Signaling pathways and expansion of hematopoietic stem cells in zebrafish

斑马鱼造血干细胞的信号通路和扩增

基本信息

批准号：
7884567
负责人：
Jill L de Jong
金额：
$ 12.91万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7884567
关键词：
Affect Anemia Anterior Biology Blood Blood Cells Blood Island Bone Marrow Transplantation Bypass C.I. Solvent Yellow 56 Cell Transplants Cells Chemicals Complex Defect Development Dianisidine Disease Dose Embryo Embryonic Development Erythroid Progenitor Cells Fishes Gene Expression Genes Genetic Models Genetic Screening Hematopoiesis Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Hemoglobin Homing Human Incubated Individual Label Lead Microarray Analysis Monitor Multipotent Stem Cells Mus Pancytopenia Partner in relationship Pathway interactions Pattern Phenotype Phylogeny Process Production Retinoic Acid Receptor Role Signal Pathway Signal Transduction Site Staining method Stains Stem Cell Development Syndrome Testing Therapeutic Transplantation Tretinoin Up-Regulation Yolk Sac Zebrafish chemical genetics embryonic stem cell human disease in vivo inhibitor/antagonist mutant progenitor receptor expression research study response small molecule tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) are formed during embryogenesis by a complex process that is largely conserved across vertebrate phylogeny. The zebrafish mutant kugelig (kgg) has a severe defect in hematopoiesis, as well as altered anterior-posterior patterning and abnormal hox gene expression. The mutant kgg phenotype is caused by a deletion in the cdx4 gene, known to regulate hox gene expression in early development, including hoxb4 and other genes that activate early HSCs. Microarray analysis of kgg mutants compared to wildtype (WT) revealed an upregulation of raldh2, the final enzymatic step for retinoic acid (RA) production. RA exposure decreases blood cells in zebrafish embryos, while a chemical that blocks activity of raldh2 restores blood formation in kgg mutants. To examine the hypothesis that cdx4 affects blood formation by altering RA signaling, Specific Aim 1 proposes to characterize the RA signaling pathway in the zebrafish cdx4 mutant by studying retinoic acid receptor expression and function, and RA inhibitors. To test whether the loss of cdx4 is a cell autonomous defect in the formation of HSCs, labeled WT HSCs will be transplanted into cdx4 mutant embryos. Transplanted cells will be monitored for homing to known sites of definitive hematopoiesis and production of circulating differentiated blood cells. In Specific Aim 2, we propose a chemical genetic screen to test the hypothesis that other molecules can rescue or bypass the requirement of cdx4 for blood formation in vivo. Heterozygous kgg fish will be mated, and their embryos, both mutant and WT, will be incubated with individual or pooled chemicals. When the chemicals are washed out, embryos will be scored for increased blood formation by staining of hemoglobin with o-dianisidine. "Positive hit" chemicals will be tested for dose-response effects, as well as alteration of transcription factors known to regulate hematopoiesis. Chemicals will then be tested on mouse ES cells and yolk sac blood island cultures to examine expansion of multipotent progenitor (CFU-GEMM) colonies. Characterization of signaling pathways affected by the "positive hit" chemicals will increase the understanding of transcription factors that control these early progenitors. The screen should identify chemicals that expand erythroid progenitor cells or multipotent HSCs, which could have a monumental impact on human diseases involving paucity of blood cells, such as certain anemias, bone marrow failure syndromes, and diseases requiring HSC transplantation.

描述（由申请人提供）：造血干细胞（HSC）是在胚胎发生过程中通过一个复杂的过程形成的，该过程在脊椎动物系统发育中很大程度上是保守的。斑马鱼突变体 kugelig (kgg) 具有严重的造血缺陷，以及前后模式改变和异常的 hox 基因表达。 kgg 突变表型是由 cdx4 基因缺失引起的，该基因已知在早期发育中调节 hox 基因表达，包括 hoxb4 和其他激活早期 HSC 的基因。与野生型 (WT) 相比，kgg 突变体的微阵列分析显示 raldh2 上调，这是视黄酸 (RA) 生产的最后一个酶促步骤。 RA 暴露会减少斑马鱼胚胎中的血细胞，而阻断 raldh2 活性的化学物质会恢复 kgg 突变体中的血液形成。为了检验 cdx4 通过改变 RA 信号传导影响血液形成的假设，Specific Aim 1 提出通过研究视黄酸受体表达和功能以及 RA 抑制剂来表征斑马鱼 cdx4 突变体中的 RA 信号传导途径。为了测试 cdx4 的缺失是否是 HSC 形成过程中的细胞自主缺陷，标记的 WT HSC 将被移植到 cdx4 突变胚胎中。将监测移植细胞归巢至已知的确定性造血作用和循环分化血细胞的产生。在具体目标 2 中，我们提出了一种化学遗传筛选来测试其他分子可以挽救或绕过 cdx4 对体内血液形成的要求的假设。杂合的公斤鱼将被交配，它们的胚胎（突变体和野生型）将与单独的或混合的化学品一起孵化。当化学物质被洗掉后，将通过用邻联茴香胺对血红蛋白进行染色来对胚胎的血液形成增加进行评分。 “积极打击”化学物质将被测试剂量反应效应，以及已知调节造血功能的转录因子的改变。然后将在小鼠 ES 细胞和卵黄囊血岛培养物上测试化学物质，以检查多能祖细胞 (CFU-GEMM) 集落的扩张。受“正命中”化学物质影响的信号通路的表征将增加对控制这些早期祖细胞的转录因子的理解。筛选应识别出能够扩增红系祖细胞或多能造血干细胞的化学物质，这些化学物质可能对涉及血细胞缺乏的人类疾病产生巨大影响，例如某些贫血、骨髓衰竭综合征和需要造血干细胞移植的疾病。