An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes.
An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes.
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DOI：
10.1371/journal.pgen.0020016
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发表时间：
2006-02
期刊：
影响因子：
4.5
通讯作者：
Michelson AM
中科院分区：
文献类型：
--
作者：
Estrada B;Choe SE;Gisselbrecht SS;Michaud S;Raj L;Busser BW;Halfon MS;Church GM;Michelson AM
在理解控制特定器官形成的机制方面，一个重要但很大程度上尚未解决的挑战是破译感兴趣的组织内细胞类型多样性所表现出的复杂和动态的遗传程序。在这里，我们使用一个集成的遗传，基因组和计算策略，全面确定不同的成肌细胞亚群内的果蝇胚胎中胚层的细胞命运最初指定的时间的分子身份。通过流式细胞术从适当分期的野生型胚胎和12种基因型中纯化的原代中胚层细胞产生了基因表达谱的纲要，其中肌生成被选择性地和可预测地干扰。基于基因表达的预期趋势和每种基因型对已知肌肉基因检测的相对贡献，对这些合并样本进行统计荟萃分析，暂时将数百个差异表达基因分配给特定的成肌细胞亚型。然后使用全胚胎原位杂交来验证这些预测中的大多数，从而使微阵列数据的真阳性检测率得以估计。这种综合分析表明，成肌细胞表现出比以前认识到的更大的基因表达异质性和总体复杂性。此外，它暗示了大量的未表征的，差异表达的基因在生肌规范和随后的形态发生的参与。这些发现也强调了产生不同的成肌细胞身份的相当大的监管特异性的要求。最后，为了说明如何新发现的成肌细胞基因的发育功能，可以有效地调查，一个快速的RNA干扰试验，可以在活的胚胎进行评分，并适用于选定的基因。这种用于检查胚胎基因表达和功能的综合策略为进一步研究该模型发育系统提供了实质上扩展的框架。动物的发育需要复杂器官中的细胞获得不同的身份。在果蝇体壁肌肉组织的发育过程中，一群明显相同的细胞产生了两种类型的肌肉前体，这两种前体都是功能性肌肉出现所必需的。这些身份依赖于基因表达的广泛程序。作者试图通过整合遗传学、基因组学和信息学中的现代方法来剖析定义这两种不同细胞类型的表达基因的互补物。通过从正常胚胎和干扰肌肉发育的突变体中纯化信息细胞，分析其全基因组基因表达程序，并将实验统计学结合起来，他们已经确定了比以前怀疑的特征细胞类型多五倍的创始人特异性基因。在整个胚胎中检查了数百个基因的表达模式，以测试统计预测，使作者能够估计还有多少细胞类型特异性基因有待发现。最后，对这些方法突出显示的几十个基因进行了测试，以确定它们是否直接参与肌肉发育，并报道了这一过程中的几个新参与者。这里使用的整合策略可以推广到研究其他复杂组织中的遗传程序。
An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets—based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes—provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system. Animal development requires cells in complex organs to acquire distinct identities. During the development of the body wall musculature of the fruit fly, a pool of apparently identical cells gives rise to two types of muscle precursors, both of which are required for the appearance of functioning muscles. These identities depend on broad programs of gene expression. The authors attempt to dissect the complements of expressed genes that define these two different cell types by integrating modern methods in genetics, genomics, and informatics. By purifying informative cells from normal embryos and mutants that perturb muscle development, assaying their genomewide gene expression programs, and combining experiments statistically, they have identified fivefold more founder-specific genes than were previously suspected to characterize this cell type. The expression patterns of hundreds of genes were examined in whole embryos to test the statistical predictions, permitting the authors to estimate how many more cell type–specific genes remain to be discovered. Finally, dozens of the genes highlighted by these methods were tested for direct involvement in muscle development, and several new players in this process are reported. The integrated strategy used here can be generalized for studying genetic programs in other complex tissues.
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