Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages

Abstract

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

Keywords: endoderm; hepatoblasts; liver; mesothelium; organogenesis; scRNA-seq; single-cell; sinusoidal endothelium; stellate cells.

Figure 1.

Tracking emergent parenchymal and non-parenchymal liver lineages using scRNA-seq. (A) Schematic of experimental approach. (B) Overview of murine liver development. (C) UMAP visualization of all cells sequenced. Each dot represents a single-cell that is color-coded by cell type. (D) Cell type composition by developmental stage. (E) Heatmap depicting select differentially-expressing genes for all cell types identified. STM, septum transversum mesenchyme; FACS, fluorescently-activated cell sorting. See also Figure S1 and Table S2.

Figure 2.

Sinusoidal and vascular endothelia diverge by E8.75 from a common endothelial progenitor. (A) Force-directed layouts displaying timepoint and annotated cluster contribution of endothelial and E7.5 hematopoietic progenitors (n = 2569). (B) Force-directed layouts displaying pseudotime and differentiation potential in endothelial lineages from an Etv2^hi ‘start cell’ (solid arrowhead) to terminal states (hollow arrowheads). Arrow indicates second local differentiation potential maximum, representing HSEC specification from the vascular endothelium. (C) Dot plot displaying known markers for hematopoietic, hemangioblast, and endothelial cell identities, including expression of immature and mature HSEC markers. Size of the dot represents proportion of the population that expresses each gene. Color indicates level of expression. (D) Heatmap representing gene trends over pseudotime from hemangioblast ‘start cell’ to HSEC and endothelial terminal states. (E) Differentiation potential as a function of pseudotime for hematopoietic, endothelial, and HSEC trajectories, where each dot represents a single-cell that is color-coded by embryonic timepoint as in (A). Arrowhead denotes endothelial cells at E8.75, E9.5, and E10.5 that are specified to the HSEC terminal state. HSEC, hepatic sinusoidal endothelial cell. See also Figure S2 and Table S2.

Figure 3.

Septum transversum and liver mesenchymal heterogeneity in the early liver. (A) Force-directed layouts displaying timepoint and annotated cluster contribution of mesenchymal progenitors (n = 3691). (B) Dot plot displaying known and previously uncharacterized markers for STM, mesothelial, and HSC identities. Size of the dot represents proportion of the population that expresses each gene. Color indicates level of expression. (C) ALCAM, SOX9, and ISL1 immunostaining of (i) ventral and (ii) anterior STM in E9.5 Afp-GFP livers. (D) ALCAM, SOX9, and ISL1 immunostaining of (i) mesenchyme at distal tip of caudal lobe, (ii) mesothelium separating rostral and caudal lobes, and (iii) mesothelium of rostral lobe in E10.5 Afp-GFP livers. Scale bars, 100µm. STM, septum transversum mesenchyme; HSC, hepatic stellate cells. See also Figure S3 and Table S2.

Figure 4.

A hepatomesenchymal hybrid progenitor exists within the early liver (A) Force-directed layouts displaying timepoint and annotated cluster contribution of hepatic parenchymal progenitors (n = 2332). (B) Dot plot displaying known and previously uncharacterized markers for endodermal, migrating hepatoblast, hepatoblast, and hepatomesenchymal cell types. Size of the dot represents proportion of the population that expresses each gene. Color indicates level of expression. (C) Heatmap and force-directed layouts showing average hepatic, mesenchymal, and epithelial gene profiles of all early liver parenchymal cells captured. Heatmap columns are labeled based on timepoint and cluster color schemes in (A). See also Figure S4 and Tables S2 and S3.

Figure 5.

Hepatoblasts and hepatomesenchymal cells emerge via distinct migratory mechanisms (A) Force-directed layouts displaying pseudotime and differentiation potential in hepatic parenchymal lineages from an Alb^lo ‘start cell’ (solid arrowhead) to terminal states (hollow arrowheads). Arrows indicate two potential trajectories to attain a hepatomesenchymal terminal state. (B) Heatmap representing gene trends over pseudotime from endodermal ‘start cell’ to hepatoblast and hepatomesenchymal terminal states. (C) Force-directed layouts displaying expression levels of cell type markers identified. See also Figure S4.

Figure 6.

The signaling niche of the primitive sinusoid. (A) Dotplot displaying putative ligand-receptor interactions between hepatoblast, HSEC, endothelial, hepatomesenchymal, and HSC lineages captured at E10.5. Size of the dot represents statistical significance of the indicated interactions. Color indicates the means of the average expression level of the ligand from cluster 1 and the receptor from cluster 2. (B) Circle plots depicting links between (1) ligands and (2) their predicted target genes for HSECs and hepatomesenchymal cells. Link colors indicate ligand sender populations, whereas width and opacity of links correlate to ligand-receptor interaction weights and ligand activity scores, respectively. HSCs, hepatic stellate cells; HSECs, hepatic sinusoidal endothelial cells. See also Figure S5 and Tables S4 and S5.