Liao W, Bisgrove B W, Sawyer H, Hug B, Bell B, Peters K, Grunwald D J, Stainier D Y
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0554, USA.
Development. 1997 Jan;124(2):381-9. doi: 10.1242/dev.124.2.381.
The zebrafish cloche mutation affects both the endothelial and hematopoietic lineages at a very early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W., Zon, L. I. and Fishman, M. C. (1995). Development 121, 3141-3150). The most striking vascular phenotype is the absence of endocardial cells from the heart. Microscopic examination of mutant embryos reveals the presence of endothelial-like cells in the lower trunk and tail regions while head vessels appear to be missing, indicating a molecular diversification of the endothelial lineage. Cell transplantation experiments show that cloche acts cell-autonomously within the endothelial lineage. To analyze further the role of cloche in regulating endothelial cell differentiation, we have examined the expression of flk-1 and tie, two receptor tyrosine kinase genes expressed early and sequentially in the endothelial lineage. In wild-type fish, flk-1-positive cells are found throughout the embryo and differentiate to form the nascent vasculature. In cloche mutants, flk-1-positive cells are found only in the lower trunk and tail regions, and this expression is delayed as compared to wild-type. Unlike the flk-1-positive cells in wild-type embryos, those in cloche mutants do not go on to express tie, suggesting that their differentiation is halted at an early stage. We also find that the cloche mutation is not linked to flk-1. These data indicate that cloche affects the differentiation of all endothelial cells and that it acts at a very early stage, either by directly regulating flk-1 expression or by controlling the differentiation of cells that normally develop to express flk-1. cloche mutants also have a blood deficit and their hematopoietic tissues show no expression of the hematopoietic transcription factor genes GATA-1 or GATA-2 at early stages. Because the appearance of distinct levels of flk-1 expression is delayed in cloche mutants, we examined GATA-1 expression at late embryonic stages and found some blood cell differentiation that appears to be limited to the region lined by the flk-1-expressing cells. The spatial restriction of blood in the ventroposterior-most region of cloche mutant embryos may be indicative of a ventral source of signal(s) controlling hematopoietic differentiation. In addition, the restricted colocalization of blood and endothelium in cloche mutants suggests that important interactions occur between these two lineages during normal development.
斑马鱼的cloche突变在非常早期的阶段就影响内皮细胞和造血细胞谱系（Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W., Zon, L. I. 和 Fishman, M. C. (1995). Development 121, 3141 - 3150）。最显著的血管表型是心脏中没有心内膜细胞。对突变胚胎的显微镜检查显示，在躯干下部和尾部区域存在内皮样细胞，而头部血管似乎缺失，这表明内皮细胞谱系存在分子分化。细胞移植实验表明，cloche在内皮细胞谱系中以细胞自主方式起作用。为了进一步分析cloche在调节内皮细胞分化中的作用，我们检测了flk - 1和tie的表达，这两个受体酪氨酸激酶基因在内皮细胞谱系中早期且顺序表达。在野生型鱼中，flk - 1阳性细胞遍布整个胚胎，并分化形成新生的脉管系统。在cloche突变体中，flk - 1阳性细胞仅在躯干下部和尾部区域发现，并且与野生型相比，这种表达延迟。与野生型胚胎中的flk - 1阳性细胞不同，cloche突变体中的那些细胞不继续表达tie，这表明它们的分化在早期阶段就停止了。我们还发现cloche突变与flk - 1没有连锁关系。这些数据表明，cloche影响所有内皮细胞的分化，并且它在非常早期的阶段起作用，要么通过直接调节flk - 1的表达，要么通过控制正常发育以表达flk - 1的细胞的分化。cloche突变体也有血液不足的情况，并且它们的造血组织在早期阶段没有造血转录因子基因GATA - 1或GATA - 2的表达。因为在cloche突变体中flk - 1不同水平表达的出现延迟，我们在胚胎后期检测了GATA - 1的表达，发现一些血细胞分化似乎仅限于由flk - 1表达细胞排列的区域。cloche突变体胚胎最腹后侧区域血液的空间限制可能表明存在控制造血分化的腹侧信号源。此外，cloche突变体中血液和内皮细胞的有限共定位表明在正常发育过程中这两个谱系之间发生了重要的相互作用。
