摘要
研究背景
造血过程是各种血细胞的发生、发育、成熟的过程,是一个多阶段、多步骤、受到多个因子调控涉及多个造血解剖位置的复杂而又有序的动态过程,并贯穿于生命体的一生。尽管各种血细胞组分的生理功能不同,但是它们有着同一祖先——造血干细胞(hematopoietic stem cells,HSCs)。从HSCs向成熟血细胞分化的过程中,经历了造血祖细胞阶段和造血前体细胞阶段,最终分化生成所有成熟血细胞。
经典的模式生物如线虫、果蝇等与人类谱系相距甚远,小鼠则是公认最好的模式生物之一。然而,由于小鼠等哺乳类动物繁殖速度较慢,且体积相对较大,胚胎子宫内发育,难以对早期造血的表型改变进行快速连续观察,因此限制了其作为高通量化学遗传学造血缺陷突变体的筛选与基因鉴定等方面的应用。斑马鱼作为脊椎动物,在基因组、蛋白质组、胚胎发育以及疾病发生机制等方面均表现出与人类高度一致的特点。此外,斑马鱼还具有其他模式生物所没有的独特生物学优势：1)个体小(体长3-4 cm),可在较小的空间内大量养殖；2)体外受精且胚胎透明,其发育过程可在解剖体视镜下直接观察；3)产卵数量多(每条雌性成鱼每周100-300枚)；4)胚胎发育较快,受精后24小时多数重要的生命器官即开始发育,72小时左右小鱼即可出膜游泳；5)性成熟期短(3个月左右),此时即可以交配产卵；6)精子可冷冻保存；7)斑马鱼有30000多个基因,87%与人类具有同源性；8)随着世界上使用斑马鱼的实验室及学者的增多,有关斑马鱼的各项技术操作也日趋成熟完善,并且有专门的斑马鱼网站等等。在对造血系统进行研究时,其发育早期身体透明,有利于在体视镜下直接观察血液系统的发生发展：如血液的循环、心脏的跳动、血细胞的数量等；并且血细胞的谱系及调节血细胞的基因与哺乳动物高度保守；并且斑马鱼心血管系统早期发育与人类极为相似,而血液和心血管系统有缺陷的突变体仍然可以存活数天,为该领域研究提供了极为有利的条件。鉴于这些优点,斑马鱼成为了造血系统研究的最佳模式生物,尤其特别适宜于N—乙基—N—亚硝基脲(N-ethyl-N-nitrosourea, ENU)化学诱变的大规模造血系统正向遗传学突变体的筛选。
与哺乳动物类似,斑马鱼血细胞生成也分为原始造血(primitive hematopoiesis)和定向造血(definitive hematopoiesis)两个阶段,产生的分化细胞组分与哺乳动物中发现的大多数成熟血细胞谱系类似。斑马鱼原始红细胞起源于5体节时期后部侧向中胚层(posterior lateral mesoderm, PLM)的一对双边条纹,最终在20体节(somite)时期形成对应于哺乳动物卵黄囊的中央细胞团结构(intermediate cell mass, ICM)。这里是红细胞进一步分化、成熟,进入血液循环,并于受精后5—7天(day post fertilization, dpf)成熟的地方。斑马鱼原始髓系细胞来源于10体节时期前部侧向中胚层(anterior lateral mesoderm, ALM),主要产生巨噬细胞和中性粒细胞。早期永久HSCs于受精后26—30小时(hours post fertilization, hpf)在等同于哺乳动物AGM的背主动脉腹壁侧(ventral wall of dorsal aorta, VDA)产生。随后这些HSCs在2dpf从背主动脉腹壁侧向体后血岛(posterior blood island, PBI)迁移。最终造血干细胞在5dpf定居在肾脏,这是成年斑马鱼的造血器官。
斑马鱼胚胎血管系统发育起始于6hpf原肠胚形成(gastrulation)时期,此时具有双向分化功能的血液血管母细胞在腹侧中胚层分化(ventral mesoderm,VM)形成。随后,其向中轴线迁移,在12hpf到达侧向中胚层(lateral plate mesoderm, LPM),分化成为成血管细胞(angioblast)。成血管细胞约16hpf在位于内胚层背侧和脊索腹侧之间的中轴线处聚集形成索样结构,称为血管索(vasclar cord, VC),从VC到成熟血管形成还需经历：空腔管道形成(lumen formation),动、静脉分化选择,血流形成以及之后的血管重塑过程中细胞之间、细胞与细胞外基质连接等复杂过程。
本研究运用的化学诱变剂为N—乙基—N—亚硝基脲(ENU)。ENU是一种DNA羟基化试剂,它通过对DNA碱基的烷基化修饰,诱导DNA在复制时发生错配而产生突变,它主要诱发单碱基突变,比较符合大多数的人类遗传性疾病的基因突变情况。ENU的突变效率非常高,可以达到0.9‰,随机突变,不具有倾向性,所以适用于饱和诱变分析斑马鱼的功能基因组。ENU诱导雄性野生型AB斑马鱼(F0代),繁殖F1和F2代。待F2代长大,挑选F2代一雄一雌成鱼同家族内自交,收集F3代胚胎,采用整体原位杂交(whole mount in situ hybridization, WISH)的方法进行大规模遗传学方法筛选红系细胞标记物globinβel基因表达缺失突变体,并初步研究了βe1基因表达缺失突变体在造血过程(原始造血和永久造血)中各个谱系标记物的表达情况,旨在分析βe1基因表达缺失突变体中其他相关造血谱系情况,以期进一步明确造血过程的相关分子调控机制,同时也对βe1基因功能的全面了解、造血过程调节机制和基因定位克隆提供线索和依据。
研究内容共分为两个部分：第一部分ENU突变以及繁殖；第二部分βe1表达缺失突变体的筛选及初步研究。
第一部分ENU突变以及繁殖
1.目的
本部分将介绍斑马鱼培育和繁殖方法、ENU化学诱变以及后期大规模正向遗传学突变体筛选,目的是筛选造血系统发育缺陷的斑马鱼突变体,用于后续研究工作。
2.方法
采用香港科技大学温子龙教授常规ENU突变方法诱变野生型AB雄性斑马鱼,依照本实验室标准斑马鱼培育技术饲养突变后成活雄鱼(F0代),并繁殖到F2代。同家族内挑选一雄一雌F2代鱼自交,收集所产胚胎,于胚胎发育5dpf时期以红系细胞标记物βe1为探针,应用WISH技术筛选βe1基因表达缺失突变体。
3.结果
ENU突变后F0代总共24条鱼存活,其与野生型AB交配产下F1子代2000条,F1代家族间杂交产生F2代,F2代同家族内自交,收集F3代5.0dpf时期的胚胎,应用βel为探针进行WISH。
第二部分βe1表达缺失突变体的筛选及初步研究
1.目的
根据第一部分叙述,本研究在ENU突变基础上,以红系造血细胞标记物βe1为探针,筛选βe1表达缺失突变体。本章节详述βel表达缺失突变体筛选过程及初步研究,目的是了解筛选的具体策略以及初步了解βe1突变体表型,为深入分析βe1基因功能提供线索和依据。
2.方法
首先,采用分子生物学实验技术方法制备原位杂交RNA探针；其次,运用斑马鱼WISH技术检测βe1基因表达；之后,同样通过WISH技术分别检测了20hpf的红系βel、36hpf的造血干细胞c-myb、5.0dpf的淋系rag1和5.0dpf的髓系lyc等基因表达情况；又运用特殊染色方法如O-dianisidine染色法检测原始造血阶段的血红蛋白合成、Neutral Red和Sudan Black B分别检测原始造血阶段的巨噬细胞和粒细胞
3.结果
我们所筛选到的4对突变体分别编号为336-2、350-1、373-6和483-2.结果显示：βe1表达缺失突变体大体形态与发育过程同野生型相比没有明显差别,但是约有近1／4的胚胎(该实验重复三次,每次突变体数目／总体胚胎数目=1／4)βe1基因表达缺失。并初步分三个不同的时间段对其造血系统系统进行检测：原始造血阶段、造血干细胞阶段和定向造血阶段。其中,22hpfβel检测原始红系、36hpf c-myb检测造血干细胞、3.0dpf时用特异性染色剂O-dianisidine对原始造血阶段的血红蛋白进行检测、3.5dpf时的特异性染色剂中性红(NR)和苏丹黑B(SB)分别对原始造血阶段的巨噬细胞和粒细胞进行检测、5.0dpfrag1检测定向淋系、5. Odpfβel验证定向红系、5. Odpf lyc检测定向造血的髓系观察,具体结果如下：我们筛选到的4对突变体,大致可以分为两类：类为定向造血过程中红系和淋系表达均有缺失：373-6及483-1；另一类为定向造血过程中只有红系表达缺失：336-2和350-1。前一类的两对突变体,在对其进行的7项检测指标中,5. Odpf rag1出现较为明显的改变：373-6在70枚鱼卵中出现了16枚表达缺失,而483-1在82枚卵中出现21枚表达缺失,均基本符合1／4的比例。其他的检测指标未发现明显的表达缺失。
4.结论
1.用ENU诱导斑马鱼突变并筛选红系造血突变体的方法是新颖而可靠。
2.我们筛选到的四对突变体：其中336-2和350-1两对突变体的突变位点仅仅只影响定向造血的红系,而另两对突变体373-6和483-2的突变位点应该是同时可以影响定向造血的红系和T淋巴细胞
Background
Hematopoiesis by definition is a complicated and dynamic process that produces all types of blood cells throughout the lifetime of the animal and is involved with a number of hematopoietic anatomical locations. Despite their functional diversities, all these different types of hematopoietic cells arise from a common ancestor known as hematopoietic stem cells (HSCs). During the process of HSCs maturing to blood cells, it experiences a phase containing hematopoietic progenitor cells and hematopoietic precursor cells, and finally differentiates to all the mature blood cells. Therefore, hematopoiesis embraces two aspects:the development of the first HSC from non-hematopoietic tissues in embryonic and fetal life, as well as its later specialization, proliferation and differentiation. Despite extensive studies in the past, the genetic programs governing the specification, migration, and survival of HSCs in these hematopoietic compartments and the mechanisms of hematopoietic development remain poorly understood.
Although many critical concepts and valuable insights have been derived from the study of nematodes, fruit flies in the past, the usage of these conventional systems in dissecting hematopoietic program is limited by their own inherited attributes. Although the development of hematopoiesis has been best characterized in the mouse model, the slow reproduction and relatively large size of mouse, and its embryonic development in uterus are not suitable for the early hematopoietic phenotype observation, especially for the forward genetic analysis. To complement these traditional models, zebrafish, a fresh water tropical fish that is noted to integrate the features suitable for both ENU mutagenesis and large-scale forward genetic screening, has recently been emerged as an excellent vertebrate system to study developmental mechanisms of hematopoietic system. A large number of zebrafish could be maintained in a relatively small space and hundreds of progenies could be collected at weekly intervals. The rapid growth and external development of zebrafish embryos also make it well suitable for embryological observation and manipulation. As hematopoietic program is highly conserved between fish and mammals, the study on zebrafish hematopoiesis would be also contributed to our understanding of this process in higher organisms.
Similarly to mammals, zebrafish hematopoiesis also consists of primitive and definitive programs, and generates differentiated cells analogous to most of the mature blood lineages found in mammals. Zebrafish primitive erythropoiesis originates from the posterior lateral mesoderm (PLM) as a pair of bilateral stripes at 5-somite stage. These stripes subsequently extend anteriorly and posteriorly, and converge in the midline at 20-somite stage to form the main structure of the intermediate cell mass (ICM) where the erythroid progenitors further proliferate and differentiate, enter the blood circulation, and finally mature at around 5-7 days post fertilization (dpf). Zebrafish primitive myelopoiesis arises from the anterior lateral mesoderm (ALM) at 10-somite stage and produces mainly macrophages and neutrophils. HSCs in zebrafish are believed to initiate at 26—30 hours postfertilization (hpf) from the ventral wall of DA (VDA), an equivalence of the mouse AGM. By 2 days postfertilization (dpf), these HSCs in the ventral wall of DA migrate to the posterior blood island (PBI) (also referred to as caudal hematopietic tissue) located between caudal artery and caudal vein, and finally home to kidney, the adult hematopoietic organ in zebrafish, by 5dpf. Thus function analogies of AGM, FL, and BM in zebrafish are very likely to be represented by the ventral wall of DA, PBI, and kidney respectively.
Zebrafish vascular system originates from the ventral mesoderm (VM) as bi-potential cells-hemangioblast at 6hpf stage in gastrulation. Then by 12hpf these cells migrate to lateral plate mesoderm (LPM) where they differentiate into angioblast. By 16hpf, these angioblasts in the LPM converge in the midline of vascular cord located between dorsal ectoderm and notochord. Some complex processes lead to the formation of a functional vascular network, such as sprouting, branching, artery and vein differentiation, lumen formation. The latter remodelling process requires the recruitment of supporting cells, such as smooth muscle cells and pericytes, and the formation of cell-cell and cell-extracellular matrix junctions.
The chemical mutagen N-ethyl-N-nitrosourea (ENU) is used to treat healthy wild type male fish (AB stain, FO). Subsequently the surviving ENU-treated male fish is mated with wild type female fish to generate F1, and further F2. Once the F2 fish mature, they are ready to use for forward genetic screen. In this study, we carried out the forward genetic screen to search forβel-deficient zebrafish mutants. The adult F2 fish will intercross within each F2 family and the resulting F3 embryos from each crossing will be collected at 5dpf and subjected to whole mount in situ hybridization (WISH) with theβel probe. We have got fourβel-deficient fishes which we called 336-2、350-1、373-6 and 483-2 mutants respectively were given for further detailed characterizations of hematopoiesis process. The analysis of the function ofβel gene in hematopoiesis will help us to understand the molecular regulation of hematopoiesis. On the other hand, from the clinical point of view, a basic understanding of molecular regulation mechanisms of hematopoiesis is essential for finding etiopathogenisis, clinical diagnosis and developing new therapeutic approaches for treatment of hematopoietic diseases.
There are two chapters in this study. Chapter one, ENU mutagenesis and generate F1, F2 family. Chapter two, forward genetic screen forβel -deficient mutant and these putative mutants were characterized with different hematopoiesis markers.
Chapter one ENU mutagenesis and generate F1, F2 family
Objective:To use chemical mutagen ENU to treat AB stain male fish and generate F1, F2 family
Method:The mutagenesis process was carried out with chemical mutagen ENU according to Dr. Zilong Wen, HKUST. Subsequently, surviving ENU-treated male fish was used to generate F1, F2 family. The genetic screen was carried out between F2 families to search forβel-deficient zebrafish mutant by WISH.
Results:16 surviving ENU-treated male fishes were used to generate 2000 F1 family. Finally we selected fourβel-deficient zebrafish by WISH. And we have already found 4 putative mutants
Chapter two, forward genetic screen forβel-deficient mutant and these putative mutants were characterized with different hematopoiesis markers.
Objective:To search forβel-deficient mutants and isolate these mutants for further study
Method:From theβel-deficient mutants we isolated, I gave these mutants for further study. The WISH,O-dianisidine staining of hemoglobin, Neutral Red staining for macrophage and Sudan Black B for granulocyte were performed to analyze the putative mutants.
Results:There are no morphological changes between wild type embryos and putative mutants except for the abolished expression ofβel gene. And then four different hematopoiesis markers and three stainings were performed on the fourβel-deficient mutants.The results show that these mutants had not affected in primitive hematopoietic stage, hematopoietic stem cell and definitive myelopoiesis. However, there are no expression of ragl which is specific marker of T lymphocyte in thymus at 5dpf in 336-2 and 350-1 mutants, and 373-6 and 483-2 mutants have similar ragl signal with wild type.
Conclusion:1 The chemical mutagen ENU based on mutagenesis is an excellent method for screening hematopoiesis-deficent mutant and erythroid lineage markerβel globin serve as a reliable marker
2 We have isolated fourβel-deficient mutants by WISH. The two of four mutants, 336-2 and 350-1, show erythroid defects during definitive hematopoiesis, whereas the others,373-6 and 483-2, present defective both in erythriod lineage and lymphocytes development
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