Primitive Hepatoblasts Driving Early Liver Development

Abstract

The embryonic development of the liver is initiated by the emergence of hepatoblasts, originating from the ventral foregut endoderm adjacent to the heart. Here, we identify and characterize a previously unrecognized population of early hepatoblasts at the ventroposterior part of the emerging liver bud, traced from Cdx2-positive endoderm progenitors, which we term primitive hepatoblasts. Mouse and human single-cell transcriptomics reveals the expression of both canonical hepatoblast markers TBX3, FGB, and KRT8/18 and primitive-specific mesenchymal markers ID3, VIM, and GATA4. Lineage tracing revealed the notable contribution up to 12.6% of LIV2+ hepatoblasts at E11.5 but diminishes in late fetal and postnatal development. Epigenetic and functional perturbation studies further uncover that primitive hepatoblast emergence is primed by WNT-suppression on RA-permissive CDX2+FOXA2+ progenitors. Furthermore, human pluripotent stem cell-derived primitive hepatoblast-like cells secrete pleiotrophin and midkine to amplify hepatoblast populations and develop epithelial-mesenchymal hybrid tissues in vivo. Our results provide a new framework for understanding lineage heterogeneity during early hepatogenesis and offer revised insights into strategies to model normal and abnormal liver development.

Figure 1.. Primitive hepatoblasts in the ventroposterior edge of the developing liver co-expressing mesenchyme markers.

a. Wholemount immunostaining of E8.5 embryo showed CDX2 expression in midgut and lateral endoderm progenitor (LEP) of the anterior intestinal portal lip. b. Schematic of the dual-lineage tracing strategy using Sox2^CreER and Cdx2^DreER mice crossed with dual-reporter alleles Rosa26^LSL-ZsGreen and H11^RSR-tdTomato. c. Flow cytometry analysis of Sox2^CreER and Cdx2^DreER dual lineage-traced embryo at E11.5, gated on LIV2+ hepatoblasts. d. Immunostaining of E11.5 embryos revealed tdTomato+ cells within LIV2+ hepatoblasts, indicating early contribution from the Cdx2 lineage. e. Low-magnification (left) and high-magnification (right) immunofluorescence images at E11.5 show tdTomato-positive cells co-expressing hepatoblast markers TBX3, DLK1, KRT8/18, and ID3, along with the mesenchymal marker VIM. f. scRNA-seq analysis of mouse E10.5 liver (left) and human embryonic liver at weeks 5–6 (right) identified four distinct hepatoblast clusters per species. Two clusters (P1 and P2) exhibited primitive hepatoblast signatures with high Id3/ID3 and Vim/VIM expression; P1 showed enriched Ptn/PTN expression compared to P2. Definitive endoderm clusters were separated by differential Mki67/MKI67 expression into proliferative (D⁺) and non-proliferative (D⁻) subtypes. g. Venn diagram showing overlap of differentially expressed genes (DEGs) between previously reported hepatomesenchyme (Cell, 2020) and ID3⁺ hepatoblasts (Cell Research, 2020).

Figure 2.. Temporal fate of Cdx2-derived primitive hepatoblasts during liver development.

a. Dre-rox–mediated lineage tracing with 4OHT induction from E7.5 to E10.5, followed by analysis at E11.5, revealed peak recombination of Cdx2-derived hepatoblasts upon induction at E8.0. b. Time-course analysis of tdTomato⁺ hepatoblasts by histology from E14.5 to 8 weeks postnatally after E8.0 induction. c. Flow cytometry gating strategy at E18.5 revealed that ~2.5% of E-cadherin⁺ EpCAM⁻ hepatocytes were tdTomato⁺ and lacked DLK1 expression, indicating maturation. d. Quantification of tdTomato⁺ hepatoblasts/hepatocytes: cells were gated as E-cadherin⁺ DLK1⁺ until E15.5, E-cadherin⁺ EpCAM⁻ at E18.5, and ASGR⁺ hepatocytes at 8 weeks. The Cdx2-derived hepatoblast population decreased to ~3.0% by E14.5, ~2.5% by E18.5, and ~1% of adult hepatocytes retained tdTomato labeling at 8 weeks.

Figure 3.. Posterior gut identity promotes primitive hepatoblast specification in boundary organoids model.

a. Schematic of the differentiation strategy to generate anterior gut (AG) and posterior gut (PG) lineages from hPSCs, followed by fusion to model the foregut–midgut boundary. b. Day 11 boundary organoids derived from PROX1::mScarlet hPSCs exhibited both PROX1+PDX− hepatic progenitors and PROX1+PDX1+ pancreatic progenitors. Immunofluorescence markers: PROX1::mScarlet (endogenous reporter), SOX2 (magenta), CDX2 (cyan), DAPI (white), PDX1 (yellow), FOXA2 (blue). c. Bulk RNA sequencing revealed increased expression of primitive and definitive hepatoblast markers in PG compared to AG. d. Gene set enrichment analysis showed significant enrichment of hepaticobiliary system development pathways in PG versus AG. e. Lineage tracing using nanoparticle-mediated DyLight647 labeling in ESH1-PROX1::mScarlet-derived boundary organoids revealed the emergence of PROX1⁺ cells from the PG region. f. scRNA-seq of day 12 boundary organoids revealed primitive-like clusters characterized by ID3 and VIM expression, alongside a smaller definitive-like cluster.

Figure 4.. Epigenetic priming of primitive hepatoblast identity in posterior gut.

a. Normalized enrichment scores of H3K4me3 (active mark) and H3K27me3 (suppressive mark) at D7AG- and D7PG-upregulated gene loci, confirming region-specific epigenetic priming. b. Primitive hepatoblast-associated genes such as TBX3 and GATA4 were marked by H3K27me3 (suppressive) enrichment in AG, while PG exhibited H3K4me3 enrichment (active) at genes including ID3, VIM, and TBX3. c. Gene ontology (GO) analysis revealed suppression of Wnt signaling in PG, supported by increased H3K4me3 (active) enrichment at Wnt pathway activators in AG and H3K27me3 (suppressive) enrichment at Wnt pathway activators in PG. d. Members of the FZD family of Wnt receptors (FZD1, FZD2, FZD5, and FZD7) showed active chromatin (H3K4me3) in AG, but were repressed (H3K27me3) in PG, consistent with epigenetic suppression of Wnt signaling in the posterior gut. e. Pharmacological activation of Wnt signaling using CHIR99021 impaired HHEX induction specifically when activating Wnt in PG, suggesting that Wnt inhibition is required for HBP specification.

Figure 5.. Primitive lineage-derived Pleiotrophin and Midkine signaling mediates hepatoblasts expansion.

a. Quantification of progenitor populations pre-spheroid formation, including FOXA2+SOX2+ foregut progenitors, FOXA2+SOX2−CDX2− definitive hepatoblast progenitors, and FOXA2+CDX2+ primitive hepatoblast progenitors, at D5 and D7 in AG and PG cultures. Fusing day 5 (D5) AG and PG spheroids enhanced hepatic induction, with 34.1% of FOXA2+ cells expressing PROX1::mScarlet by D14, compared to 19.8% from D7 AG–PG fusions. b. CellChat analysis of scRNA-seq from mouse embryonic liver, human fetal liver and boundary organoids identified Ptn/PTN and Mdk/MDK signaling pathways enriched in primitive and definitive cross talk. c. PTN was predominantly expressed in the P1 primitive cluster, while both P1 and P2 clusters expressed MDK. Receptors such as Ncl/NCL were enriched in the definitive hepatoblast clusters suggesting paracrine signaling between hepatoblast subtypes. d. Inhibition of PTN/MDK signaling using the NCL-targeting aptamer AS1411 significantly reduced the area of PROX1+ area, demonstrating that this signaling axis is essential for progenitor expansion. e. Schematic illustrating the proposed lineage relationships between primitive hepatoblast progenitors (CDX2+) and definitive hepatoblast progenitors (SOX2−CDX2−) during early hepatic specification. f. Diagram depicting temporal changes in the abundance of primitive and definitive hepatoblast populations, alongside CD45+ hematopoietic cells and CD31+CD45− endothelial cells, highlighting dynamic shifts in cell composition during organoid development.