Leaps and lulls in the developmental transcriptome of Dictyostelium discoideum

Development of the soil amoeba Dictyostelium discoideum is triggered by starvation. When placed on a solid substrate, the starving solitary amoebae cease growth, communicate via extracellular cAMP, aggregate by tens of thousands and develop into fully integrated multicellular organisms. Early phases of the developmental program are often studied in cells starved in suspension while cAMP is provided exogenously. Previous studies revealed massive shifts in the transcriptome under these developmental conditions and a close relationship between gene expression and morphogenesis, but were limited by the sampling frequency and the resolution of the methods. Here, we combine the superior depth and specificity of RNA-seq with high frequency sampling during filter development and cAMP pulsing in suspension. We found that the developmental transcriptome exhibits mostly gradual changes interspersed by a few instances of large changes. Considering the whole transcriptome as a single phenotype, we treated each time point as an independent entity and were able to characterize development as groups of similar time points separated by gaps. The grouped time points represent gradual changes in mRNA abundance and the gaps represent times during which many genes are differentially expressed rapidly. Comparing development on solid substrate to development in suspension revealed that gene expression in filter developed cells lagged behind cells treated with exogenous cAMP in suspension. The high sampling frequency revealed many genes whose regulation is reproducibly more complex than indicated by previous studies. Gene Ontology enrichment analysis showed that the transition to multicellularity at the mound stage coincided with rapid accumulation of transcripts associated with mitosis and DNA replicative processes. Later development included the up-regulation of organic signaling molecules and co-factor biosynthesis. We observed multiple instances of enrichment of oxidation-reduction and reactive oxygen related terms. Our analysis also demonstrated a high level of synchrony between the developing structures throughout development. Our data describe D. discoideum development as a series of coordinated cellular and multicellular activities. Coordination occurred within fields of aggregating cells and between multicellular bodies, such as mounds or migratory slugs that experience both cell-cell contact and various soluble signaling regimes. These time courses, sampled at the highest temporal resolution to date in this system, provide a comprehensive resource for future studies of developmental gene expression. Dictyosteloium discoideum gene expression (mRNA) profiles from two experimental time courses: development on filters and cAMP signaling in suspension. Filter development consists of 2 replicates of 19 time point samples, while cAMP in suspension contains 3 replicates of 10, 10 and 9 samples each.

土壤变形虫盘基网柄菌（Dictyostelium discoideum）的发育由饥饿信号触发。当被接种至固体基质上时，处于饥饿状态的单个变形虫会停止增殖，通过细胞外环腺苷酸（extracellular cAMP）进行信号交流，随后数以万计的个体发生聚集，最终发育为高度整合的多细胞生物体。
发育程序的早期阶段通常在悬浮饥饿培养的细胞中开展研究，此时通过外源途径提供环腺苷酸。既往研究表明，该发育条件下转录组会发生大规模表达重塑，且基因表达与形态发生密切相关，但受限于采样频率与实验方法的分辨率。
本研究将RNA测序（RNA-seq）优异的测序深度与特异性，与滤膜发育及悬浮液中环腺苷酸脉冲刺激过程中的高频采样相结合。我们发现，发育转录组整体以渐进式变化为主，其间穿插少量剧烈的表达波动。
将整个转录组视为单一表型，我们将每个时间点作为独立样本进行分析，最终可将发育过程划分为若干由间隙分隔的相似时间点集群。这些集群对应mRNA丰度的渐进式变化，而间隙则代表大量基因快速发生差异表达的阶段。
对比固体基质上的发育过程与悬浮培养的发育过程发现，滤膜培养细胞的基因表达滞后于悬浮外源环腺苷酸处理的细胞。
高频采样揭示了大量调控模式较既往研究更为复杂且具备可重复性的基因。
基因本体（Gene Ontology）富集分析显示，在丘期向多细胞性转变的过程中，与有丝分裂及DNA复制相关的转录本快速积累。发育后期则包含有机信号分子及辅因子生物合成相关基因的上调。我们还多次观察到氧化还原与活性氧相关术语的富集。
本分析同时证实，在整个发育过程中，发育中的结构始终保持高度同步性。本研究将盘基网柄菌的发育描述为一系列协同的细胞及多细胞活动过程。这种协同既发生于聚集细胞群体内部，也存在于多细胞结构体之间——如既存在细胞间接触、又受多种可溶性信号调控的丘体或迁移蛞蝓阶段。
本研究的时间序列采样在该体系中达到了目前最高的时间分辨率，可为后续发育基因表达研究提供全面的参考资源。
本数据集包含两组实验时间序列的盘基网柄菌基因表达（mRNA）谱：滤膜发育组与悬浮液中环腺苷酸信号处理组。其中滤膜发育组包含2组生物学重复，共计19个时间点样本；悬浮环腺苷酸处理组包含3组生物学重复，每组分别包含10、10及9个时间点样本。