The human sarcomeric myosin heavy chain gene MYH14 contains an intronic microRNA, miR-499. Our previous studies demonstrated divergent genomic organization and expression patterns of myh14/miR-499 among teleosts; however, the regulatory mechanism is partly known. In this study, we report the regulation of myh14 expression in zebrafish, Danio rerio. Zebrafish myh14 has three paralogs, myh14-1, myh14-2, and myh14-3. Detailed promoter analysis suggested that a 5710-bp 5′-flanking region of myh14-1 and a 5641-bp region of myh14-3 contain a necessary regulatory region to recapitulate specific expression during embryonic development. The 5′-flanking region of zebrafish myh14-1 and its torafugu ortholog shared two distal and a single proximal conserved region. The two distal conserved regions had no effect on zebrafish myh14-1 expression, in contrast to torafugu expression, suggesting an alternative regulatory mechanism among the myh14 orthologs. Comparison among the 5′-flanking regions of the myh14 paralogs revealed two conserved regions. Deletion of these conserved regions significantly reduced the promoter activity of myh14-3 but had no effect on myh14-1, indicating different cis-regulatory mechanisms of myh14 paralogs. Loss of function of miR-499 resulted in a marked reduction in slow muscle fibers in embryonic development. Our study identified different cis-regulatory mechanisms controlling the expression of myh14/miR-499 and an indispensable role of miR-499 in muscle fiber–type specification in zebrafish.

人肌节肌球蛋白重链基因MYH14含有内含子 microRNA miR-499。我们之前的研究证明了硬骨鱼类中myh14 /miR-499 的不同基因组组织和表达模式；然而，监管机制是部分已知的。在这项研究中，我们报告了斑马鱼Danio rerio中myh14表达的调节。斑马鱼myh14具有三个旁系同源物，myh14-1、myh14-2和myh14-3。详细的启动子的分析表明，一个5710-bp的5'侧翼的区域myh14-1和的5641-bp的区域myh14-3包含一个必要的调控区域来概括胚胎发育过程中的特定表达。斑马鱼myh14-1的 5'-侧翼区域及其 torafugu 直向同源物共享两个远端和一个近端保守区域。与 torafugu 表达相反，这两个远端保守区域对斑马鱼myh14-1表达没有影响，表明myh14直向同源物中存在替代调节机制。myh14旁系同源物的 5'-侧翼区域之间的比较揭示了两个保守区域。删除这些保守区域显着降低了myh14-3的启动子活性，但对myh14-1没有影响，表明不同的顺式-myh14旁系同源物的调节机制。miR-499 功能的丧失导致胚胎发育中慢肌纤维的显着减少。我们的研究确定了控制myh14 /miR-499表达的不同顺式调节机制，以及 miR-499 在斑马鱼肌肉纤维类型规范中不可或缺的作用。