Signaling pathways and expansion of hematopoietic stem cells in zebrafish

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，该过程在脊椎动物的胚胎发生中很大程度上是保守的。斑马鱼突变体kugelig（kgg）在造血方面有严重缺陷，同时前后模式改变和hox基因表达异常。突变型kgg表型是由cdx 4基因缺失引起的，已知cdx 4基因在早期发育中调节hox基因表达，包括hoxb 4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揭示了raldh 2的上调，raldh 2是维甲酸（RA）生产的最后酶促步骤。RA暴露会减少斑马鱼胚胎中的血细胞，而一种阻断raldh 2活性的化学物质会恢复kgg突变体中的血液形成。为了检验cdx 4通过改变RA信号传导来影响血液形成的假设，具体目标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cdx 4突变体中的RA信号传导途径。为了测试cdx 4的缺失是否是HSC形成中的细胞自主缺陷，将标记的WT HSC移植到cdx 4突变胚胎中。将监测移植的细胞归巢至确定造血的已知位点和循环分化血细胞的产生。在特定目标2中，我们提出了一种化学遗传筛选来测试其他分子可以拯救或绕过cdx 4在体内血液形成的假设。将杂合kgg鱼交配，并将其胚胎（突变体和WT）与单独或合并的化学品一起孵育。当化学物质被洗掉时，通过用邻联茴香胺染色血红蛋白来对胚胎的血液形成增加进行评分。“积极的打击”化学品将被测试的剂量反应效应，以及已知的调节造血转录因子的改变。然后将在小鼠ES细胞和卵黄囊血岛培养物上测试化学品，以检查多能祖细胞（CFU-GEMM）集落的扩增。表征受“积极打击”化学物质影响的信号通路将增加对控制这些早期祖细胞的转录因子的理解。筛选应确定扩大红系祖细胞或多能HSC的化学物质，这可能对涉及血细胞缺乏的人类疾病产生巨大影响，如某些贫血症，骨髓衰竭综合征和需要HSC移植的疾病。

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.