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A single-cell transcriptome atlas of marsupial embryogenesis and X inactivation

A Publisher Correction to this article was published on 19 January 2021

A Publisher Correction to this article was published on 16 October 2020

This article has been updated

Abstract

Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of X dosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160 million years ago. They exhibit distinctive developmental features, including late implantation1 and imprinted X chromosome inactivation2, which is associated with expression of the XIST-like noncoding RNA RSX3. Here we perform a single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in a marsupial, the grey short-tailed opossum (Monodelphis domestica). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian–marsupial divergence. RSX coating and inactivation of the X chromosome occurs early and rapidly. This observation supports the hypothesis that—in organisms with early X chromosome inactivation—imprinted X chromosome inactivation prevents biallelic X silencing. We identify XSR, an RSX antisense transcript expressed from the active X chromosome, as a candidate for the regulator of imprinted X chromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and X dosage compensation.

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Fig. 1: Opossum embryogenesis and single-cell clustering.
Fig. 2: Opossum lineage formation.
Fig. 3: Ontogeny of opossum X-dosage compensation.
Fig. 4: XCI in the EPI and characterization of XSR.

Data availability

All cytological data and R markdown files in this study are included in this published Article, and its Supplementary Information (Supplementary Information 36). Sequence data have been deposited at ArrayExpress (accession code E-MTAB-7515).

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Acknowledgements

This work was supported by European Research Council (CoG 647971) and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001193), UK Medical Research Council (FC001193) and Wellcome Trust (FC001193). We thank the Francis Crick Institute Biological Research, Advanced Sequencing and R. Goldstone, and Light Microscopy facilities and D. Bell for their expertise; H. Niwa for the POU5F3 antibody; D. Page for the MAB1980 (opossum Y chromosome) probe; and members of the J.M.A.T. and K. Niakan laboratories for comments on the manuscript.

Author information

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Authors

Contributions

J.M.A.T. and S.K.M. conceived the project. S.K.M. performed and optimized embryo collection, single-cell collection, RNA extraction, cDNA synthesis, RNA FISH, DNA FISH, immunofluorescence, RT–PCR and MiSeq. M.N.S. performed computational analysis. T.H. advised on data representation and generated figures. J.M.A.T., S.K.M. and M.N.S. wrote the manuscript.

Corresponding author

Correspondence to James M. A. Turner.

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The authors declare no competing interests.

Additional information

Peer review information Nature thanks Takashi Sado and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Illustration of late opossum embryo development and single-cell clustering.

a, Schematic of opossum embryos at E8–E12.5. b, Single-cell correlation coefficients. c, Principal component analysis.

Extended Data Fig. 2 Embryonic genome activation, Y gene expression and clustering by unsupervised 3D t-SNE.

a, Y gene expression in scRNA-seq data derived from oocytes (n = 6 cells) and presumptive male embryos at E1.5 (n = 7 embryos) and E2.5 (n = 5 embryos), and E3.5 to E7.5 (n = 20 embryos). Y transcripts cannot be maternally deposited, because the Y chromosome is inherited from the father. Their appearance must therefore signify the initiation of embryonic transcription. b, Immature and mature (asterisk) nucleolar morphology in embryos at E2.5 and E3.5, respectively. Images are maximum projections. Scale bars, 10 μm. c, Percentage of multi-allelic X- and autosomally encoded SNP variants in male bulk RNA-seq data (n = 28 embryos). When multiple SNPs for X-encoded RNAs are detected, maternal RNA is still present. However, when single SNPs are detected, it has been degraded. d, Unsupervised clustering of scRNA-seq data. e, Gene ontology analysis for genes in d.

Extended Data Fig. 3 Identification of lineage-specific genes.

a, Pseudotemporal ordering from Fig. 2c in 3D. b, Pseudotemporal ordering from Fig. 2c separated by sex. c, Pseudotemporal ordering from Fig. 2c, with cells coloured according to developmental age. d, Full heat map of genes defining cluster 1, cluster 2, early EPI, EPI, PrE, early TE and TE. e, Gene ontology analysis for cluster 1 and cluster 2, early EPI and early TE.

Extended Data Fig. 4 Lineage marker gene expression and immunostaining.

a, Expression of representative genes in cluster 1, early EPI, EPI, PrE, early TE and TE. b, Expression of GATA6 and TEAD4 at E5.5. All cells are TEAD4-negative, even those that are not expressing GATA6 (encircled). Images are maximum projections. Scale bar, 10 μm.

Extended Data Fig. 5 Lineage clusters in eutherians.

ac, Semi-supervised 3D t-SNE (left) and heat map (right) of mouse (a), macaque (b) and human (c) embryos, with single cells coloured according to cluster.

Extended Data Fig. 6 Eutherian and marsupial lineage conservation.

a, Genes expressed in EPI, PrE and TE in mouse, macaque and human. b, Heat map showing expression of conserved genes in opossum. c, Expression of opossum genes, the orthologues of which are associated with naive and primed pluripotency in macaque. d, Expression heat map of opossum genes with mouse orthologues that are expressed specifically in mouse EPI at E6.5 (Coro1a) or E4.5 (all other genes).

Extended Data Fig. 7 Analysis of X inactivation.

a, RSX expression in female opossums. b, Dual RSX and MSN RNA FISH in embryos at E2.5 (16 embryos = 68 cells). c, Dual RSX and MSN RNA FISH (left), followed by Y chromosome DNA FISH (right) in male embryos at E4.5; blastomeres magnified in insets. d, Y gene versus RSX expression. e, MSN RNA FISH followed by Y chromosome DNA FISH in opossum spermatids. f, Dual RSX and MSN RNA FISH in female opossum brain cells. Images are maximum projections. Scale bars, 10 μm. g, Percentage of multi-allelic X SNP variants in female (n = 15 embryos) and male (n = 28 embryos) bulk RNA-seq data from E3.5 to E7.5. Mann–Whitney U-test was used to calculate P values. For RNA FISH data, quantification is shown below the images. h, SNP analysis showing expression of 11 genes from the paternal X chromosome in embryos at E3.5 (n = 3) and E4.5 (n = 2). The location of RSX is shown. Asterisks denote instances in which informative SNPs were not present. ATRX is the only gene that exhibits no expression from the paternal X chromosome at E3.5 (star): expression from the maternal X chromosome at this age could represent maternal products, with activation of embryonic ATRX initiating at E4.5.

Extended Data Fig. 8 Further analysis of X inactivation.

a, RSX localization to the inactive X chromosome (identified using MSN DNA FISH) in dividing cells. Images are maximum projections. Scale bar, 10 μm. b, X chromosome-to-autosome ratios in female clusters at E6.0–E7.5. c, Reverse transcription PCR analysis of RSX, XSR, TFE3Y and GAPDH in female and male embryos at E5.5. d, RSX and XSR expression in adult female and male tissues. e, Dual RSX and XSR RNA FISH in female and male embryos at E3.5. The explanation for why the RSX and XSR FISH signals are the same colour is given in the Methods.

Extended Data Table 1 Opossum embryos and single cells used for RNA-seq
Extended Data Table 2 RNA FISH for RSX and MSN in the preimplantation opossum embryo

Supplementary information

Supplementary Information 9

Supplementary discussion. Interrogation of the pluriblast cell population in opossum embryos.

Reporting Summary

Supplementary Table 1

. Differential expression test results of lineage markers. Relates to Fig.2a, b, d and Extended Data Figure 3.

Supplementary Table 2

. Eutherian - marsupial lineage conservation analysis. Relates to Extended Data Figure 6.

Supplementary Information 3

. R markdown html file for Monodelphis domestica analysis.

Supplementary Information 4

. R markdown html file for Macaca fascicularis analysis.

Supplementary Information 5

. R markdown html file for Homo sapiens analysis.

Supplementary Information 6

. R markdown html file for Mus musculus analysis.

Supplementary Table 7

. X chromosome genes primers. The 11 X genes primers used in identifying parental SNPs.

Supplementary Table 8

. RSX and XSR MiSeq reads with SNPs - Parent-of-origin expression of RSX and XSR in whole embryos at E3.5 and E5.5 opossum embryos.

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Mahadevaiah, S.K., Sangrithi, M.N., Hirota, T. et al. A single-cell transcriptome atlas of marsupial embryogenesis and X inactivation. Nature 586, 612–617 (2020). https://doi.org/10.1038/s41586-020-2629-6

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