27166 (2009).</li><li>Birsoy, B., 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=XPACE4+is+a+localized+pro-protein+convertase+required+for+mesoderm+induction+and+the+cleavage+of+specific+TGFbeta+proteins+in+Xenopus+development.">XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFbeta proteins in Xenopus development.</a> Development. 132 (3), 591-602 (2005).</li><li>Mimoto, M. S., Christian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+gene+function+in+Xenopus+laevis.">Manipulation of gene function in Xenopus laevis.</a> Methods Mol Biol. 770, 55-75 (2011).</li><li>Nelsen, S., Berg, L., Wong, C., Christian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proprotein+convertase+genes+in+Xenopus+development.">Proprotein convertase genes in Xenopus development.</a> Developmental Dynamics. 233 (3), 1038-1044 (2005).</li><li>Constam, D. B., Robertson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+bone+morphogenetic+protein+activity+by+pro+domains+and+proprotein+convertases.">Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases.</a> Journal of Cell Biology. 144 (1), 139-149 (1999).</li><li>Sopory, S., Nelsen, S. M., Degnin, C., Wong, C., Christian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+bone+morphogenetic+protein-4+activity+by+sequence+elements+within+the+prodomain.">Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain.</a> Journal of Biological Chemistry. 281 (45), 34021-34031 (2006).</li><li>Sopory, S., Christian, J. L., Whitman, M. A., Whitman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of Growth Factor Signaling in Embryos. , 38-55 (2006).</li><li>Kim, H. S., McKnite, A., Christian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+Activation+of+Bmps:+Analysis+of+Cleavage+in+Xenopus+Oocytes+and+Embryos.">Proteolytic Activation of Bmps: Analysis of Cleavage in Xenopus Oocytes and Embryos.</a> Methods in Molecular Biology. 1891, 115-133 (2019).</li><li>Degnin, C., Jean, F., Thomas, G., Christian, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavages+within+the+prodomain+direct+intracellular+trafficking+and+degradation+of+mature+bone+morphogenetic+protein-4.">Cleavages within the prodomain direct intracellular trafficking and degradation of mature bone morphogenetic protein-4.</a> Molecular Biology of the Cell. 15 (11), 5012-5020 (2004).</li></ol>

这里描述的协议的主要优点是，它可以相对较快地完成，不需要昂贵的试剂，并提供在生理条件下浓缩Tgfí产品的来源。另一个优点是，它允许人们分析表位标记的蛋白质，从而规避缺乏商业上可用的抗体，识别大多数Tgfí家族前列腺素。虽然也可以分析转染培养哺乳动物细胞中表位标记的 Tgf+ 前体蛋白的，但使用X . laevis有几个优点。早期X.laevis胚胎表达所有四个成员的PC家族是内源?...

转化生长因子 +（Tgf í） 超级家庭的成员被合成为非活性、变暗的前体蛋白质。然后，前体由蛋白转化酶 （PC） 家族的成员切割，无论是在分泌途径内还是在细胞外。这创造了一个活跃的，脱硫粘结的配体二元和两个前列蛋白片段1。虽然30多年来人们一直知道，Tgfí家族前体的前列质需要产生一个活跃的配体2，但关于前列质如何促进配体功能的理解是不完整的。
虽然对Tgfí家庭成员的蛋白解激活过程的理解仍然不完整，但人们越来越有兴趣了解哪些 PC 共识主题在体内被切开，是否发生在特定的细胞外或细胞外隔间，以及亲体是否与裂开的配体3有共性或非共性联系。多项研究表明，前列腺素不仅能引导4 、 5前配体折叠，还能影响生长因子稳定性和作用范围6 、 7 、 8 、 9 ，驱动同质体或异质体10的形成，将配体锚定在细胞外基质中，以维持配体延迟11 ，在某些情况下，作为配体的功能本身的权利激活异质性 信号12.Tgfí家族许多成员的前列肌内的异质性突变与人类的眼睛、骨骼、肾脏、骨骼或其他缺陷有关。这些发现突出了前列膜在产生和维持活性配体方面的关键作用，并强调了识别和破译 Tgf+ 家族前体蛋白解成熟期间开发的产品的作用的重要性。
在这里，我们描述了一个详细的协议，用于吸入在 Tgf 1 家族前体成熟期间产生的产品，这些前体来自X . laevis胚胎的胚芽，然后在免疫膨胀上分析它们。此协议可用于确定前体蛋白质中的一个或多个 PC 共识主题是否在体内10、13中裂开，确定切开每个主题的内源 PC （s）13，14、比较Tgf+家族同质体与异质体10的体内形成，或分析Tgf+前体中人类疾病相关点突变是否影响其形成功能性二元配体的能力。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#223;-mercaptoethanol</td>
			<td>Fisher</td>
			<td>AC125470100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Fisher</td>
			<td>B392-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Nitrate</td>
			<td>Fisher</td>
			<td>C109-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pellet Pestle/Tissue Grinder</td>
			<td>Fisher</td>
			<td>12-141-364</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Sigma Aldrich</td>
			<td>F9378-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary, 1 X 90 mm</td>
			<td>Narshige</td>
			<td>G-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin</td>
			<td>Sigma Aldrich</td>
			<td>CG10-10VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection Syringe, 1 mL</td>
			<td>Fisher</td>
			<td>8881501368</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 26 G</td>
			<td>Fisher</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Picoliter Microinjector</td>
			<td>Warner Instruments</td>
			<td>PLI-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>Narashige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Membrane</td>
			<td>Sigma Aldrich</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine-S</td>
			<td>Pentair</td>
			<td>TRS5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of transforming growth factor &#223; family cleavage products secreted into the blastocoele of xenopus laevis embryos

以Tgfí/Nodal或骨形态遗传蛋白（Bmp）配体为代表的转化生长因子+（Tgf+）超级家庭的两只手臂分别在胚胎发育和成人平衡中起着至关重要的作用。Tgf í 家族的成员被制成不活跃的前体，在内质视网膜内变暗并折叠。前体随后被切成配体和前列体碎片。虽然只有二元配体可以与Tgfí受体接触并激活下游信号，但人们越来越认识到，原体的浮躁有助于配体活动。本文描述了一个协议，可用于识别在激活 Tgf+ 前体蛋白质期间产生的产品。RNA编码Tgf+前体首先微注射到 X.莱维斯 胚胎中。第二天，产品从胃肠阶段胚胎的胚芽中采集，并在西方的斑点上进行分析。此协议可以相对较快地完成，不需要昂贵的试剂，并在生理条件下提供浓缩 Tgf + 产品的来源。

下面描述的实验目标是确定Bmp4和Bmp7是否形成异质体（由一个Bmp4配体和一个Bmp7配体组成的迪默），同质体（由两个Bmp4或两个Bmp7配体组成），还是在X.laevis中共同表达它们时的混合体。图2中显示的数据摘自先前发表的研究10。图 2A是一个示意图，显示 Bmp4 或 Bmp7 同质前体或 Bmp4 / 7 异质前体的蛋白解成熟产生的产品。Bmp4 在普罗?...

Watch this Scientific Journal Video about Analysis of Transforming Growth Factor ÃŸ Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos at JoVE.com

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

当转化生长因子 +家族前体蛋白在 Xenopus laevis 胚胎中异位表达时，它们变暗、裂开并分泌到从晚期到早期胃肠阶段的胚芽。我们描述了一种从气穴中吸气产品的方法，用于免疫腹腔分析。

转变生长因子的分析 - 家族产品分泌到 异种胚胎 的爆炸

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

此协议可用于研究在蛋白质裂解后将更广泛的分离前体蛋白（包括 TGF-beta 家庭成员）转化为活性蛋白质的过程。此协议的主要优点是，它提供了非常快速和廉价的方法，以获得高度浓缩的 TGF - β产品在体内可视化条件下。首先，在第二天注射后，取出Ficoll溶液和任何死亡或垂死的胚胎，然后用MBS冲洗胚胎一两次，并在室温下或在16摄氏度的培养器中将MBS中的胚胎培养在长凳上，以减缓发育。使用具有所需设置的微管拉拔器加热和拉玻璃毛细血管到精细点。使用钳子，从拔针尖上剪下来。为了防止堵塞，爆破吸入针的开口应大于微喷针。将吸入针插入连接到微喷射器的针架中，并将针架连接到微操纵器上。将早期到中期胃肠阶段胚胎放到充满 MBS 的注射托盘或盘中。将针头插入动物杆附近的胚胎表面下方。通过解剖显微镜观察针头和胚胎时，按微喷管上的填充按钮。几秒钟后，针头的透明液体水平上升，胚胎崩溃并凹陷。脉冲注射按钮一次或多次，以弹出任何多云的白色物质进入针含有碎片或蛋白酶。要检测免疫腹胀上的产品，根据抗体，吸气 10 至 20 个或以上的胚胎的胚泡液。将一微升无核水撒在注射托盘上的石蜡片上。将针头浸入水中，按下注射按钮，将爆破液排入水中。或者，要将液体直接喷射到副膜上并防止其变平，则脉冲注射按钮，以在较低压力下排出液体。将收获的爆破液转移到冰上无菌微中心管中，并加入无核水，将最终体积调整为 30 微升。为了检测TGF-beta前体蛋白，将爆炸性液体耗尽的胚胎转移到冰上单独的管子中。去除多余的 MBS，并添加 200 微升预冷冻胚胎裂解缓冲器。要使胚胎完全同质化，移液器上下10至20次，直到没有团块存在。将同质化胚胎以1万倍G为10000G的冷藏微中心中离心10分钟，然后用P200移液器取出160微升超分子，并转移到冰上的新管子上，小心避免管下半部分的白蛋黄蛋白和其他细胞碎片。重复一次微中心计算，将128微升的透明超高超高音调剂转移到冰上的新管子上。此时，清除的胚胎解解液和收集的胚泡液可以储存在零下80摄氏度，只要需要。通过使用 PNGase F 通过跨戈尔吉网络修改的爆破液中的裂解蛋白脱脂，并遵循制造商的说明。去糖基化产品将迁移为 SDS 凝胶上更浓缩的带，这有助于准确识别。为了评估在胚泡和裂解物中的前列腺素片段单体和展开的蛋白质，通过在减少条件下添加5个微升，将4倍样品缓冲液添加到15个微光体爆破细胞液和15个澄清胚胎裂解物微升体中来分析蛋白质。为了评估裂开的同质或异质配体在爆破细胞中的形成，通过在其余15个微升的爆破液中加入5个不减少的4倍样品缓冲的微升来分析非减少条件下的蛋白质，然后加热5分钟，将其放在冰上。使用此协议后，蛋白质被SDS-PAGE分离，免疫膨胀物被探测到识别myc表位标签的抗体。在减少条件下，在只表达Bmp4或Bmp7的胚胎的裂解物中，检测到与裂开的Bmp4单体对应的单个带和与裂开的Bmp7单体对应的较慢的迁移带。这两个带子在共同表达Bmp4和Bmp7的胚胎中被检测到。当蛋白质在非减少条件下分离时，在胚胎中检测到单个成熟的Bmp4和7个中间移动的异质体带，以及共同表达Bmp4和Bmp7的微量Bmp4同质体。 当Bmp7蛋白质水平高时，也观察到Bmp4和Bmp7异质体的形成。然而，在这种情况下，多余的Bmp7形成了同质体和胚胎共同表达Bmp4和Bmp7。这些结果表明，Bmp4和7在异种胚胎中共同表达时优先形成异质体。在这个实验中，收集纯爆破球菌是需要精确针头处理经验的最重要步骤。所以，只要继续尝试和修复错误。此过程测试 BMP 异质体是否形成，哪些氨基酸对此很重要。下一步，可以生成敲击鼠标，询问这些氨基酸在哺乳动物发育过程中是否具有重要功能。

Research

JoVE Journal

Developmental Biology

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

染色质监管机构和各国的全基因组快照<em&gt;爪</em&gt;通过胚胎的ChIP-SEQ

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

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 ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

转变生长因子的分析 - 家族产品分泌到 异种胚胎 的爆炸

转变生长因子的分析 - 家族产品分泌到异种胚胎的爆炸