A stem cell zoo uncovers intracellular scaling of developmental tempo across mammals

Abstract

Differential speeds in biochemical reactions have been proposed to be responsible for the differences in developmental tempo between mice and humans. However, the underlying mechanism controlling the species-specific kinetics remains to be determined. Using in vitro differentiation of pluripotent stem cells, we recapitulated the segmentation clocks of diverse mammalian species varying in body weight and taxa: marmoset, rabbit, cattle, and rhinoceros. Together with mouse and human, the segmentation clock periods of the six species did not scale with the animal body weight, but with the embryogenesis length. The biochemical kinetics of the core clock gene HES7 displayed clear scaling with the species-specific segmentation clock period. However, the cellular metabolic rates did not show an evident correlation. Instead, genes involving biochemical reactions showed an expression pattern that scales with the segmentation clock period. Altogether, our stem cell zoo uncovered general scaling laws governing species-specific developmental tempo.

Keywords: allochrony; developmental tempo; segmentation clock; stem cell zoo.

Graphical abstract

Figure 1

Recapitulation of the segmentation clock using stem cells from diverse mammalian species (A) Schematic illustration of the differentiation of mammalian PSCs toward iPSM. Cells differentiated under similar culture conditions show species-specific segmentation clock periods. Average adult body weight of each species is displayed. (B) Bright-field images of PSCs and iPSM cells from each species. Scale bars are 100 μm. (C) Representative histogram of flow cytometry analysis of a PSM marker TBX6 in PSCs (gray) and iPSM cells (colored) of each species. The average percentage of cells expressing TBX6 compared with the PSC control and the time of collection are shown. (D) Normalized HES7 reporter activity in iPSM cells of each species. Shading indicates mean ± SD (n = 3). The signal has been detrended and amplitude-normalized (see STAR Methods). (E) Oscillatory periods estimated from Figures S2C to S2E. Error bars indicate mean ± SD (n = 3). Human and mouse data (striped bars) are from Matsuda et al.

Figure 2

Correlations between the segmentation clock period and animal characteristics (A) Scatterplot showing the relationship between the log₁₀ average adult body weight and the segmentation clock period. (B) Scatterplot showing the relationship between the gestation length and the segmentation clock period. (C) Scatterplot showing the relationship between the embryogenesis length and the segmentation clock period. Rhinoceros is missing as it lacks embryological data. (A–C) Color scheme representing species is shown on top of the figure. Dashed lines represent linear fitting. R-squared values are shown. The values of body weight, gestation length, and embryogenesis length for all species can be found in Table S3. (D) Phylogenetic tree of the six species used in this study. The tree represents a subset of the complete mammalian tree (see STAR Methods). Names of the common clades are shown.

Figure 3

Measuring biochemical parameters of HES7 (A) Schematic representation of the negative feedback loop of HES7. Protein degradation and intron delay were measured in the indicated panels. Reporters used for these two assays are shown. NLuc, NanoLuc; FLuc, firefly luciferase. (B) HES7 protein degradation assay. The transcription of a HES7 protein fused with NLuc was halted upon the addition of doxycycline at time zero. The signal decay of NLuc was monitored. Inset represents the slope of the fitted lines used to quantify the protein half-life. (C) HES7 intron delay assay. Reporter constructs without (w/o) and with (w/) HES7 introns were measured simultaneously (top). The cross-correlation of the two reporters was calculated (bottom). (B and C) Shading indicates mean ± SD (n = 3). (D) Scatterplot showing the relationship between the segmentation clock period and the HES7 protein half-life. (E) Scatterplot showing the relationship between the segmentation clock period and the HES7 intron delay. (D and E) Color scheme representing species is the same as in Figure 2. Dashed lines represent linear fitting. R-squared values are shown. Human and mouse data (light purple and light blue) are from Matsuda et al.

Figure 4

Measuring cellular metabolic rates (A) Histogram showing the size distribution of iPSM cells. Total cell number was normalized. Shading indicates mean ± SD (n = 3). (B) Scatterplot showing the relationship between the median cell volume and the segmentation clock period across species. (C) Oxygen consumption rate measured throughout the Seahorse real-time ATP rate assay in iPSM cells. Oligomycin (Oligo) and rotenone + antimycin A (Rot + AA) were added at the marked time points. (D) Volume-specific oxygen consumption rate. (E) Volume-specific ATP production rate. (F) Volume-specific glycolytic rate of ATP production. This measurement is equivalent to the glycolic proton efflux rate as per the stoichiometry of the glycolysis reaction. (G) Volume-specific mitochondrial rate of ATP production. (C–G) Error bars indicate mean ± SD (n = 3). (H) Scatterplot showing the relationship between the segmentation clock period and the volume-specific oxygen consumption rate. (I) Scatterplot showing the relationship between the segmentation clock period and the volume-specific glycolytic rate of ATP production. (B, H, and I) Color scheme representing species is the same as in Figure 2. Dashed lines represent linear fitting. R-squared values are shown.

Figure 5

The transcriptomic signature of species-specific developmental tempo (A) Principal-component analysis (PCA) from bulk RNA-seq. Two biological replicates of PSCs (circles) and iPSM (triangles) of six species were used. Components 1 and 5 are shown. The variance explained by each component is indicated. (B) Scatterplots showing the relationship between the normalized gene expression levels in iPSM cells and the segmentation clock period across species. Color scheme representing species is the same as in Figure 2. Spearman correlation coefficients (ρ) and p values are shown in the plots. The highlighted genes are representative examples of genes with high negative/positive ρ values. (C) Enrichment map network of genes that showed correlated expression with the segmentation clock period. Each dot represents an enriched GO biological process term. Two terms are connected if they have a high overlap of genes. Related functional terms tend to cluster together. Circle size represents the number of genes in that process. Blue and red colors represent processes correlating negatively and positively with the segmentation clock period, respectively. (D) Proposed scheme from this study.