Name: visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish
Uploaded: 2016-12-21T13:00:00
Description: Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

我们感谢克里斯托夫·恩格勒特教授和Didier Stainier教授送礼的的Tg（wt1b：GFP）LI1和TG（kdrl：EGFP）S843 株，分别与台湾斑马鱼核心设施提供的Tg（kdrl：mCherry）CI5。这项研究是由赠款科技（96-2628-B-029-002-MY3，101-2313-B-029-001，102-2628-B-029-002-MY3台湾省，支持102- 2321-B-400-018）。

伦理声明：对斑马鱼的所有试验程序都经东海大学的机构动物护理和使用委员会（IRB批准文号101-12），并按照批准的指导方针进行。 1.3β-HSD酶的活性染色股票方案<ol><li>制备反式dehydroandrosterone [10毫克/毫升的二甲亚砜（DMSO）。 </li><li>制备β-烟酰胺腺嘌呤二核苷酸水合物（1.2毫克/毫升的0.1M磷酸盐缓冲液，pH 7.2）。 </li><li>准备烟酰胺（维生素B3，50毫克/毫升H 2 O）。 ...

<ol>
	<li>Hsu, H. J., Lin, G., Chung, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+early+development+of+zebrafish+interrenal+glands+and+pronephros:+differential+control+by+wt1+and+ff1b.">Parallel early development of zebrafish interrenal glands and pronephros: differential control by wt1 and ff1b.</a> Development. 130, 2107-2116 (2003).</li><li>To, T. T., 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=Pituitary-interrenal+interaction+in+zebrafish+interrenal+organ+development.">Pituitary-interrenal interaction in zebrafish interrenal organ development.</a> Mol Endocrinol. 21, 472-485 (2007).</li><li>Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrenal+organogenesis+in+the+zebrafish+model.">Interrenal organogenesis in the zebrafish model.</a> Organogenesis. 3, 44-48 (2007).</li><li>Cravioto, M. 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=A+new+inherited+variant+of+the+3+beta-hydroxysteroid+dehydrogenase-isomerase+deficiency+syndrome:+evidence+for+the+existence+of+two+isoenzymes.">A new inherited variant of the 3 beta-hydroxysteroid dehydrogenase-isomerase deficiency syndrome: evidence for the existence of two isoenzymes.</a> J Clin Endocrinol Metab. 63, 360-367 (1986).</li><li>Lachance, 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=Characterization+of+human+3+beta-hydroxysteroid+dehydrogenase/delta+5-delta+4-isomerase+gene+and+its+expression+in+mammalian+cells.">Characterization of human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase gene and its expression in mammalian cells.</a> J Biol Chem. 267, 3551 (1992).</li><li>Simard, J., 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=Molecular+biology+of+the+3beta-hydroxysteroid+dehydrogenase/delta5-delta4+isomerase+gene+family.">Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.</a> Endocr Rev. 26, 525-582 (2005).</li><li>Lin, J. 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=Two+zebrafish+hsd3b+genes+are+distinct+in+function,+expression,+and+evolution.">Two zebrafish hsd3b genes are distinct in function, expression, and evolution.</a> Endocrinology. 156, 2854-2862 (2015).</li><li>Grassi Milano, E., Basari, F., Chimenti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+and+adrenomedullary+homologs+in+eight+species+of+adult+and+developing+teleosts:+morphology,+histology,+and+immunohistochemistry.">Adrenocortical and adrenomedullary homologs in eight species of adult and developing teleosts: morphology, histology, and immunohistochemistry.</a> Gen Comp Endocrinol. 108, 483-496 (1997).</li><li>Chai, C., Liu, Y. W., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ff1b+is+required+for+the+development+of+steroidogenic+component+of+the+zebrafish+interrenal+organ.">Ff1b is required for the development of steroidogenic component of the zebrafish interrenal organ.</a> Dev Biol. 260, 226-244 (2003).</li><li>Liu, Y. W., Gao, W., Teh, H. L., Tan, J. H., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prox1+is+a+novel+coregulator+of+Ff1b+and+is+involved+in+the+embryonic+development+of+the+zebra+fish+interrenal+primordium.">Prox1 is a novel coregulator of Ff1b and is involved in the embryonic development of the zebra fish interrenal primordium.</a> Mol Cell Biol. 23, 7243-7255 (2003).</li><li>Chai, C., Liu, Y. W., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ff1b+is+required+for+the+development+of+steroidogenic+component+of+the+zebrafish+interrenal+organ.">Ff1b is required for the development of steroidogenic component of the zebrafish interrenal organ.</a> Dev. Biol. 260, 226-244 (2003).</li><li>Chou, C. W., Zhuo, Y. L., Jiang, Z. Y., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hemodynamically-regulated+vascular+microenvironment+promotes+migration+of+the+steroidogenic+tissue+during+its+interaction+with+chromaffin+cells+in+the+zebrafish+embryo.">The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.</a> PLoS One. 9, e107997 (2014).</li><li>Liu, Y. W., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium+is+required+for+the+promotion+of+interrenal+morphogenetic+movement+during+early+zebrafish+development.">Endothelium is required for the promotion of interrenal morphogenetic movement during early zebrafish development.</a> Dev Biol. 297, 44-58 (2006).</li><li>Jin, S. W., Beis, D., Mitchell, T., Chen, J. N., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Proulx, K., Lu, A., Sumanas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+vasculature+in+zebrafish+forms+by+angioblast+cluster-derived+angiogenesis.">Cranial vasculature in zebrafish forms by angioblast cluster-derived angiogenesis.</a> Dev Biol. 348, 34-46 (2010).</li><li>Perner, B., Englert, C., Bollig, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wilms+tumor+genes+wt1a+and+wt1b+control+different+steps+during+formation+of+the+zebrafish+pronephros.">The Wilms tumor genes wt1a and wt1b control different steps during formation of the zebrafish pronephros.</a> Dev Biol. 309, 87-96 (2007).</li><li>Chou, C. W., Chiu, C. H., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+mediates+correct+positioning+of+the+interrenal+organ+in+zebrafish.">Fibronectin mediates correct positioning of the interrenal organ in zebrafish.</a> Dev Dyn. 242, 432-443 (2013).</li><li>Chiu, C. H., Chou, C. W., Takada, S., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+fibronectin+signaling+requirements+of+the+zebrafish+interrenal+vessel.">Development and fibronectin signaling requirements of the zebrafish interrenal vessel.</a> PLoS One. 7, e43040 (2012).</li></ol>

的3β-HSD活性的信号强度增加了反应过程。 4小时从28 HPF向前阶段反应后，检测所述3β-HSD活性的明确信号。然而，反应的持续时间，需要经验确定，这取决于测定的目的。在染色要求夜间处理的情况下，略微偏蓝的背景往往对样品开发。这个问题可以通过3β-HSD活性染色之前增加注视时间（ 例如 ，在在RT 4％PFAT 4小时）来克服。相比之下，overfixation，如固定过夜与RT 4％PFAT，导致了一个非常清...

肾上腺，下丘脑 - 垂体 - 肾上腺轴的重要组成部分，分泌类固醇和类固醇协调平衡和身体应激反应。肾上腺包括外皮层，可分泌类固醇在特定区域的方式，和内髓质，其中合成儿茶酚胺。在硬骨鱼的interrenal腺是在哺乳动物中肾上腺的对应，并且由类固醇合成interrenal和嗜铬细胞，这是分别1-3肾上腺皮质和髓质，功能等价物。已经使用斑马鱼模型进行的研究报告，这两个类固醇和嗜铬细胞谱系是由分子和细胞机制高度类似于那些在哺乳动物1,2-形成。因此，斑马鱼是研究的遗传性疾病，神经内分泌控制和下丘脑 - 垂体 - 肾上腺（interrenal）轴的系统生物学潜在强大的模型。 4,5-转换，和雄烯二酮。 3β-HSD为生物合成的激素类固醇，即孕酮，糖皮质激素，盐皮质激素，雄激素，和雌激素的所有类重要的。两个人3β-HSD同工酶HSD3B1和HSD3B2差异表达6。 HSD3B1在胎盘和外周组织中表达，而HSD3B2在肾上腺皮质和性腺表达。人类HSD3B1和HSD3B2是斑马鱼HSD3B1，这是在interrenal组织和成人性腺表达的共同的直向同源物;斑马鱼hsd3b2是一个母系基因表达的转录的器官前7消失。在整个安装3β-HSD酶活性测定斑马鱼的方案，通过修改利维的方法发达，■通过米兰等描述。上八硬骨鱼类8种冰冻切片。因为组织渗透性和显影斑马鱼的光学透明的，整个安装3β-HSD组织化学可成功地用于固定斑马鱼胚胎和幼虫和具体划定分化interrenal组织。 这个敏感和快速的测定法已应用于各种突变体和morphants展示不同类型interrenal dysmorphogenesis的。所述interrenal3β-HSD活性在其中interrenal组织的规范是通过Ff1b转录因子的特定击倒打乱并作为interrenal分化由Ff1b coregulator Prox1的9,10的击倒影响被降低的胚胎不存在。值得注意的是，3β-HSD活性可在重症早期缺陷的突变体进行检测，如独眼针头和眯 ，其中3β-HSD组织化学界定了interrenal细胞迁移是如何影响11。所述interrenal组织的分化不即使在完全不存在的血液和血管损害。因此，内皮源性信号是如何塑造发展interrenal机关可确定12,13。总体而言，此组织化学测定法已被成功地用于在斑马鱼模型学习规范，分化和类固醇合成的细胞的迁移。因此，它应该是一个高效率和为任何遗传或化学屏幕定位肾上腺和interrenal器官障碍的可靠工具。

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Confocal microscope</td>
			<td style="width:187px;">Carl Zeiss&#160;</td>
			<td style="width:72px;">LSM510&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D8418&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>USB</td>
			<td>US16374</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Fetal Calf Serum&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30073</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Nicotinamide adenine dinucleotide hydrate</td>
			<td>Sigma</td>
			<td>N1636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Nitro blue tetrazolium</td>
			<td>Promega</td>
			<td>S380C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nusieve GTG</td>
			<td>Lonza</td>
			<td>50081</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylthiourea</td>
			<td>Sigma&#160;</td>
			<td>P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>P4417-100Tab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PYREX Spot Plate</td>
			<td>Corning</td>
			<td>7220-85</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reef Salt</td>
			<td>AZOO</td>
			<td>AZ28001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trans-Dehydroandrosterone</td>
			<td>Sigma</td>
			<td>D4000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma</td>
			<td>P9416</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000M</td>
		</tr>
	</tbody>
</table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

要确定如何与pronephric肾小球及新生血管的生成类固醇interrenal组织codevelops中，3β-HSD酶活性测定的双转的Tg（wt1b：GFP）进行LI1;的Tg（kdrl：mCherry）CI5胚胎在34 HPF （ 图1）。在这个阶段，3β-HSD活性阳性类固醇合成组织位于右向中线，并立即尾鳍到pronephric肾小球，而一些类固醇合成细胞开始跨越中线迁移并形成从背视图一个突出边缘?...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

在斑马鱼的interrenal腺是哺乳动物的肾上腺的硬骨鱼类的对应。此协议引入了如何执行3-β羟基类固醇脱氢酶（Δ5-4异构酶;3β-HSD）的酶活性测定法，其检测在显影斑马鱼分化类固醇合成的细胞。

可视化Interrenal类固醇合成组织及其血管微环境斑马鱼

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

Research

JoVE Journal

Developmental Biology

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Dissection of Larval Zebrafish Gonadal Tissue

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em&gt;果蝇</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Live Imaging of Mouse Secondary Palate Fusion

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

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 ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

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

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...