Use of somatic mutations to quantify random contributions to mouse development

Abstract

Background: The C. elegans cell fate map, in which the lineage of its approximately 1000 cells is visibly charted beginning from the zygote, represents a developmental biology milestone. Nematode development is invariant from one specimen to the next, whereas in mammals, aspects of development are probabilistic, and development exhibits variation between even genetically identical individuals. Consequently, a single defined cell fate map applicable to all individuals cannot exist.

Results: To determine the extent to which patterns of cell lineage are conserved between different mice, we have employed the recently developed method of "phylogenetic fate mapping" to compare cell fate maps in siblings. In this approach, somatic mutations arising in individual cells are used to retrospectively deduce lineage relationships through phylogenetic and-as newly investigated here-related analytical approaches based on genetic distance. We have cataloged genomic mutations at an average of 110 mutation-prone polyguanine (polyG) tracts for about 100 cells clonally isolated from various corresponding tissues of each of two littermates of a hypermutable mouse strain.

Conclusions: We find that during mouse development, muscle and fat arise from a mixed progenitor cell pool in the germ layer, but, contrastingly, vascular endothelium in brain derives from a smaller source of progenitor cells. Additionally, formation of tissue primordia is marked by establishment of left and right lateral compartments, with restricted cell migration between divisions. We quantitatively demonstrate that development represents a combination of stochastic and deterministic events, offering insight into how chance influences normal development and may give rise to birth defects.

Figure 1

Somatic polyG mutation profiles of two mouse littermates. (A) Histogram showing how many different mutant alleles are identified for each marker across all of the approximately 100 cells genotyped for each mouse. An average of 5 mutant alleles was observed on each polyG marker in both mice, yielding an average mutation rate as 0.5 mutant alleles/polyG locus/cell. (B) Histogram showing the number of polyG marker mutations (from all approximately 110 markers) detected per cell for each mouse. For mouse 1, somatic mutations were observed in 36.7% of approximately 110 polyG markers for each single cell clone on average, while in mouse 2, an average of 34.4% were observed. (C) Average genetic distance of different types of tissues to the zygote for each mouse.

Figure 2

Lineage relationships inferred from methods based on genetic distance. (A) Modified eBURST analysis, showing “population snapshot” of single cell clones in mouse 1. Clusters of related single cell clones and individual unlinked clones are displayed as a single modified eBURST diagram by using the distance value D=0.2 as cut-off. Clusters of linked single cell clones correspond to complexes that share highly similar mutational profiles. Each single cell clone is represented as a dot with color indicative of its tissue origin. (Mouse 2 shown in Figure S1.) (B) Network representation depicting mutational similarities among single cell clones between both mice. Significant similarities between single cell clones for mouse 1 are shown with grey connecting lines. Each single cell clone is depicted as a dot with different colors indicative of tissue origin while the layout on the graph reflects relative anatomical location on the anteroposterior axis. The diameter of the circles correlates with the average distance within tissues. Orange lines show relationships that are conserved in mouse 2. (C) Scatter plot of distance between equivalent pairs of tissue, comparing mouse 1 to mouse 2. Distances of specific tissues to the zygote are colored orange; a trend line indicates their correlation. Among these comparisons, the distances between individual tissues to the zygote are largely conserved between the two mice.

Figure 3

Phylogenetic reconstruction of tissues and single cell clones in both mice. (A) Phylogenetic tree of tissues, with mouse 1 in black and mouse 2 in orange, overlaid. Numbers at bifurcations indicate Bayesian posterior probabilities. (B) Phylogenetic tree of single cell clones in mouse 1. Only branch structures with larger than 50% posterior probability are shown. Pairs of single cell clones from the same parental cell are marked with asterisks. (Mouse 2 shown in Additional file 8: Figure S2) (C) Distribution of N-bar symmetry statistic for mouse 1 tissue trees with highest posterior probabilities compared to random trees with the same number of branches, showing a symmetric nature of the actual tissue trees. Examples of symmetric (left, N-bar = 4) and asymmetric trees (right, N-bar = 7.43) are shown. (Equivalent distribution for the I_c symmetry statistic are shown in Additional file 8: Figure S3).

Figure 4

Developmental model. As gastrulation begins at around E6.5, the primitive streak forms and extends through the midline, establishing the anteroposterior body axis. During the process, mesoderm ingresses and begins to migrate to its ultimate position, where it will give rise to fibroblasts in muscle, preadipocytes, and endothelium. The progenitors of muscle fibroblasts and preadipocytes might arise earlier when mesoderm forms, starting from a pool of cells with fewer cell divisions (ranging from 6 to 8 divisions), while progenitors of brain endothelial cells could arise later from a few cells with a lengthier cell division history (ranging from 12 to 15 divisions). Once progenitors are established, those tissues may require similar numbers of further cell divisions to mature and develop into left and right compartments. The differing genetic identities and relative size of the progenitors for fibroblasts and preadipocytes are represented by differently colored spheres, and cell division history is indicated during mesoderm formation by color gradient, in which cells with fewer divisions appear more darkly colored. Numbers of cell divisions were calculated from the average genetic distance summarized in Table 1 using the mutation rate 0.010 for mouse 1 and 0.013 for mouse 2 as observed in this study. Schematic adapted from [46].