SCREEN FOR LYMPHOID MUTANTS

筛查淋巴细胞突变体

• 批准号: 6629110
• 负责人: NIKOLAUS S TREDE
• 金额: $ 12.49万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629110
• 关键词: artificial chromosomes; chromosome walking; developmental genetics; developmental immunology; gene mutation; genetic mapping; genetic screening; histogenesis; in situ hybridization; lymphopoiesis; nonmammalian vertebrate embryology; pharynxs; regulatory gene; thymus; zebrafish

During lymphopoiesis a pluripotent stem cell develops into T or B lymphocytes. The thymus provides a specialized environment for terminal T lymphocyte differentiation. By studying natural mouse mutants and "knock-out" mice, several of the important genes in lymphocyte development and thymic organogenesis have been recently characterized. A full understanding of lymphopoiesis would also require the identification and hierarchical ordering of very early regulatory genes. To achieve this understanding we propose to take a novel approach using the zebrafish (Danio rerio) as a genetic model system. The zebrafish combines genetic and embryologic advantages and thus offers an ideal model system to study lymphopoiesis. A mutagenesis screen was done using chemical mutagenesis and ionizing radiation. F2 progeny from mutagenized fish were screened with probes for the recombination activating gene-1 (Rag-1) and alpha embryonic globin by whole mount in-situ hybridization. Of 17 potential mutants with absent Rag-1 staining in the thymus, eight were confirmed to date by F2 incrossing. One of these mutants, CZ-3, is an autosomal recessive mutation which results in microcephaly, microphthalmia and abnormalities of the pharyngeal arches. Lymphoid markers, including the earliest gene in lymphoid ontogeny Ikaros, are totally absent in thymi from CZ-3. Histologic sections show a rudimentary thymus and complete absence of lymphocytes by ultrastructural analysis. Other hematopoietic lineages are normal in CZ-3. I have undertaken a positional cloning strategy to identify the CZ-3 gene. I have mapped the mutated gene with a closely linked marker, z10517 (0.33 cM based on 9/2744 meiotic recombinants) and have established a YAC contig distal to z10517. The zebrafish homolog of whn, the nude mouse gene, colocalizes with z10517 on the same YAC clone. whn is an attractive candidate for the gene mutated in CZ-3, as the immune deficiency in the nude mouse is the result of a rudimentary thymus. A specific aim of this proposal is to test if CZ-3 and whn are identical. If they are not, I will continue the chromosomal walk and clone CZ-3 by genetic and functional means. If CZ-3 and whn are identical I will map the remaining mutants. I will select two mutants with interesting phenotypes (which fall into two groups: those with normal and with abnormal pharyngeal arches) for fine-mapping. Cloning of these genes will further our understanding of phylogeny of the immune system as well as of molecular events in both normal T lymphopoiesis and diseases such as immune deficiencies and leukemias and will be instrumental in devising novel therapeutic strategies.

在淋巴生成过程中，多能干细胞发育成T或B淋巴细胞。胸腺为终末T淋巴细胞分化提供了专门的环境。通过对自然突变小鼠和“基因敲除”小鼠的研究，最近已经确定了几个与淋巴细胞发育和胸腺器官发生有关的重要基因。对淋巴生成的充分了解还需要对非常早期的调控基因进行鉴定和分级排序。为了实现这一理解，我们建议采用一种新的方法，使用斑马鱼(Danio Rerio)作为遗传模型系统。斑马鱼结合了遗传学和胚胎学的优势，因此为研究淋巴生成提供了一个理想的模型系统。采用化学诱变和电离辐射的方法进行诱变筛选。以重组激活基因-1(RAG-1)和α胚胎珠蛋白为探针，通过整装原位杂交法筛选诱变鱼的F2代。在17个胸腺中没有RAG-1染色的潜在突变体中，有8个通过F2杂交得到确认。其中一个突变，CZ-3，是一种常染色体隐性突变，导致小头畸形、小眼球和咽弓异常。淋巴标记物，包括淋巴个体发育中最早的基因Ikaros，在CZ-3的胸腺中完全缺失。组织切片超微结构分析显示胸腺发育不全，淋巴细胞完全缺失。在CZ-3中，其他造血血统是正常的。我已经采取了定位克隆策略来鉴定CZ-3基因。我已经用紧密连锁的标记z10517(基于9/2744减数分裂重组子的0.33 cM)定位了突变基因，并在z10517远端建立了YAC重叠群。裸鼠基因Whn的斑马鱼同源物与Z10517共定位在同一个YAC克隆上。WHN是CZ-3中突变基因的一个有吸引力的候选基因，因为裸鼠的免疫缺陷是胸腺发育不全的结果。这项提议的一个具体目的是测试CZ-3和WHN是否相同。如果不是，我将继续进行染色体行走，并通过遗传和功能手段克隆CZ-3。如果CZ-3和WHN是相同的，我会映射其余的突变体。我将选择两个具有有趣表型的突变体(分为两组：咽弓正常和异常)进行精细定位。克隆这些基因将加深我们对免疫系统的系统发育以及正常T淋巴细胞生成和免疫缺陷和白血病等疾病的分子事件的了解，并将有助于设计新的治疗策略。