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Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer

Abstract

The concept of germ layers has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years (refs 1, 2, 3). 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 evolve1,4,5. The origin of the ectoderm and endoderm germ layers, however, remains unclear, with models supporting the antecedence of each as well as a simultaneous origin4,6,7,8,9. 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.

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Figure 1: Determining the expression profiles of the C. elegans embryonic founder cell lineages.
Figure 2: Dynamics of germ-layer gene expression throughout development.
Figure 3: The endoderm expression program precedes the ectoderm program in diverse species.
Figure 4: The germ layers exhibit distinct gene ages and functional category enrichments.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The complete data set has been deposited in the National Center for Biotechnical Information Gene Expression Omnibus database under accession number GSE50548.

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Acknowledgements

We acknowledge the contribution of computational analyses by D. H. Silver, L. Anavy and F. Wagner in an early stage of this project. We also acknowledge advice from B. Degnan, A. Cole, M. Adamska and A. Polsky. We thank the Technion Genome Center for technical assistance. This work was supported by a European Research Council grant (EvoDevoPaths) and the EMBO Young Investigator Program.

Author information

Authors and Affiliations

Authors

Contributions

T.H. and I.Y. designed the experiment. T.H. performed the experiments. M.L. contributed whole-embryo data. M.F. performed the initial analysis on the RNA-sequencing data. I.Y. analysed the data with help from T.H. and M.F. T.H., B.K.H. and I.Y. wrote the manuscript.

Corresponding author

Correspondence to Itai Yanai.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 In vitro culturing of the C. elegans embryonic founder blastomeres.

The cells are separated as shown in the left schematic and then cultured in embryonic growth medium11 as shown in the micrographs on the right. The numbers indicate the stages at which the cells were collected for transcriptome analysis. Six of the 11 stages are shown in the micrographs.

Extended Data Figure 2 A transcriptomic survey of C. elegans embryonic founder cell lineages.

a, Replicates of the embryonic blastomere time courses. The heat maps show the correlations among the replicates for each blastomere lineage at each of the eleven examined stages. For three blastomere stages there were no replicates. The median correlation coefficient is 0.9. Samples were collected in triplicates. Only samples with at least 750,000 reads were used, which has been previously shown to be of sufficient sequencing depth for CEL-seq12. Supplementary Table 3 provides the sequencing statistics for each sample. b, Expression profiles of the 3,910 dynamic genes across the blastomere lineage time courses. See Methods for definition of dynamic genes. c, Correlation coefficients between samples of the whole-embryo time course. Each of the 50 samples comprises a single embryo, collected at the indicated minutes past the four-cell stage. Again, only samples with at least 750,000 reads were used and Supplementary Table 3 provides the sequencing statistics for each sample. d, The expression profiles of the 1,664 genes with differentiated expression analysed in Fig. 1c. Each profile was ‘standardized’ by subtracting its mean and dividing by its standard deviation. e, Comparison of the blastomere time courses to the EPIC data set15. For 115 genes, we could compare gene expression to previously published embryonic expression profiles generated by microscopic lineaging until the 300-cell stage15,39. Of these, 75% of our profiles had consistent localized expression (Supplementary Table 1). Of those, 54% matched completely, and 21% of the genes expressed in all of the lineages in our data set had some missing expression in the EPIC data set because the lineaging was not performed until the end of the developmental process. The remaining genes have some overlap in expression. Such differences in expression could be caused by the transgene in the EPIC data set not recapitulating the profile of the endogenous gene, or missing signals between cells in the blastomere data set, as is seen from the whole-embryo/blastomere expression level ratio (see Supplementary Table 1, ratios defined as equal, slightly higher/lower or much higher/lower). Expression profile compared with the EPIC data set deviates more when expression in the blastomeres is low compared with the whole embryo, but the blastomere data set has the advantage that all genes are assayed simultaneously, no transgenes are used, maternal transcripts are seen and downregulation of genes is observable.

Extended Data Figure 3 Lineage-restricted gene expression identifies genes dependent upon coherence of the lineages and tissue specificity.

a, Expression profiles of genes involved in pharynx specification. The left and right panels correspond to the two Notch signalling events. The top and bottom images correspond to the expected regulatory patterns in the whole embryo and isolated blastomeres, respectively. The tbx-37 gene is not shown since it is identical to tbx-38 in expression profile. b, Comparison of the overall sum of expression between the two time courses, plotted on a log2 scale (black). Genes ‘missing’ in the separated lineage time course were manually added to the graph at −3. The additional plots indicate the same measure for dynamically expressed genes (blue) and constitutive genes (red). c, Idealized expression profiles used to identify gene expression clusters. d, The gene expression profiles for the temporally restricted gene expression profiles. Each profile was ‘standardized’ by subtracting its mean and dividing by its standard deviation. e, Average expression profiles of ten clusters of dynamically expressed genes determined on the basis of the whole-embryo expression data (see Methods). f, The number of dynamic genes in each temporal period. In each group, the genes not expressed in the lineage time course (b) are marked in red.

Extended Data Figure 4 The first principal component correlates with developmental time.

Principal component analysis as described in Fig. 2a. Colour codes are the same as in Fig. 1. PC1, PC2 and PC3 capture 18%, 12% and 11%, respectively, of the variation in the expression, in the 1,320 dynamically expressed genes with no expression in the first stage (to exclude genes with maternal expression).

Extended Data Figure 5 Germ-layer-specific expression.

Expression profiles of the germ-layer-specific genes in each of the lineages. The x and y axes are the 11 examined temporal stages and individual genes, respectively. Germ-layer-specific genes were identified by hierarchical clustering based upon correlation among dynamically expressed genes (see Methods).

Extended Data Figure 6 Robustness of gene age analysis.

a, Same format as Fig. 4a but with the definition of old genes as those present in at least 25% of the examined eukaryotes (see Methods) that are not ophisthokonts. b, Same as Fig. 4a with a definition of ‘old’ as those present in 25% of the examined organisms that are not eukaryotes (Eubacteria and Archaea).

Extended Data Figure 7 Truncated endoderm gene set control.

To exclude the possibility that general genes were included as ‘endoderm-specific’ because the endoderm program is induced earlier, we excluded temporal clusters 8, 9 and 10 from the endoderm genes and repeated the relevant analyses. We found that there was no marked change in the results. The results are shown in the same format as Figs 3 and 4b, c.

Extended Data Table 1 The fates of the progeny of each blastomere in vivo and in isolated cultured blastomeres
Extended Data Table 2 Description of the developmental stages queried in this study
Extended Data Table 3 Tissue-specific gene sets

Supplementary information

Supplementary Tables

This file contains Supplementary Tables 1-3 (PDF 841 kb)

Supplementary Table 4

This file contains endoderm, ectoderm, and mesoderm germ layer gene sets. (XLSX 617 kb)

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Hashimshony, T., Feder, M., Levin, M. et al. Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer. Nature 519, 219–222 (2015). https://doi.org/10.1038/nature13996

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