Ding Yi, Colozza Gabriele, Zhang Kelvin, Moriyama Yuki, Ploper Diego, Sosa Eric A, Benitez Maria D J, De Robertis Edward M
Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA.
Department of Biological Chemistry, University of California, Los Angeles, CA 90095, USA.
Dev Biol. 2017 Jun 15;426(2):176-187. doi: 10.1016/j.ydbio.2016.02.032. Epub 2016 Mar 23.
RNA sequencing has allowed high-throughput screening of differential gene expression in many tissues and organisms. Xenopus laevis is a classical embryological and cell-free extract model system, but its genomic sequence had been lacking due to difficulties arising from allotetraploidy. There is currently much excitement surrounding the release of the completed X. laevis genome (version 9.1) by the Joint Genome Institute (JGI), which provides a platform for genome-wide studies. Here we present a deep RNA-seq dataset of transcripts expressed in dorsal and ventral lips of the early Xenopus gastrula embryo using the new genomic information, which was further annotated by blast searches against the human proteome. Overall, our findings confirm previous results from differential screenings using other methods that uncovered classical dorsal genes such as Chordin, Noggin and Cerberus, as well as ventral genes such as Sizzled, Ventx, Wnt8 and Bambi. Complete transcriptome-wide tables of mRNAs suitable for data mining are presented, which include many novel dorsal- and ventral-specific genes. RNA-seq was very quantitative and reproducible, and allowed us to define dorsal and ventral signatures useful for gene set expression analyses (GSEA). As an example of a new gene, we present here data on an organizer-specific secreted protein tyrosine kinase known as Pkdcc (protein kinase domain containing, cytoplasmic) or Vlk (vertebrate lonesome kinase). Overexpression experiments indicate that Pkdcc can act as a negative regulator of Wnt/ β-catenin signaling independently of its kinase activity. We conclude that RNA-Seq in combination with the X. laevis complete genome now available provides a powerful tool for unraveling cell-cell signaling pathways during embryonic induction.
RNA测序使得在许多组织和生物体中对差异基因表达进行高通量筛选成为可能。非洲爪蟾是一种经典的胚胎学和无细胞提取物模型系统，但由于异源四倍体带来的困难，其基因组序列一直缺失。目前，联合基因组研究所（JGI）发布的完整非洲爪蟾基因组（9.1版）引发了广泛关注，它为全基因组研究提供了一个平台。在此，我们利用新的基因组信息，展示了非洲爪蟾早期原肠胚胚胎背唇和腹唇中表达的转录本的深度RNA测序数据集，并通过与人蛋白质组的比对搜索对其进行了进一步注释。总体而言，我们的研究结果证实了之前使用其他方法进行差异筛选所得到的结果，这些方法揭示了经典的背侧基因，如脊索蛋白、头蛋白和Cerberus，以及腹侧基因，如Sizzled、Ventx、Wnt8和Bambi。我们呈现了适用于数据挖掘的完整全转录组mRNA表格，其中包括许多新的背侧和腹侧特异性基因。RNA测序具有很高的定量性和可重复性，使我们能够定义可用于基因集表达分析（GSEA）的背侧和腹侧特征。作为一个新基因的例子，我们在此展示了一种组织者特异性分泌蛋白酪氨酸激酶的数据，该激酶被称为Pkdcc（含蛋白激酶结构域，胞质）或Vlk（脊椎动物孤独激酶）。过表达实验表明，Pkdcc可以独立于其激酶活性，作为Wnt/β-连环蛋白信号通路的负调节因子。我们得出结论，RNA测序与现有的非洲爪蟾完整基因组相结合，为揭示胚胎诱导过程中的细胞间信号通路提供了一个强大的工具。
