SCREEN FOR LYMPHOID MUTANTS

在淋巴细胞形成过程中，多能干细胞发育成T淋巴细胞或B淋巴细胞。胸腺为T淋巴细胞分化提供了一个特殊的环境。通过研究自然小鼠突变体和“敲除”小鼠，最近已经确定了淋巴细胞发育和胸腺器官发生中的几个重要基因。对淋巴系统的全面了解还需要对非常早期的调节基因进行鉴定和等级排序。为了实现这一理解，我们建议采取一种新的方法，使用斑马鱼（Danio rerio）作为遗传模型系统。斑马鱼结合了遗传和胚胎学的优势，因此为研究淋巴系统提供了理想的模型系统。利用化学诱变和电离辐射进行诱变筛选。用重组激活基因-1 （Rag-1）和α胚珠蛋白探针进行全座原位杂交，筛选诱变鱼的F2后代。在胸腺中缺失Rag-1染色的17个潜在突变体中，有8个通过F2内交证实。其中一种突变，CZ-3，是一种常染色体隐性突变，可导致小头畸形、小眼症和咽弓异常。在CZ-3的胸腺中完全没有淋巴标记，包括最早的淋巴个体发育基因Ikaros。组织学切片显示胸腺发育不全，超微结构分析显示淋巴细胞完全缺失。其他造血系在CZ-3正常。我已经采取了定位克隆策略来鉴定CZ-3基因。我已经用一个紧密相连的标记绘制了突变基因，z10517（基于9/2744减数分裂重组的0.33 cM），并建立了一个远至z10517的YAC组。裸鼠基因whn的斑马鱼同源基因与z10517在同一个YAC克隆上共定位。当它是CZ-3突变基因的一个有吸引力的候选者，因为裸小鼠的免疫缺陷是胸腺发育不全的结果。这个提议的一个具体目的是测试CZ-3是否和何时相同。如果不是，我将继续染色体行走，通过基因和功能手段克隆CZ-3。如果CZ-3和whn是相同的，我将绘制剩下的突变体。我将选择两个具有有趣表型的突变体（分为两组：咽弓正常的和咽弓异常的）进行精细定位。这些基因的克隆将进一步加深我们对免疫系统的系统发育以及正常T淋巴生成和免疫缺陷和白血病等疾病中的分子事件的理解，并将有助于制定新的治疗策略。

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.