The Role of the Homeobox Gene Hex in Hematopoietic Differentiation

描述(申请人提供)：造血涉及一系列严格调控的分子事件。控制多能干/祖细胞决定自我更新或分化的机制还不是很清楚。许多最初的发育决定受转录因子同源框家族的调控。同源异型盒基因Hex是研究早期造血分化的重要候选基因。拟议研究的目的是加强我们目前对十六进制在造血谱系承诺中所起作用的理解。Hex在胚胎干细胞分化为造血祖细胞中所起的作用将通过三个特定的目标来检测：1)定向下调Hex基因的表达，以阐明Hex是否是造血承诺所必需的。2)Hex的过表达以确定Hex是否足以诱导造血承诺。3)基因芯片分析以确定Hex基因下游介导的造血分化靶点。ES细胞为这些研究提供了理想的模型系统。在体外，它们概括了早期造血系和所有髓系的发展。采用Era和Witte建立的小鼠ES细胞体外共培养体系进行造血模型的建立。在正常情况下，未分化的ES细胞维持在LIF中。当ES细胞在OP-9基质细胞上培养时，第5天分化为血管母细胞。继续与OP-9细胞共培养可使ES细胞分化为造血祖细胞(第8天)，并在第14天分化为成熟的、终末分化的谱系。然后造血细胞可以用流式细胞仪进行细胞表面标志的分析，并用细胞学方法进行形态观察。在ES细胞中使用四环素诱导系统(Tet-Off)，允许人们严格调控十六进制表达的时间和数量。本实验的目的是研究Hex转录活性和Hex基因敲除对造血细胞存活、凋亡、增殖和分化的影响。总体而言，这些实验旨在更清楚地阐明调控造血干细胞分化的分子途径。这项工作将为包括癌症、贫血和免疫疾病在内的许多血细胞疾病提供重要的见解。

DESCRIPTION (provided by applicant): Hematopoiesis involves a series of tightly regulated molecular events. The mechanisms controlling the decision of the multi-potent stem/progenitor cell to self-renew or differentiate are not well understood. Many initial developmental decisions are regulated by the homeobox family of transcription factors. The hematopoietically expressed homeobox gene, Hex, is an interesting candidate gene in the study of early hematopoietic differentiation. The goal of the proposed studies is to enhance our current understanding of the role Hex play in hematopoietic lineage commitment. The role Hex plays in embryonic stem (ES) cell differentiation into the hematopoietic progenitor will be examined by three specific aims: 1) directed knockdown of Hex gene expression in order to elucidate whether Hex is necessary from hematopoietic commitment. 2) overexpression of Hex to determine whether Hex is sufficient to induce hematopoietic commitment. 3) microarray analysis to determine the downstream targets of Hex which mediate hematopoietic differentiation. ES cells provide an ideal model system for these studies. In vitro, they recapitulate the development of the early hematopoietic lineages and all myeloid lineages. To model hematopoiesis, an in vitro murine ES cell co-culture system was adapted from Era and Witte. Under normal conditions, undifferentiated ES cells are maintained in LIF. When cultured on OP-9 stromal cells, ES cells differentiate into hemangioblasts by day 5. Continued co-culture with OP-9 cells results in the differentiation of ES cells into hematopoietic precursors (day 8) and mature, terminally differentiated lineages by day 14. Hematopoietic cells can then be analyzed for cell surface markers by flow cytometry and morphologically by cytoprep. The use of a Tetracyline- inducible system (Tet-Off), within the ES cells, allows one to tightly regulate the timing and amount of Hex expression. The goal of the proposed experiments is to characterize the effects of Hex transcriptional activity and Hex gene knockdown on hematopoietic cell survival, apoptosis, proliferation and differentiation. Overall these experiments are designed to more clearly elucidate the molecular pathways that regulate hematopoietic stem cell differentiation. This work will provide important insights relevant to a number of blood cell disorders including cancer, anemias and immune disorders.