印度斑马鱼:斑马鱼的介绍

JoVE Science Education
Biology II: Mouse, Zebrafish, and Chick
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JoVE Science Education Biology II: Mouse, Zebrafish, and Chick
An Introduction to the Zebrafish: Danio rerio

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08:31 min
April 30, 2023

Overview

印度斑马鱼是一种小型淡水鱼类,用作生物医学研究中的模式生物。这种鱼类的优点包括它们和人类基因高度保守的相似,以及它们的维护简单、低廉。另外,基因表达在斑马鱼的胚胎中很容易控制,而且它们的透明体征允许我们观察它们的发育过程。在介绍斑马鱼的优点之前,本视频将首先介绍斑马鱼的基本生物学,包括系统发育、生命周期以及自然环境。本视频还将概述斑马鱼的研究历史,将回顾其中的主要发现,包括从早期建立有效的遗传学筛选方法到为人类疾病发现新型的治疗方法,例如癌症。最后,还将讨论许多斑马鱼实验中的一些,包括免疫学和发育学方面的研究。

Procedure

印度斑马鱼,或称斑马鱼,是一种小型鱼类但却在生物医学研究中作出了巨大的贡献。斑马鱼可以产成百上千的卵,它们在体外发育。这样科学家可以进行遗传操作并监测这个复杂生物体中的早期表型。因为斑马鱼和人类的大部分基因组相同,所以对它的的研究能够帮助我们理解并治疗人类的疾病。本短片将全面介绍斑马鱼,斑马鱼成为极佳的模型动物的独特之处,以及一些在今天实验室中使用斑马鱼的方法。

在讨论科学原理之前,让我们先了解一下斑马鱼。如同小鼠和人类,斑马鱼也是脊椎动物,意思是它们都有脊椎。

具体来讲,斑马鱼是多骨鱼,属辐鳍鱼类,特点是在它们的鳍上有辐射型的骨质。更准确的说,斑马鱼属于一类最大的脊椎动物家族:鲤鱼科,它包含有可爱的金鱼在内的共2400多种鱼类。

斑马鱼是这个家族最小的成员之一。成年鱼体长30到40毫米,或者1.5英寸。斑马鱼的名字来源于它们貌似斑马。不是长得像斑马,而是因为在它们鱼雷一样的身体的外表面有纵贯全身的条纹。

斑马鱼起源于喜马拉雅地区,它们通常可以在缓慢流动的淡水水体中找到。但是其实你不必作很远的旅行去寻找它们,因为这种鱼能够适应多种环境,它们在家庭养鱼池就可以稳定生长。

斑马鱼的生命周期主要由4个发育阶段构成:胚胎期,幼虫期,仔鱼期和成年期。当卵子和精子从交配的成鱼体内释放时,周期开始。在受精之后,发育的初期进展迅速。受精三天后,胚胎就发育成为幼虫。从这个时间点开始,直至发育成一个性成熟的成年鱼体需要两到3个月。

我们已经对野生的斑马鱼稍有了解,现在让我们来看一下为什么它们在实验室有这么高的价值。首先,斑马鱼能够高密度生长,饲养简单,比维护其他脊椎动物模型花费较低。

其次,斑马鱼繁殖能力强。成熟的母体每周产卵数百个。

斑马鱼胚胎的体外发育非常有用,因为可以通过显微注射技术来操纵它的基因表达。另外,它的胚胎是透明的,早期的发育进程可以在活体内观察到。

重要的是,斑马鱼像包括人类在内的高等脊椎动物一样,具有高度保守的遗传性状。斑马鱼的基因组含有25条染色体和15亿对碱基,大约是人体基因组的一半。不仅如此,大约70%的人体基因和80%的已知人类疾病的相关基因都可以在斑马鱼内找到至少一个对应体。

我们已经知道了为什么斑马鱼会成为优秀的模型动物,现在我们再来看一下它们是怎样赢得在实验室中的地位的。在上世纪70年代,George Streisinger首先建立了斑马鱼鱼模型。在那时侯,还有几个科研小组在探索果蝇和线虫在发育方面的遗传基础。

作为一个鱼类爱好者,Streisingery意识到斑马鱼作为脊椎动物模型在发育研究方面的潜力。Streisinger 还开创了一种制备产雌胚胎的技术,即胚胎中的遗传物质全部来自于母体,减少了获得纯和突变体所需要的时间。

直到1995年,Charles Kimmel和他的同事们在该领域内对正常斑马鱼的发育进行了彻底的鉴定。

一年以后,Christiane Nusslein-Volhard, Mark Fishman 和 Wolfgang Driever公布了第一次大规模脊椎动物的遗传筛选结果。这些工作是在马萨诸塞州的波士顿和德国的图宾根完成的。继Nusslein-Volhard在果蝇上的建模工作之后,斑马鱼的筛选是用于鉴别胚胎发育所需要的基因。这些结果包括了一个含有两千多个斑马鱼突变体的目录。从此以后,对这些突变体的分析告诉了我们大量的关于我们自身生物体的知识。

2005年,Keith Cheng和他的同事克隆了基因:slc24a5。该基因负责编码金色斑马鱼突变体内不正常的色素沉着。这种金色表型启发了Cheng,继而发现这个特殊的基因在为斑马鱼和人类皮肤细胞内黑色素合成所必需。它对蛋白质的修饰与人类皮肤颜色的变化紧密相关。

在2011年,Leonard Zon实验室的研究人员使用斑马鱼胚胎发现了黑色素瘤的新的治疗方法。在筛选化学药物过程中,他们发现了包括来氟米特在内的一类药物,可以减缓导致黑色素瘤的细胞的生长。在现在的临床试验中,来氟米特是唯一一个以斑马鱼为模型的高通量筛选中发现的新型治疗药物。

您已经知道了斑马鱼模型的价值,现在让我们看一下斑马鱼在当今实验室中的一些用途。

首先,斑马鱼作为人类遗传性疾病的模型非常有用。通过向早期胚胎中进行显微注射可以很容易地复制疾病表型,从而改变蛋白质的表达。这一点通过基因突变也可以实现,例如杜兴氏肌肉营养不良症,表现的症状是对触摸反应异常。

因为斑马鱼自身的免疫系统在受精后的头几天开始发育,所以斑马鱼的胚胎对于感染性疾病的研究也很有帮助。在这个研究中,细菌被注射进入转基因株系的血流,该株系携带有荧光标记的巨噬细胞。这样就可以实时观察宿主的反应。

由于斑马鱼身体透明,它的胚胎还被用于最前沿的神经科学技术,称为光遗传学。研究者们设计了一种胚胎,可以表达来自分离的神经元的蛋白质。这样可以使得他们能够光学激活这些细胞从而在神经通路中确定它特定的功能。

您刚观看的是JoVE对斑马鱼介绍的短片。在本视频中,我们阐述了斑马鱼作为一种独特的脊椎动物模型具有很多脊椎动物系统的优点。将来,在提高我们对于人类疾病的理解和发现临床治疗的新方法上,斑马鱼将会扮演重要的角色。感谢观看!

Transcript

Danio rerio, or zebrafish, are small fish that are making a big splash in biomedical research. Zebrafish lay hundreds of eggs that develop externally, allowing scientists to perform genetic manipulations and monitor early phenotypes in a complex organism. Since they share much of their genome with humans, zebrafish research is helping us on our way to understanding and treating human disease. This video will provide an overview of the zebrafish, the features that make them great models, and some of the ways in which they are used in labs today.

Before we talk about all that fishy science, let’s get to know the zebrafish. Like mice and humans, zebrafish are vertebrates, meaning they possess a backbone.

Specifically, zebrafish are bony fish in the class Actinopterygii, characterized by the presence of bony rays in their fins. More precisely, zebrafish belong to the single largest vertebrate family: Cyprinidae, which contains over 2,400 species, including the loveable goldfish.

Danio rerio are among the smallest members of this family, with adults measuring 30 – 40 millimeters, or about 1.5 inches, long. Zebrafish get their name because they resemble zebras. No, not quite like that. The name derives from the stripes running the length of their torpedo-shaped bodies.

Zebrafish originate from the Himalayan region, where they are found in slow-moving bodies of fresh water. However, you don’t need to travel very far to find them, as Danios are hardy fish that are staples of home aquariums.

The zebrafish life cycle advances through 4 major developmental stages: Embryo, larva, juvenile and adult. The cycle begins when eggs and sperm are released by a mating pair. After fertilization, the initial stages of development progress rapidly, with embryos hatching into larvae by 3 days post fertilization, or dpf. From this point, progression into a sexually mature adult requires an additional two to three months.

Now that we know a little bit about zebrafish in the wild, let’s review why they are so valuable in the lab. First, zebrafish can be housed at high density and are simple to care for, making them less expensive to maintain than other vertebrate models.

Next, zebrafish are extremely fertile. Mature females can lay hundreds of eggs on a weekly basis.

The external development of zebrafish embryos is extremely convenient, because of the ease with which gene expression can be manipulated by microinjection techniques. Additionally, since embryos are transparent, early developmental processes can be observed within the living organism.

Importantly, zebrafish also possess a high degree of genetic conservation with higher vertebrates, including humans. The zebrafish genome contains 25 chromosomes and 1.5 billion base pairs, which is about half the size of the human genome. Nevertheless, approximately 70% of all human genes, and 80% of all known human disease related genes have at least one zebrafish counterpart.

Now that you know why zebrafish make great model organisms, let’s take a look at how they’ve earned their stripes in the lab. In the 1970s, George Streisinger pioneered the establishment of the zebrafish model. At the time, several groups were investigating the genetic basis of development in flies and worms. As a fish hobbyist, Streisinger recognized the potential of zebrafish as a vertebrate model of development. Streisinger developed techniques for making “gynogenetic” embryos, whose genetic material derives entirely from the mother, thus reducing the generation time required to obtain homozygous mutants.

It wasn’t until 1995 that Charles Kimmel and colleagues contributed a thorough characterization of normal zebrafish development to the field.

One year later, Christiane Nusslein-Volhard, Mark Fishman and Wolfgang Driever published the results of the first large-scale vertebrate genetic screen, which was conducted in Boston, Massachusetts and Tubingen, Germany. Modeled after Nusslein-Volhard’s work in Drosophila, this zebrafish screen was designed to identify genes required for embryonic development. The results included a catalog of more than 2,000 mutant zebrafish. Analysis of these mutants has since taught us a great deal about our own biology.

In 2005, Keith Cheng and colleagues cloned slc24a5: the gene responsible for abnormal pigmentation in the golden zebrafish mutant. The golden phenotype inspired Cheng’s discovery that this particular gene is required in fish and human skin cells for synthesis of the pigment melanin, and that modifications in the protein are tightly linked to natural variations in human skin color.

In 2011, researchers in Leonard Zon’s lab used zebrafish embryos to identify a novel therapeutic for melanoma. In a chemical screen, they discovered a class of drugs, including Leflunomide, that slowed the growth of cells that contribute to melanoma. Now in clinical trials, Leflunomide is but one example of the novel therapeutics likely to be discovered in high-throughput zebrafish screens.

Now that you have a feel for the value of the zebrafish model, let’s look at some of the ways in which fish are used in labs today.

To begin, zebrafish are very useful for modeling heritable human diseases. Disease states can be easily reproduced by microinjection of early embryos to alter protein expression. This can also be achieved by genetic mutants, such as this model of Duchenne muscular dystrophy, which exhibits an abnormal response to touch.

Since their innate immune system develops during the first few days post fertilization, zebrafish embryos are also useful for infectious disease research. In this study, bacteria were injected into the bloodstream, and the host response was visualized in real time using transgenic lines with fluorescent macrophages.

Thanks to their transparency, zebrafish embryos are also amenable to a cutting edge neuroscience technique called optogenetics. These researchers engineered an embryo that expresses a protein in isolated neurons, which allows them to optically activate the cell and determine its specific function in a neural circuit.

You’ve just watched JoVE’s introduction to the zebrafish, Danio rerio. In this video, we’ve demonstrated that zebrafish are a unique vertebrate model organism with many of the advantages of invertebrate systems. In the future, zebrafish are likely to play a significant role in improving our understanding of human disease and our discovery of clinically useful therapeutics. Thanks for watching!