A single-cell time-lapse of mouse prenatal development from gastrula to birth

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4–8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.


Statistics
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Software and code
Policy information about availability of computer code Data collection No software was used except Illumina RTA basecalling at this stage.
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Data Policy information about availability of data
All manuscripts must include a data availability statement.This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The data generated in this study can be downloaded in raw and processed forms from the NCBI Gene Expression Omnibus under accession number GSE186069 and nature research | reporting summary April 2020 GSE228590.The data are also available at https://omg.gs.washington.edu/,together with a browser that enables its visual exploration.The published datasets analyzed for this study were retrieved from either the GEO repository (GSE44183, GSE100597, GSE109071), https://github.com/MarioniLab/EmbryoTimecourse2018, or https://db.cngb.org/stomics/mosta/and re-processed.Published ISH images were obtained from the MGI website (https:// www.informatics.jax.org/).Mouse reference genome (mm10) and gene annotations (GENCODE VM12) were used for read alignment and gene count matrix generation.

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Sample size
No statistical methods were used to predetermine sample size.Our previous study (Qiu et al., 2022), which profiled 154,313 cells from 12 mouse embryos at early somitogenesis stage, successfully identified the same 30 cell types as those identified in E8.5 data by Pijuan-Sala et al. (2019).The extensive data, along with the separate processing of individual somite-resolved embryos, enabled the detection of significant substructures, such as A-P floor plates and various hindbrain segmentations.In this study, we applied the same technology to profile single nuclei from mouse embryos, identifying over 200 distinct cell types and focusing on several specific tissues and organs.This comprehensive analysis suggests that our sample size is adequate for investigating cell states and developmental trajectories during mouse organogenesis.In addition, we experimentally quantified the total DNA of staged embryos and estimated that the embryo grows 3,000-fold from E8.5 to P0.Therefore, despite the large number of nuclei profiled, our cellular coverage remains limited, ranging from 0.5-fold for early stages (summing 6 embryos, somite counts 7-12) to 0.002-fold immediately before birth (summing 6 embryos, E17.5-E18.75).
Data exclusions When we took a first round of cell-embedding, we noticed that one mouse embryo at E14.5 had a grossly reduced proportion of neuronal cells.This particular sample had been divided during pulverization, and we suspect that large portions of the frozen embryo did not make it into the experiment.We removed cells from this E14.5 embryo.

Replication
Firstly, we performed 15 sci-RNA-seq3 experiments, and the data from each experiment overlapped well, demonstrating high replicability.We have employed various methods to confirm the data quality.Secondly, to validate our findings regarding posterior embryos, we generated an independent validation dataset comprising somites 8-21, and the findings were validated.Thirdly, to validate our observations of abrupt transcriptional changes before and after birth, we generated a new "birth-series" dataset, and the findings were validated.Finally, for the spatial mapping analysis, we utilized publicly available ISH images to verify our cell-type annotations within the lateral plate mesoderm.
Randomization From a total of 523 embryos staged at the Jackson Laboratory, we selected 75 for whole embryo scRNA-seq, targeting one embryo for every somite count from 0 to 34 (2-hr increments), and one embryo for every 6-hr bin from E10 to P0.Embryos used in experiments were randomly selected from each timepoint before sample preparation.

Blinding
In this study, investigators were blinded to group allocation during sample collection and data generation/analysis: embryo collection and sci-RNA-seq3 data generation/analysis were performed by different researchers in different locations.
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