Spatiotemporal embryonic transcriptomics reveals the evolutionary history of the endoderm germ layer. Caenorhabditis elegans

The concept of germ layers has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years. Of the three germ layers, the mesoderm is found in bilaterian animals but is absent in species in the phyla Cnidaria and Ctenophora, which has been taken as evidence that the mesoderm was the final germ layer to evolve. The origin of the ectoderm and endoderm germ layers, however, remains unclear, with models supporting the antecedence of each as well as a simultaneous origin. Here we determine the temporal and spatial components of gene expression spanning embryonic development for all Caenorhabditis elegans genes and use it to determine the evolutionary ages of the germ layers. The gene expression program of the mesoderm is induced after those of the ectoderm and endoderm, thus making it the last germ layer both to evolve and to develop. Strikingly, the C. elegans endoderm and ectoderm expression programs do not co-induce; rather the endoderm activates earlier, and this is also observed in the expression of endoderm orthologues during the embryology of the frog Xenopus tropicalis, the sea anemone Nematostella vectensis and the sponge Amphimedon queenslandica. Querying the phylogenetic ages of specifically expressed genes reveals that the endoderm comprises older genes. Taken together, we propose that the endoderm program dates back to the origin of multicellularity, whereas the ectoderm originated as a secondary germ layer freed from ancestral feeding functions. Overall design: Two temporal assays of Caenorhabditis elegans embryonic development, starting at the zygote: (a) Embryos collected at fixed (~10 minute) time intervals. (b) Embryo segregates, up to five lines of blastomeres, isolated in reference to mitotic events. There were 184 samples in total, representing 100 distinct data points (50 in each assay).

胚层（germ layers）概念作为发育生物学、分类学、系统学与演化生物学领域的核心组织原则之一，已沿用150年之久。在三类胚层中，中胚层（mesoderm）存在于两侧对称动物中，但在刺胞动物门（Cnidaria）和栉水母动物门（Ctenophora）的物种中缺失，这一现象曾被视为中胚层是最晚演化出现的胚层的证据。然而，外胚层（ectoderm）与内胚层（endoderm）的起源仍尚无定论，现有研究模型分别支持二者之一更早出现，亦或二者同步起源。本研究对秀丽隐杆线虫（Caenorhabditis elegans）所有基因的胚胎发育全过程基因表达的时空特征进行解析，并以此推断各胚层的演化年龄。结果显示，中胚层的基因表达程序晚于外胚层与内胚层被诱导激活，由此表明其既是演化最晚也是发育最晚出现的胚层。值得注意的是，秀丽隐杆线虫的外胚层与内胚层表达程序并非同步激活：内胚层的激活更早，这一现象在热带爪蟾（Xenopus tropicalis）、海葵（Nematostella vectensis）以及澳洲大堡礁海绵（Amphimedon queenslandica）的胚胎发育过程中，其对应内胚层同源基因的表达模式中均得到验证。通过查询特异性表达基因的系统发育年龄，我们发现内胚层由更为古老的基因构成。综上，我们提出：内胚层的基因表达程序可追溯至多细胞生物的起源之时，而外胚层则作为脱离祖先摄食功能的次生胚层演化而来。总体实验设计：针对秀丽隐杆线虫的胚胎发育开展两项时序检测，均以受精卵为起始点：(a) 以约10分钟的固定时间间隔收集胚胎样本；(b) 依据有丝分裂事件分离胚胎裂球谱系，最高可至5个细胞裂阶段。本研究共包含184个样本，对应100个独立数据点（两项检测各50个）。