Altmann C R, Bell E, Sczyrba A, Pun J, Bekiranov S, Gaasterland T, Brivanlou A H
Laboratory of Molecular Vertebrate Embryology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Dev Biol. 2001 Aug 1;236(1):64-75. doi: 10.1006/dbio.2001.0298.
In order to examine transcriptional regulation globally, during early vertebrate embryonic development, we have prepared Xenopus laevis cDNA microarrays. These prototype embryonic arrays contain 864 sequenced gastrula cDNA. In order to analyze and store array data, a microarray analysis pipeline was developed and integrated with sequence analysis and annotation tools. In three independent experimental settings, we demonstrate the power of these global approaches and provide optimized protocols for their application to molecular embryology. In the first set, by comparing maternal versus zygotic transcription, we document groups of genes that are temporally regulated. This analytical approach resulted in the discovery of novel temporally regulated genes. In the second, we examine changes in gene expression spatially during development by comparing dorsal and ventral mesoderm dissected from early gastrula embryos. We have discovered novel genes with spatial enrichment from these experiments. Finally, we use the prototype microarray to examine transcriptional responses from embryonic explants treated with activin. We selected genes (two of which are novel) regulated by activin for further characterization. All results obtained by the arrays were independently tested by RT-PCR or by in situ hybridization to provide a direct assessment of the accuracy and reproducibility of these approaches in the context of molecular embryology.
为了在脊椎动物胚胎发育早期全面研究转录调控，我们制备了非洲爪蟾cDNA微阵列。这些胚胎微阵列原型包含864个已测序的原肠胚cDNA。为了分析和存储阵列数据，我们开发了一个微阵列分析流程，并将其与序列分析和注释工具整合在一起。在三个独立的实验设置中，我们展示了这些全局方法的强大功能，并为其在分子胚胎学中的应用提供了优化方案。在第一组实验中，通过比较母源转录与合子转录，我们记录了在时间上受到调控的基因群。这种分析方法导致发现了新的在时间上受到调控的基因。在第二组实验中，我们通过比较从早期原肠胚胚胎中分离出的背侧和腹侧中胚层，研究了发育过程中基因表达的空间变化。我们从这些实验中发现了具有空间富集性的新基因。最后，我们使用微阵列原型来检测用激活素处理的胚胎外植体的转录反应。我们选择了受激活素调控的基因（其中两个是新基因）进行进一步表征。通过阵列获得的所有结果均通过逆转录聚合酶链反应（RT-PCR）或原位杂交进行独立测试，以便在分子胚胎学背景下直接评估这些方法的准确性和可重复性。
