可视化Interrenal类固醇合成组织及其血管微环境斑马鱼
Summary
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。
Abstract
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-β-Hydroxysteroid dehydrogenase /Δ5-4 isomerase (3β-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 3β-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 3β-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.
Introduction
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。
<p class ="“jove_content”">在肾上腺，3β-HSD催化孕酮从孕烯醇酮，17α羟从17α-hydroxypregnelolone从脱氢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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Confocal microscope | Carl Zeiss | LSM510 |
| DMSO | Sigma | D8418 |
| Glycerol | USB | US16374 |
| Hyclone Fetal Calf Serum | GE Healthcare Life Sciences | SH30073 |
| Nicotinamide | Sigma | N0636 |
| β-Nicotinamide adenine dinucleotide hydrate | Sigma | N1636 |
| 4-Nitro blue tetrazolium | Promega | S380C |
| Nusieve GTG | Lonza | 50081 |
| Paraformaldehyde | Sigma | P6148 |
| Phenylthiourea | Sigma | P7629 |
| Phosphate buffered saline | Sigma | P4417-100Tab |
| PYREX Spot Plate | Corning | 7220-85 |
| Reef Salt | AZOO | AZ28001 |
| trans-Dehydroandrosterone | Sigma | D4000 |
| Triton X-100 | Sigma | T8787 |
| Tween 20 | Sigma | P9416 |
| Vibratome | Leica | VT1000M |
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