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Extensive phylogenies of human development inferred from somatic mutations

Abstract

Starting from the zygote, all cells in the human body continuously acquire mutations. Mutations shared between different cells imply a common progenitor and are thus naturally occurring markers for lineage tracing1,2. Here we reconstruct extensive phylogenies of normal tissues from three adult individuals using whole-genome sequencing of 511 laser capture microdissections. Reconstructed embryonic progenitors in the same generation of a phylogeny often contribute to different extents to the adult body. The degree of this asymmetry varies between individuals, with ratios between the two reconstructed daughter cells of the zygote ranging from 60:40 to 93:7. Asymmetries pervade subsequent generations and can differ between tissues in the same individual. The phylogenies resolve the spatial embryonic patterning of tissues, revealing contiguous patches of, on average, 301 crypts in the adult colonic epithelium derived from a most recent embryonic cell and also a spatial effect in brain development. Using data from ten additional men, we investigated the developmental split between soma and germline, with results suggesting an extraembryonic contribution to primordial germ cells. This research demonstrates that, despite reaching the same ultimate tissue patterns, early bottlenecks and lineage commitments lead to substantial variation in embryonic patterns both within and between individuals.

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Fig. 1: Phylogenies of clonal populations.
Fig. 2: Developmental phylogenies and embryonic asymmetries.
Fig. 3: Embryonic mosaicism in tissues and organs.
Fig. 4: Patterns of mutations in early embryogenesis.

Data availability

The DNA sequencing data are deposited in the European Genome-Phenome Archive (EGA) with the accession codes EGAD00001006641 (whole-genome sequencing) and EGAD00001006643 (targeted sequencing).

Code availability

The bespoke R scripts used for analysis and visualization in this study are available online from GitHub (https://github.com/TimCoorens/PanBody_Phylogenies).

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Acknowledgements

We thank the staff of the Wellcome Sanger Institute Sample Logistics, Genotyping, Pulldown, Sequencing and Informatics facilities for their contribution, especially L. O’Neill, C. Latimer and K. Roberts for their support with sample management and laboratory work; and S. Behjati, Y. S. Ju, S. Park, F. Abascal, J. Ijaz, P. Nicola and G. Collord for helpful discussions or critical review of the manuscript. This experiment was primarily funded by Wellcome (core funding to the Wellcome Sanger Institute and PhD studentship to T.H.H.C.; 203943/Z/16/Z). L.M. is a recipient of a Cancer Research UK (CRUK) Clinical PhD fellowship (C20/A20917) and the Jean Shank/Pathological Society of Great Britain and Ireland Intermediate Research Fellowship (grant reference no. 1175). T.J.M. is supported by CRUK and the Royal College of Surgeons (C63474/A27176). I.M. is funded by CRUK (C57387/A21777) and the Wellcome Trust. R.R. is funded by CRUK (C66259/A27114).

Author information

Affiliations

Authors

Contributions

T.H.H.C., L.M., R.R. and M.R.S. conceived the study design. T.H.H.C. wrote the scripts and performed the analyses with help or input from R.S., J.C., M.D.C.N., M.S.C. and I.M. L.M., P.S.R., A.C. and T.R.W.O. performed the microdissections with support from Y.H. M.J.P. and A.R.J.L. called and analysed mitochondrial variants. T.J.M., A.N. and R.C.F. aided in sample procurement. M.R.S. oversaw the study. T.H.H.C. and M.R.S. wrote the manuscript with input from all other authors.

Corresponding author

Correspondence to Michael R. Stratton.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Nuria Lopez-Bigas, Aaron Mckenna, David Posada and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Extended data figures and tables

Extended Data Fig. 1 VAF distributions reflect clonality of LCM sample.

a, Schematic of three different progenitor or stem cell contributions to the eventual sample. Monoclonal samples consist of the progeny of one cell, while oligoclonal and polyclonal are derived from a few and many progenitors, respectively. bd, VAF histograms and binomial decompositions for a monoclonal (b), oligoclonal (c) and polyclonal (d) sample. The red and blue dashed lines indicate clonal decomposition through a binomial mixture model, with the estimated peak VAF of clones indicated in the legend. The number indicated in the title of each histogram is the SNV burden.

Extended Data Fig. 2 Model of early embryogenesis and ABC.

a, Heat maps showing the results of estimates of the early embryonic bottleneck obtained through approximate Bayesian computation, with and without cell death rate as a parameter (Methods). The darkness of the colour indicates the frequency of the observed bottleneck in the accepted simulations. b, Estimates of the mutation rate per cell per division before and after zygotic genome activation (ZGA); the dot indicates the mean of the 20,000 accepted simulations and the line spans the 95% confidence interval.

Extended Data Fig. 3 The most recent common ancestors of tissues and completeness of early lineages.

ad, Phylogenetic trees with unit branch lengths for PD28690, showing the coalescence (red) of all samples from four tissues types: thyroid follicles (a), seminiferous tubules (b), small bowel crypts (c) and bronchial epithelium (d). The most recent common ancestor for all these tissues is the root of the tree. e, Sum of mean VAFs of branches of the same generation per bulk sample in PD28690 (n = 33), PD43850 (n = 1) and PD43851 (n = 2). The solid black line indicates the mean value across tissue samples. A total sum of mean VAFs approximating 0.5 indicates that all cells belong to one of the lineages of that generation and are accounted for, that is, no lineages are missing from the phylogeny. This is mostly the case for generations 1 and 2, but the total VAF of generation 3 indicates missing lineages.

Extended Data Fig. 4 Embryonic patch size in the colon.

a, Kernel smoothed 2D histogram of the linear distance (in number of crypts) and the number of shared SNVs between any two crypts from the same biopsy. The red line is shown at a shared SNV burden of 15, above which crypts were taken to be from the same embryonic patch. b, Histogram of the number of SNVs shared between all pairs of crypts showing a bimodal distribution on either side of an SNV burden of 15 (red line). c, Density plot of the prior distribution of the embryonic patch size radius. d, Plot of the radius versus the Euclidean distance in summary statistics between the simulations and our observed data. The red dots indicate those within the 5% closest simulations and are accepted. e, Density plot of the prior distribution (dashed line), the posterior distribution from the rejection method (black line) and the posterior distribution from the neural network regression (red line) of the embryonic patch size radius. f, A QQ-plot of the residuals of the neural network regression.

Extended Data Fig. 5 Clonal expansions later in life.

a, Phylogenetic tree for appendiceal crypts in PD28690, with annotated cancer driver mutations. An asterisk indicates that the two neighbouring crypts were taken as biological replicates of one another. Within the clade of crypts that acquired the BRAF mutation, the mutation burdens are Poisson distributed, consistent with a molecular clock (P = 0.99, dispersion test). Accordingly, we can estimate that the BRAF mutation was acquired before 23 years of age. b, c, Phylogeny (b) and sampling overview (c) for prostatic acini in PD28690, showing widespread benign prostatic hyperplasia in one biopsy. d, Histology and sampling overview alongside the phylogeny for a microscopic polyp in the colon of PD28690. e, Phylogeny of seminiferous tubules from PD42034, where a frameshift deletion in MEIOB was acquired after only six post-zygotic SNVs. Parts of the figure are composed of pictures from Servier Medical Art (https://smart.servier.com/). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

Extended Data Fig. 6 Decomposition of polyclonal samples.

Phylogenetic trees with unit branch lengths for four polyclonal samples of the epidermis from PD28690, showing the contribution (blue) of early embryonic progenitors in the phylogeny to the sample. These samples were not used for the reconstruction of the phylogeny because of their lack of a dominant clone, but can still be decomposed into the contributing embryonic lineages that give rise to these polyclonal aggregates. For example, ‘SKN2_D2’, while somatically polyclonal, seems to be derived from a single early lineage.

Extended Data Fig. 7 Targeted resequencing in PD28690.

Cladogram of PD28690 with contribution to 84 bulk samples (none derived from testes) as assessed through targeted resequencing of embryonic and spermatogonia-specific variants. The colour of the branch indicates the mean VAF of substitutions on that branch across all bulk samples. Nodes that gave rise to only seminiferous tubules are annotated with an asterisk. Branches coming from those nodes do not contribute to the bulk samples, confirming that the segregation of primordial germ cell lineages coincides with the observed branching point on the phylogenies.

Extended Data Fig. 8 Early embryogenesis and bottlenecks.

a, Overview of lineage commitments in the early human embryo, up until gastrulation and early organogenesis. The blue arrows indicate the putative contribution of extraembryonic cells to embryonic lineages (for yolk sac haematopoiesis and intercalation of the endoderm) or lineages with an unknown origin (primordial germ cells). b, Schematic of the possible influence of multiple, successive bottlenecks on the eradication of a specific lineage in a certain population of cells. The two daughter lineages of the zygote are coloured in red and blue. Note that this is a toy example merely for illustration and the relative cell numbers or size of the bottlenecks need not represent reality.

Extended Data Fig. 9 Patterns of mitochondrial and nuclear SNVs.

ad, Phylogenies of nuclear SNVs with the VAF of mitochondrial mutations overlaid on them, showing a late shared SNV (a), an SNV that was heteroplasmic in the zygote (b), an SNV that is consistent with a shared subclone or stromal contamination (c) and an SNV recurrently acquired in samples from different tissues (d). e, Mutational spectrum and decomposition of early embryonic nuclear SNVs.

Extended Data Fig. 10 Loss of the Y chromosome.

a, b, Scatterplots showing the ratio between the mean Y-chromosomal coverage and autosomal coverage against the mean autosomal coverage for all samples from PD28690 (a) and PD43851 (b). The dashed red lines indicate the 95% confidence interval around an expected ratio of 0.5. The red dots indicate samples with significant evidence of loss of the Y chromosome. c, Phylogeny of PD28690 with samples exhibiting loss of the Y chromosome marked in red, indicating that all loss of the Y chromosome events are acquired independently.

Supplementary information

Supplementary Methods

This Supplementary Methods file includes Supplementary Tables 7–10 and Supplementary Fig. 1, and has the following sections: Proportion of SNVs filtered at each stage; Validation of phylogenies; Validation of non-monophyly of tissues; Recurrent SNVs and the infinite sites model; and Mutation rate in early embryogenesis.

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Coorens, T.H.H., Moore, L., Robinson, P.S. et al. Extensive phylogenies of human development inferred from somatic mutations. Nature 597, 387–392 (2021). https://doi.org/10.1038/s41586-021-03790-y

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