Transcriptional Regulation of Myogenesis

确定决定细胞命运的基因表达级联是理解发育的关键因素。全基因组微阵列的可获得性，再加上几乎纯的细胞类型种群的分离，使人们能够开始定义与特定细胞命运相关的转录组。肌肉细胞由于易于分离和培养以及在人类病理学中的重要性，一直是成熟动物进行此类研究的有吸引力的目标。然而，在活体早期确定胚胎肌源性基因表达模式更加困难，这将为肌肉发育的调控提供洞察力。为此，我们将Miller实验室合作者开发的一种技术--秀丽线虫微阵列分析(MAPCeL)应用于从由myo-3基因的肌球蛋白重链启动子(myo-3：：gfp)标记的发育中的线虫胚胎中提取的肌肉细胞群。 用荧光激活细胞分选技术(FACS)从分离的线虫早期胚胎中分离出myo-3：：GFP阳性的肌细胞及其培养的衍生物。对这些细胞群体中基因表达的微阵列分析确定了6,693个表达基因，其中700个基因在myo-3：：GFP阳性细胞群体中丰富。通过与已知的肌肉标记、独立获得的表达数据和转基因菌株中的GFP报告进行比较，验证了肌肉丰富的基因集。结果证实了MAPCeL用于细胞类型特异性表达谱的有效性和能力，并揭示了一组600个基因，这些基因很可能在胚胎发育过程中在非咽部肌肉细胞中特异富含。本研究证明了MAPCeL在确定发育中线虫肌肉细胞体内基因表达谱方面的能力和实用性。这些结果提供了肌肉细胞转录组及其部署的初步特征，这些转录组是肌纤维组装和功能的基础。 我们也已经开始研究体内的靶基因，这些基因是由生肌调节因子HLH-1激活的，HLH-1是脊椎动物MyoD家族的同源物。我们先前已经证明，hlh-1在线虫早期胚胎中的异位表达足以将大多数卵裂球转化为类似命运的体壁肌肉。为了确定作为肌肉细胞命运指定和分化基础的HLH-1的转录靶点，我们使用了染色质免疫沉淀(CHIP)和探测全基因组分枝微阵列(CHIP)。结果开始揭示这种转录因子在体内的结合部位，使我们能够将DNA结合与早期胚胎中的基因表达联系起来。我们希望这种方法能够更详尽地描述肌肉细胞发育过程中的转录级联。

Defining the gene expression cascade underlying cell fate determination is a key element in understanding development. The availability of whole genome microarrays, coupled with the isolation of nearly pure cell type populations, allows one to begin to define the transcriptome associated with specific cell fates. Muscle cells have been attractive targets for such studies in mature animals due to their ease of isolation andor culture and their importance in human pathologies. It has been more difficult, however, to determine early in vivo embryonic myogenic gene expression patterns that would give insights into the regulation of muscle development. To this end, we have applied Micro-Array Profiling of C. elegans (MAPCeL), a techinque developed by our collaborators in the Miller lab, to muscle cell populations extracted from developing nematode embryos that have been marked by the myosin heavy chain promoter from the myo-3 gene (myo-3::gfp). Fluorescence Activated Cell Sorting (FACS) was used to isolate myo-3::gfp-positive muscle cells, and their cultured derivatives, from dissociated early C. elegans embryos. Microarray analysis of gene expression in these cell populations identified 6,693 expressed genes, 700 of which are enriched in the myo-3::gfp positive cell population. The muscle-enriched gene set was validated by comparisons to known muscle markers, independently derived expression data, and GFP reporters in transgenic strains. The results confirm the validity and power of MAPCeL for cell type-specific expression profiling and reveal a set of 600 genes that are very likely to be specifically enriched in non-pharyngeal muscle cells during embryogenesis. This study demonstrated the power and utility of MAPCeL in defining the in vivo gene expression profile of developing C. elegans muscle cells. The results provide an initial characterization of the muscle cell transcriptome and its deployment that underlies myofiber assembly and function. We have also begun to study the in vivo target genes activated by the myogenic regulator, HLH-1, a homolog of the vertebrate MyoD family. We have previously shown that ectopic expression of hlh-1 in early C. elegans embryos is sufficient to convert most blastomeres to a body wall muscle like fate. To define the transcriptional targets of HLH-1 that underlie muscle cell fate specification and differentiation, we have use chromatin immunoprecipitation (ChIP) followed by probing whole genome tilling microarrays (Chip). The results are beginning to reveal the in vivo binding sites for this transcription factor, allowing us to correlate DNA binding with gene expression in the early embryo. Our hope is that this approach will allow a more exhaustive description of the transcriptional cascade that play out during muscle cell development.