Chen Yau-Hung, Wang Yun-Hsin, Chang Min-Yen, Lin Cheng-Yung, Weng Chih-Wei, Westerfield Monte, Tsai Huai-Jen
Graduate Institute of Life Sciences, Tamkang University, Tamsui, Taiwan.
BMC Dev Biol. 2007 Jan 3;7:1. doi: 10.1186/1471-213X-7-1.
Myf5 is one member of the basic helix-loop-helix family of transcription factors, and it functions as a myogenic factor that is important for the specification and differentiation of muscle cells. The expression of myf5 is somite- and stage-dependent during embryogenesis through a delicate regulation. However, this complex regulatory mechanism of myf5 is not clearly understood.
We isolated a 156-kb bacterial artificial chromosome clone that includes an upstream 80-kb region and a downstream 70-kb region of zebrafish myf5 and generated a transgenic line carrying this 156-kb segment fused to a green fluorescent protein (GFP) reporter gene. We find strong GFP expression in the most rostral somite and in the presomitic mesoderm during segmentation stages, similar to endogenous myf5 expression. Later, the GFP signals persist in caudal somites near the tail bud but are down-regulated in the older, rostral somites. During the pharyngula period, we detect GFP signals in pectoral fin buds, dorsal rostral myotomes, hypaxial myotomes, and inferior oblique and superior oblique muscles, a pattern that also corresponds well with endogenous myf5 transcripts. To characterize the specific upstream cis-elements that regulate this complex and dynamic expression pattern, we also generated several transgenic lines that harbor various lengths within the upstream 80-kb segment. We find that (1) the -80 kb/-9977 segment contains a fin and cranial muscle element and a notochord repressor; (2) the -9977/-6213 segment contains a strong repressive element that does not include the notochord-specific repressor; (3) the -6212/-2938 segment contains tissue-specific elements for bone and spinal cord; (4) the -2937/-291 segment contains an eye enhancer, and the -2937/-2457 segment is required for notochord and myocyte expression; and (5) the -290/-1 segment is responsible for basal transcription in somites and the presomitic mesoderm.
We suggest that the cell lineage-specific expression of myf5 is delicately orchestrated by multiple modules within the distal upstream region. This study provides an insight to understand the molecular control of myf5 and myogenesis in the zebrafish.
Myf5是转录因子基本螺旋-环-螺旋家族的成员之一，它作为一种生肌因子，对肌肉细胞的特化和分化至关重要。在胚胎发育过程中，Myf5的表达通过精细的调控依赖于体节和发育阶段。然而，Myf5这种复杂的调控机制尚未完全清楚。
我们分离出一个156 kb的细菌人工染色体克隆，它包含斑马鱼Myf5上游80 kb区域和下游70 kb区域，并构建了一个携带与绿色荧光蛋白（GFP）报告基因融合的156 kb片段的转基因品系。我们发现在体节形成阶段，在最前端的体节和前体节中胚层有强烈的GFP表达，类似于内源性Myf5的表达。之后，GFP信号在尾芽附近的尾侧体节中持续存在，但在较老的头侧体节中表达下调。在咽胚期，我们在胸鳍芽、头背侧肌节、轴下肌节以及下斜肌和上斜肌中检测到GFP信号，这种模式也与内源性Myf5转录本很好地对应。为了鉴定调控这种复杂动态表达模式的特定上游顺式元件，我们还构建了几个在上游80 kb片段内包含不同长度片段的转基因品系。我们发现：（1）-80 kb / -9977片段包含一个鳍和头肌元件以及一个脊索抑制子；（2）-9977 / -6213片段包含一个强抑制元件，不包括脊索特异性抑制子；（3）-6212 / -2938片段包含骨骼和脊髓的组织特异性元件；（4）-2937 / -291片段包含一个眼增强子，-2937 / -2457片段是脊索和肌细胞表达所必需的；（5）-290 / -1片段负责体节和前体节中胚层的基础转录。
我们认为Myf5的细胞谱系特异性表达是由远端上游区域内的多个模块精心编排的。这项研究为理解斑马鱼中Myf5和肌肉发生的分子调控提供了见解。
