Signaling pathways and expansion of hematopoietic stem cells in zebrafish

描述(由申请人提供)： 造血干细胞(HSCs)是在胚胎发育过程中通过一个复杂的过程形成的，在脊椎动物的系统发育过程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，前后部构型改变，HOX基因表达异常。突变的kgg表型是由cdx4基因的缺失引起的，cdx4基因在发育早期调节HOX基因的表达，包括HOXB4和其他激活早期HSCs的基因。对kgg突变体和野生型(Wt)进行的微阵列分析显示，raldh2上调，这是生产维甲酸(RA)的最后一个酶步骤。RA暴露会减少斑马鱼胚胎中的血细胞，而一种阻止raldh2活性的化学物质可以恢复kgg突变体的血液形成。为了验证cdx4通过改变RA信号来影响血液形成的假说，特定目的1建议通过研究维甲酸受体的表达和功能以及RA抑制剂来表征斑马鱼cdx4突变体中的RA信号通路。为了测试cdx4的缺失是否是HSCs形成中的细胞自主缺陷，标记的WT HSCs将被移植到cdx4突变的胚胎中。移植的细胞将被监测是否归巢到已知的最终造血点和产生循环分化的血细胞。在特定的目标2中，我们提出了一种化学遗传筛选来检验这样的假设，即其他分子可以挽救或绕过体内cdx4对血液形成的要求。杂合的kgg鱼将进行交配，它们的胚胎，无论是突变的还是WT的，都将与单独或混合的化学品孵化。当这些化学物质被洗掉后，通过用邻二苯甲胺对血红蛋白进行染色，胚胎将被标记为增加了血液形成。将测试“阳性”化学物质的剂量-反应效应，以及已知调节造血的转录因子的变化。然后，将在小鼠ES细胞和卵黄囊血岛培养上测试化学物质，以检查多潜能祖细胞(CFU-GEMM)克隆的扩张。对受“阳性撞击”化学物质影响的信号通路进行表征将增加对控制这些早期前体细胞的转录因子的理解。筛查应该识别能够扩大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，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。

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.