The Role of the Homeobox Gene Hex in Hematopoietic Differentiation

描述（由申请人提供）：造血涉及一系列严格调控的分子事件。控制多能干/祖细胞自我更新或分化的决定的机制还不清楚。许多最初的发育决策都受到转录因子同源盒家族的调节。造血表达的同源异型盒基因Hex是研究早期造血分化的重要候选基因。提出的研究的目标是提高我们目前的理解的作用Hex发挥造血谱系承诺。Hex在胚胎干（ES）细胞分化为造血祖细胞中的作用将通过三个特定的目的来检查：1）定向敲低Hex基因表达以阐明Hex是否是造血定型所必需的。2)Hex过表达以确定Hex是否足以诱导造血定型。3)微阵列分析以确定介导造血分化的Hex的下游靶标。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谱系和所有髓系谱系的发育。为了模拟造血，体外鼠ES细胞共培养系统改编自Era和Witte。在正常条件下，未分化的ES细胞维持在LIF中。当在OP-9基质细胞上培养时，ES细胞在第5天分化为成血管细胞。继续与OP-9细胞共培养导致ES细胞分化成造血前体细胞（第8天）和成熟的终末分化谱系（第14天）。然后可以通过流式细胞术分析造血细胞的细胞表面标志物，并通过cytoprep进行形态学分析。在ES细胞内使用四环素诱导系统（Tet-Off）允许严格调节Hex表达的时间和量。所提出的实验的目标是表征Hex转录活性和Hex基因敲低对造血细胞存活、凋亡、增殖和分化的影响。总的来说，这些实验旨在更清楚地阐明调节造血干细胞分化的分子途径。这项工作将提供重要的见解有关的一些血细胞疾病，包括癌症，贫血和免疫系统疾病。

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.