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。控制多能干细胞/祖细胞自我更新或分化决策的机制尚不清楚。许多最初的发育决定是由同源盒家族的转录因子调控的。造血表达的同源盒基因Hex是早期造血分化研究中一个有趣的候选基因。拟议研究的目的是加强我们目前对Hex在造血谱系承诺中的作用的理解。Hex在胚胎干细胞向造血祖细胞分化过程中所起的作用将通过三个特定目的进行研究：1)定向敲低Hex基因表达，以阐明Hex是否为造血承诺所必需。2)过表达Hex以确定Hex是否足以诱导造血承诺。3)通过微阵列分析确定Hex介导造血分化的下游靶点。胚胎干细胞为这些研究提供了理想的模型系统。在体外，他们概括了早期造血谱系和所有髓系谱系的发展。为了模拟造血，采用Era和Witte的体外小鼠胚胎干细胞共培养系统。在正常情况下，未分化的胚胎干细胞维持在LIF中。在OP-9基质细胞上培养，胚胎干细胞在第5天分化为成血管细胞。继续与OP-9细胞共培养，胚胎干细胞分化为造血前体细胞（第8天）和成熟的终末分化谱系（第14天）。造血细胞可以通过流式细胞术和细胞学分析进行表面标记。在胚胎干细胞内使用四环素诱导系统（Tet-Off），可以严格调节Hex表达的时间和数量。本实验的目的是表征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.