Yin Z, Xu X L, Frasch M
Brookdale Center for Developmental and Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Development. 1997 Dec;124(24):4971-82. doi: 10.1242/dev.124.24.4971.
The Drosophila tinman homeobox gene has a major role in early mesoderm patterning and determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Here we show that each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. We provide evidence that the early-active enhancer element is a direct target of twist, a gene encoding a basic helix-loop-helix (bHLH) protein, which is necessary for tinman activation. This 180 bp enhancer includes three E-box sequences which bind Twist protein in vitro and are essential for enhancer activity in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. We further show that the 180 bp enhancer also includes negatively acting sequences. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. We find that this repression requires the function of buttonhead, a head-patterning gene, and that buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area. Together, our results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restrict twist activity to specific areas within the twist expression domain.
果蝇tinman同源框基因在早期中胚层模式形成中起主要作用，并决定内脏中胚层、心脏祖细胞、特定体壁肌肉前体和胶质样中胚层细胞的形成。tinman的这些功能反映在其动态表达模式中，其特点是最初在躯干中胚层广泛表达，然后细化到广泛的背侧中胚层区域，最后在心脏祖细胞中表达受限。在这里，我们表明这些表达阶段中的每一个都是由一个离散的增强子元件驱动的，第一个在早期中胚层活跃，第二个在背侧中胚层活跃，第三个在成心肌细胞中活跃。我们提供的证据表明，早期活跃的增强子元件是twist的直接靶点，twist是一个编码碱性螺旋-环-螺旋（bHLH）蛋白的基因，它是tinman激活所必需的。这个180 bp的增强子包含三个E-box序列，它们在体外与Twist蛋白结合，并且对于体内增强子活性至关重要。twist在外胚层中的异位表达导致该增强子在外胚层细胞中的异位激活，表明twist是早期tinman表达的唯一中胚层特异性激活剂。我们进一步表明，180 bp的增强子还包含负向作用序列。Even-skipped与这些序列的结合似乎以周期性方式降低twist依赖性激活，从而在早期中胚层中产生条纹状的tinman模式。此外，这些序列阻止twist在产生血细胞的头部中胚层的特定部分激活tinman。我们发现这种抑制需要buttonhead的功能，buttonhead是一个头部模式形成基因，并且buttonhead对于同一区域中造血分化基因serpent的正常激活是必需的。总之，我们的结果表明，tinman受一系列离散的增强子元件控制，这些元件通过差异遗传输入相继激活，以及通过早期作用增强子内紧密相连的激活剂和抑制剂结合位点，将twist活性限制在twist表达域内的特定区域。
