Ismailoglu Ismail, Yeamans Gabriel, Daley George Q, Perlingeiro Rita C R, Kyba Michael
Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA.
Exp Hematol. 2008 Dec;36(12):1593-603. doi: 10.1016/j.exphem.2008.07.005. Epub 2008 Sep 21.
The transcription factor SCL is critically required for establishing hematopoiesis and for proper endothelial development, but not for maintenance of hematopoietic stem cells or endothelial cells in the adult. Conflicting data exists regarding the developmental function of SCL, namely, whether it acts as a master regulator, actively patterning mesoderm toward hematopoietic development at the expense of other lineages, or is merely necessary to maintain the earliest committed hematopoietic precursors. To answer this question, we have engineered murine embryonic stem cells with a conditional doxycycline-inducible SCL transgene, and evaluated the effects of SCL expression at defined time points during in vitro development. Continual SCL expression during differentiation results in markedly increased hematopoiesis. By using pulses of gene expression over a 6-day differentiation time course, we map and characterize windows of SCL responsiveness. Notably, a pulse of SCL expression during early mesodermal patterning (48 to 72 hours of differentiation) promoted Flk1+ PDGFRalphaneg presumptive extraembryonic/lateral plate mesoderm at the expense of PDGFRalpha+ Flk1neg presumptive paraxial mesoderm. Consistent with this, the early pulse of SCL expression also expanded hematopoietic colony-forming cell numbers, while concomitantly repressing expression of paraxial and cardiac markers, and inhibited development of beating cardiomyocytes. By mixing the inducible embryonic stem cells with fluorescently labeled wild-type cells in chimeric embryoid bodies, we show that these effects of SCL are cell autonomous. These data support a master-regulatory role for SCL in mesodermal patterning, in addition to its established later roles in hematopoietic differentiation.
转录因子SCL对于造血作用的建立以及内皮细胞的正常发育至关重要，但对于成体中造血干细胞或内皮细胞的维持并非必需。关于SCL的发育功能存在相互矛盾的数据，即它是作为一个主要调节因子，以牺牲其他谱系为代价积极地将中胚层导向造血发育，还是仅仅对于维持最早的定向造血前体细胞是必需的。为了回答这个问题，我们构建了带有条件性强力霉素诱导型SCL转基因的小鼠胚胎干细胞，并评估了SCL在体外发育过程中特定时间点表达的影响。分化过程中持续的SCL表达导致造血作用显著增加。通过在6天的分化时间进程中使用基因表达脉冲，我们绘制并表征了SCL反应性的窗口。值得注意的是，在早期中胚层模式形成期间（分化48至72小时）的SCL表达脉冲促进了Flk1 + PDGFRα阴性的假定胚外/侧板中胚层，同时牺牲了PDGFRα + Flk1阴性的假定轴旁中胚层。与此一致的是，SCL表达的早期脉冲也增加了造血集落形成细胞的数量，同时抑制了轴旁和心脏标志物的表达，并抑制了跳动心肌细胞的发育。通过在嵌合胚状体中将可诱导的胚胎干细胞与荧光标记的野生型细胞混合，我们表明SCL的这些作用是细胞自主性的。这些数据支持SCL在中胚层模式形成中起主要调节作用，此外它在造血分化中还具有已确定的后期作用。
