斑马鱼高分辨率整胚原位杂交实验方法与流程

整胚原位杂交技术是利用反义RNA探针检测体内mRNA表达的一项技术, 在利用模式动物研究基因时空表达方面有着重要的应用。如何使用该技术得到特异、高敏感度的表达结果, 对每一个使用该技术的实验室来说都很重要。本实验室参照常规的实验方法, 对该技术加以改进, 使之更加灵敏, 结果更加特异。文章主要以斑马鱼为例, 介绍了整胚原位杂交技术的发展历史, 并重点介绍了本实验室所用的整胚原位杂交实验流程, 同时还分析了实验结果不理想的原因及其解决方法。     

斑马鱼胚胎发育过程中TGF-1基因的表达特征分析

为研究转化生长因子 (Transforming growth factor , TGF)1对斑马鱼胚胎发育的调控作用, 通过NCBI获得TGF-1基因序列, TGF-1 cDNA全长1571 bp, 编码377个氨基酸。系统进化树分析发现, TGF-蛋白按照不同的类型严格聚类, 斑马鱼TGF-1与其他鱼类的TGF-1聚集到一个分支, 在进化中非常保守。对斑马鱼胚胎进行RT-PCR和Real-Time PCR检测显示, TGF-1基因为母源表达基因, 在分节期之前的表达水平比较低, 而从咽囊期开始持续高水平的表达。胚胎整体原位杂交发现, TGF-1基因在斑马鱼24 hpf 胚胎中开始有特异信号出现, TGF-1基因的表达主要分布在腮弓、侧线原基、耳囊、嗅觉基板、心脏和前肾等处, 表明TGF-1基因可能参与斑马鱼胚胎免疫调节、循环系统发育和侧线形成。用低氧处理斑马鱼胚胎, 发现低氧处理24h后斑马鱼胚胎发育延迟。利用Real-Time PCR和胚胎整体原位杂交检测发现, 低氧处理后发育延迟的斑马鱼胚胎中TGF-1 mRNA表达量较常氧组显著降低。以上结果表明, TGF-1基因参与斑马鱼胚胎发育调控, 并且可能与低氧处理后斑马鱼胚胎发育延迟有关。研究结果将为深入研究斑马鱼TGF-1基因的功能奠定基础。       

二甲双胍对斑马鱼骨骼发育及损伤修复的机制研究

二甲双胍(metformin,MET)是治疗糖尿病的一线药物,对骨骼疾病也有一定的治疗效果,但具体作用机制尚不明确.本研究利用斑马鱼(Danio rerio)构建骨质疏松模型,通过荧光观察、骨骼染色、半定量PCR、原位杂交及ELISA等技术方法,探究MET对斑马鱼骨骼发育及损伤修复的作用机制.首先通过胚胎致死率、骨骼矿...     

斑马鱼消化器官发育缺陷突变体的遗传筛选

目的正向遗传筛选斑马鱼肝脏、肠和胆囊发育缺陷突变体。方法 ENU诱变野生型斑马鱼并开展经典的F2代筛选,以lfabp为探针的全胚原位杂交、BES-H2O2-Ac荧光染料分别检测斑马鱼早期胚胎肝脏、肠和胆囊的表型。结果在128个突变基因组中筛选获得了源自14个F2家族的斑马鱼消化器官发育缺陷突变体品系23个,并按表型划分为6类。结论斑马鱼肝脏、肠和胆囊的发育调控机制有相似性和差异性。     

肿瘤坏死因子α突变型斑马鱼在海分枝杆菌感染中的应用研究

肿瘤坏死因子α(tumor necrosis factorα,TNF-α)作为重要的炎性细胞因子,在类风湿关节炎、感染性休克等疾病的发病中起重要作用。为探究TNF-α在宿主抵抗海分枝杆菌感染中的作用,本研究采用成簇的规律间隔的短回文重复序列及其相关蛋白9[clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9]技术,使野生型斑马鱼TNF-α基因发生突变,成功构建并筛选出含有移码突变的TNF-α突变型(TNF-α-/-)斑马鱼。利用原位杂交技术,发现TNF-α-/-斑马鱼中TNF-αmRNA表达水平显著降低。与野生型斑马鱼相比,海分枝杆菌在TNF-α-/-斑马鱼体内扩散和增殖更显著。结果提示,本研究成功构建TNF-α-/-斑马鱼,有助于深入探究TNF-α在斑马鱼抵抗海分枝杆菌感染中的作用及其免疫调节机制。     

外源性视黄酸对斑马鱼心血管系统发育的影响

目的观察不同浓度外源性视黄酸对斑马鱼早期胚胎和心血管系统发育的影响,为进一步研究视黄酸影响斑马鱼心脏前后轴(A-P轴)发育的分子机制提供形态学依据。方法选择斑马鱼胚胎孵育的3,6,9·5,12h四个时间点,用不同浓度视黄酸(1×10-6,1×10-7,4×10-8,1×10-8mol/L)处理斑马鱼胚胎,在解剖显微镜下实时观察斑马鱼胚胎心脏发育的全过程和视黄酸对斑马鱼心脏发育的影响。并采用胚胎整体原位杂交技术观察flk-1mRNA在斑马鱼胚胎的表达。结果1×10-6mol/L视黄酸可导致斑马鱼胚胎表现出多系统的严重畸形,胚胎很快死亡。在胚胎孵育的9·5、12h给与10-7~10-8mol/L浓度的视黄酸,胚胎只表现出心血管系统的畸形,其他系统无明显异常。胚胎整体原位杂交显示视黄酸对flk-1mRNA在斑马鱼胚胎血管的表达没有影响。结论视黄酸影响斑马鱼胚胎心脏发育有剂量依赖性和严格的时间窗,视黄酸影响心脏前后轴发育的关键时间是原肠胚晚期。视黄酸处理组胚胎的循环缺陷主要为心脏发育异常所致。10-7~10-8mol/L浓度视黄酸在9·5、12h处理斑马鱼胚胎可以作为研究心脏发育调控机制的动物模型。     

ripply1在斑马鱼早期胚胎背腹发育中的作用

目的探究ripply1基因在斑马鱼早期背腹轴发生过程中的作用。方法利用斑马鱼整封原位杂交技术揭示ripply1基因在斑马鱼早期胚胎发育过程中的表达模式,通过显微注射技术在胚胎1细胞期注射ripply1的mRNA来高表达Ripply1蛋白并在后期观察胚胎背腹标记基因的变化及胚胎形态变化。利用Tol2转基因技术构建ripply1启动子驱动的GFP转基因鱼。结果原位杂交结果显示ripply1基因在斑马鱼原肠早期胚盾期特异表达在胚盾处,即预定的背部。高表达ripply1后,在胚盾期背部标记基因表达范围扩大,腹部标记基因表达减弱,受精后24小时胚胎表现出严重的背部化表型:头部增大,腹部卵黄延伸减弱,尾部躯干及尾部区域减少,有的甚至形成了第二个体轴。得到的转基因鱼揭示ripply1母源表达,并且转录起始位点上游1200个碱基驱动的GFP能模拟内源基因的表达图式。结论 ripply1可能参与斑马鱼胚胎早期背腹轴的发生。     

Connexin43基因抑制对斑马鱼心血管系统发育的影响

为了研究cx43基因抑制对斑马鱼胚胎心血管系统发育的影响,针对cx43的翻译起始位点设计两个吗啉修饰的反义寡核苷酸抑制其表达,在斑弓鱼受精卵一到两细胞期混合注射并且验证其有效性.注射后用原位杂交和原位免疫荧光检测心脏标志基因的表达以及心脏的表型,同时利用显微荧光造影和原位杂交检测血管的发育情况.用心室心房的标志基因vmhc和amhc反义RNA探针进行的原位杂交结果显示,vmhc表达抑制,而amhc表达上调.原位免疫荧光显示与原位杂交一致的结果表明:心房扩张心室缩小,并且心脏环化不全.用血管标志基凶flk-1的RNA探针原位杂交和显微荧光造影表明,cx43基因抑制的斑马鱼胚胎血管无明显缺陷.此外,cx43基因抑制的斑马鱼胚胎心脏功能也有明显改变,包括心脏搏动无力,有血液回流现象.抑制cx43的表达可能通过影响两个细胞群的迁移导致斑马鱼胚胎心脏的发育缺陷,从而影响了心脏的功能,但是未发现胚胎血管系统发育的明显缺陷.     

Le 斑马鱼nanos1基因在配子发生中的原位杂交研究

利用组织原位杂交技术,以地高辛标记的反义RNA为探针,检测了斑马鱼(Danio rerio) nanos1基因在卵子发生及精子发生中的表达分布特点,初步探讨了该基因在斑马鱼配子发生中可能的功能。结果表明：在斑马鱼卵子发生中,nanos1 mRNA均匀分布于卵原细胞和各时期卵母细胞的胞质中;在卵原细胞和Ⅰ、Ⅱ期卵母细胞中,nanos1 mRNA的杂交信号十分强烈,而较晚期卵母细胞中信号明显减弱。在斑马鱼精子发生中, nanos1 mRNA可在精原细胞和初级精母细胞中检测到。nanos1 mRNA的阳性信号在精原细胞中极为强烈,在初级精母细胞中较为微弱,而精子细胞中没有阳性信号。本研究结果初步表明,斑马鱼nanos1基因对生殖干细胞-卵原细胞和精原细胞的维持和正常功能可能起着重要作用。     

合子基因mcm3在斑马鱼肝早期发育中的作用

目的研究斑马鱼合子基因mcm3是否参与了斑马鱼肝早期发育的调控。方法运用原位杂交分析mcm3在斑马鱼发育早期表达谱;运用mcm3 MO敲降mcm3基因功能,然后用原位杂交及转基因鱼胚胎分析肝的发育状况。结果 mcm3为合子表达基因,在中囊胚期后到尾牙期广泛表达,而在体节发生期及以后分别在体节、头部及内胚层组织高表达。进一步研究发现在mcm3功能敲降后肝变小,而中胚层器官血管心脏的发育并未受到明显影响。mcm3 mRNA回救实验表明mcm3特异的调控了肝的发育。结论斑马鱼合子基因mcm3参与了肝早期发育的调控。     

An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed. A. Cardona and J. Fernández have contributed equally to this work.     

Programmable in situ amplification for multiplexed imaging of mRNA expression

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization.     

Expression of zebrafish Hoxa1a in neuronal cells of the midbrain and anterior hindbrain

The expression pattern of zebrafish hoxa1a mRNA during embryonic development was studied. Herein, we show that hoxa1a mRNA is expressed in the ventral region of both the midbrain and anterior hindbrain during the developmental period from the pharyngula to the protruding-mouth stages via whole-mount in situ hybridization. Furthermore, double-labeling with anti-zHu antibody confirms that the zebrafish hoxa1a gene is expressed in neuronal cells. The observed temporal and spatial distributions of zebrafish hoxa1a mRNA differ greatly from the expression patterns of zebrafish hoxb1a and hoxb1b paralagous genes. In addition, in embryos injected with mouse ihh mRNA, hoxa1a-expressing cells increase in number with a dorsalized expression pattern in the midbrain.     

High-resolution in situ hybridization to whole-mount zebrafish embryos

The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.     

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.     

Spatial and temporal expression patterns of selenoprotein genes during embryogenesis in zebrafish

Selenium is important for embryogenesis in vertebrates but little is known about the expression patterns and biological functions of most selenoprotein genes. Taking advantage of the zebrafish model, systematic analysis of selenoprotein gene expression was performed by in situ hybridization on whole-mount embryos at different developmental stages. Twenty-one selenoprotein mRNAs were analyzed and all of them exhibited expression patterns restricted to specific tissues. Moreover, we demonstrated that highly similar selenoprotein paralogs were expressed within distinct territories. Therefore, tissue- and development-specific expression patterns provided new information for selenoproteins of unknown function.