Developmental Gene Expression In C elegans

多年来，我们的主要重点领域之一是肌肉发展。在小鼠中，横纹肌的发育需要MRF的功能，这是一套由四个密切相关的因子(MyoD、Myf-5、MRF-4、Mygenin)组成的集合，它们调节肌肉的规格和分化。线虫只有一种名为CeMyoD(HLH-1)的MRF相关因子。线虫肌肉发生的一个令人费解的方面是观察到在没有CeMyoD的情况下肌肉可以形成，就像在突变动物中所证明的那样。为了进一步确定该转录因子的确切作用，我们在早期线虫胚胎中异位表达了HLH-1/MyoD，发现它足以将所有早期的卵裂球转化为肌肉样的命运。仅HLH-1/MyoD就足以引导细胞进入肌肉谱系，这说明了它的效力，并揭示了在功能上进化保守的水平，这是以前没有人认识到的。此外，这些实验揭示了早期胚胎细胞在细胞命运选择方面需要重新编程的显著可塑性。利用三个基因的遗传缺失等位基因，我们表明，当这些因素中至少有一个存在时，肌肉可以形成。然而，这三种基因产物的消除阻碍了胚胎中的肌肉分化。这些结果将这三个转录因子定义为线虫肌肉的关键调控因子，解释了为什么肌肉可以在没有HLH-1/MyoD的情况下形成。 在过去的一年里，我们主要关注调节前中胚层规格和体壁肌肉发育的因素。在已确定的线虫细胞谱系的基础上，我们定义了涉及在单细胞水平上调节肌肉发育的转录因子的层次结构。这使得我们能够梳理出多条通路，所有这些通路都最终导致HLH-1/MyoD和UNC-120/SRF的激活，这是一组常见的体壁肌肉命运指定和分化的驱动因素。我们的结果还揭示了脊椎动物前部肌肉发育过程中途径成分的意外进化保守。 在与艾森曼博士(巴尔的摩县MD大学)的持续合作中，我们探索了由Wnt信号调节的基因表达。Wnt信号通路在后生动物的发育过程中起着基础性的作用，它调控多种过程，包括细胞命运指定、细胞迁移和干细胞更新。激活β-连环素依赖/规范的Wnt通路上调Wnt靶基因的表达，以介导细胞反应。在线虫线虫中，一个典型的Wnt信号通路调控着幼虫发育过程中的几个过程，但该通路的目标基因还很少。为了解决这一缺陷，我们使用了一种新的方法，通过过度表达激活的β-连环蛋白，在幼虫生命的特定阶段有条件地激活Wnt信号，然后使用微阵列分析来鉴定与对照动物相比表达发生变化的基因。我们鉴定了166个差异表达基因，其中104个上调。在Wnt信号减弱或增加的突变体中，上调基因的一部分被证明改变了表达；我们认为这些基因是线虫Wnt途径的真正靶点。 在与刘博士(康奈尔大学)的合作中，我们利用线虫的遗传学来确定骨形态发生蛋白(BMP)在胚胎后中胚层发育事件信号中的作用。这项工作进一步揭示了细胞膜Tetraspanins调节BMP信号的作用，提供了一种可能的机制，支持它们在癌症发展中的先前定义的作用。

One of our main focus areas over the years is muscle development. In mice, striated muscle development requires the function of the MRFs, a set of four closely related factors (MyoD, Myf-5, MRF-4, Myogenin) that regulated muscle specification and differentiation. C. elegans has a single MRF-related factor called CeMyoD (HLH-1). One of the puzzling aspects of C. elegans myogenesis was the observation that muscles could be formed in the absence of CeMyoD, as demonstrated in mutant animals. To further define the exact role of this transcription factor, we ectopically expressed HLH-1/MyoD in the early nematode embryo and found that it was sufficient to convert all early blastomeres to a muscle-like fate. The sufficiency of HLH-1/MyoD alone to direct cells into the muscle lineage illustrated its potency and revealed a level of evolutionary conservation in function that had not previously been appreciated. Moreover, these experiments revealed a remarkable degree of plasticity of the early embryonic cells to be reprogrammed with regards to cell fate choice. Using genetic deletion alleles of three genes, we showed that muscle could form when at least one of these factors was present. However, elimination of all three gene products blocked muscle differentiation in the embryo. These results defined this trio of transcription factors as the key regulators of C. elegans muscle, explaining why muscle could form in the absence of HLH-1/MyoD. This past year, we have focused on the factors regulating anterior mesoderm specification and bodywall muscle development. Captializing on the defined cell lineage of C. elegans, we have defined a hierarchy of transcription factors involved regulating muscle development at the single cell level. This has allowed us to tease apart multiple pathways all leading to the eventual activation of HLH-1/MyoD and UNC-120/SRF, the common set of drivers for bodywall muscle fate specification and differentiation. Our results also revealed an unexpected evolutionary conservation of pathway components with anterior muscle development in vertebrates. In on-going collaboration with Dr. Eisenmann (Univ of MD, Baltimore County) we have explored gene expression regulated by Wnt signaling. The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. In a collaboration with Dr. Liu (Cornell University) we capitalized on the genetics of C. elegans to identify a role of Bone Morphogentic Protein (BMP) in signaling developmental events in the postembryonic mesoderm. This work further revealed a role for membrane tetraspanins to modulate BMP signaling, providing a possible mechanism underlying their previously defined role in cancer development.