High Throughput Analysis Reveals Novel Maternal Germline RNAs Critical for PGC Preservation and Proper Migration. Xenopus laevis

During oogenesis hundreds of RNAs are selectively localized to either the animal or vegetal cortical region. These maternal RNAs include determinants of both somatic and germline fates. Although microarray analysis has contributed to identifying localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high throughput RNA-sequencing analysis to comprehensively interrogate both animal and vegetal pole RNAs in the fully-grown Xenopus laevis oocyte. We identified 411 enriched RNAs at the vegetal pole, 198 of them annotated transcripts, and 27 RNAs enriched at the animal pole, 15 annotated. Of these, 90 were novel RNAs over 4-fold enriched at the vegetal pole and 6 over 10-fold at the animal pole. Unlike mRNAs, we found that microRNAs were not asymmetrically distributed. Whole mount in situ hybridization revealed all 17 selected RNAs were localized, confirming our data set. Biological function and network analysis of vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands, RNA binding proteins and 10 transcription factors or co-factors with 5 defining hubs linking 47 genes in a network. Initial functional studies of maternal vegetally-localized RNAs show, for the first time, that sox7 plays an important role in primordial germ cell (PGC) development and efnb1(ephrinB1), known to play important roles in migration/adhesion in the nervous system, is required for proper PGC migration. Based on our findings, we propose potential pathways operating at the vegetal pole that highlight where future investigations might be most fruitful. Overall design: Examination of animal and vegetal pole samples of stg. VI X. laevis oocyte to determine vegetally enriched genes that may contribute to germ plasm and PGCs.

在卵子发生过程中，数百种RNA会被选择性定位于动物极或植物极皮层区域。这些母源RNA包含体细胞和生殖系的命运决定因子。尽管微阵列（microarray）分析曾助力定位相关决定因子，但该方法并不全面，且仅能针对已知转录本开展研究。本研究采用高通量RNA测序（high throughput RNA-sequencing）技术，对完全成熟的非洲爪蟾（Xenopus laevis）卵母细胞的动物极与植物极RNA进行了全面检测分析。我们共鉴定出411个植物极富集RNA，其中198个为已注释转录本；另有27个动物极富集RNA，其中15个为已注释转录本。其中，90个为植物极富集倍数≥4倍的新型RNA，6个为动物极富集倍数≥10倍的新型RNA。与信使RNA（mRNA）不同，我们发现微小RNA（microRNA）并未呈现不对称分布。通过整体原位杂交（whole mount in situ hybridization）实验，我们验证了所选的17个RNA均存在定位现象，证实了本数据集的可靠性。对植物极富集转录本的生物学功能与调控网络分析显示，其包含蛋白修饰酶、受体、配体、RNA结合蛋白以及10个转录因子或辅因子，并鉴定出5个核心枢纽节点，连接了该网络中的47个基因。针对母源植物极定位RNA的初步功能研究首次证实：Sox7在原始生殖细胞（primordial germ cell, PGC）发育过程中发挥关键作用；而此前已知在神经系统迁移与黏附过程中发挥功能的EFNB1（ephrinB1），则对正常PGC迁移不可或缺。基于本研究结果，我们提出了植物极潜在的调控通路，为未来的研究指明了方向。实验整体设计：对stg. VI期非洲爪蟾卵母细胞的动物极与植物极样本进行检测，以鉴定可能参与生殖质形成与原始生殖细胞发育的植物极富集基因。