The liver consists of more than 20 distinct cell types, such as hepatocytes, biliary ductal cells (cholangiocytes), and liver endothelial cells, among others (Robinson et al., 2016; MacParland et al., 2018; Aizarani et al., 2019; Yang et al., 2019). Liver dysfunction, caused by conditions such as fatty liver and liver fibrosis, can trigger a range of pathological symptoms within the human body. It has been reported that the liver plays critical and distinct roles in the fetus and adult. During mid to late gestation, the fetal liver serves as the primary site for hematopoiesis and lacks most metabolic functions, in contrast to the adult liver (Timens and Kamps, 1997). However, this claim warrants further investigation.

During embryonic development, the liver originates from the lateral domain of the endoderm within the ventral foregut (Chalmers and Slack, 2000; Tremblay and Zaret, 2005). Following the specification of foregut endoderm, hepatoblasts bud into the septum transversum, where they continue to proliferate and differentiate. During embryogenesis, hepatocytes and cholangiocytes, the two primary lineages responsible for most liver functions, arise from bipotential hepatoblasts. These progenitor cells are generally believed to originate from the definitive endoderm approximately 3–4 weeks post-coitum in humans and at embryonic day 8.5–9.0 in mice (Gordillo et al., 2015). Maturation into hepatocytes and bile duct epithelial cells continues for several weeks postnatally.

Numerous genes and signaling pathways involved in liver development have been identified. During hepatic specification, WNT signaling facilitates hepatic induction, promoting the emergence and differentiation of liver bud (Finley et al., 2003; Monga et al., 2003; Ober et al., 2006; McLin et al., 2007). At this stage, the newly specified hepatic cells, known as hepatoblasts, adopt a columnar morphology and invade the septum transversum mesenchyme, initiating liver bud formation (Bort et al., 2006). Several transcription factors, including FOXA1/2, GATA4/6, HHEX, and HNF1A/1B, play integral roles in hepatoblast specification (Gordillo et al., 2015; Villasenor and Stainier, 2017). As hepatoblasts begin budding into the surrounding mesenchyme, they continue to proliferate, driven by various cytokines and growth factors. These signaling molecules, such as Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor (TGF)-β, Tumor Necrosis Factor (TNF)-α, and Interleukin-6 (IL-6), are secreted by mesenchymal cells within the septum transversum (Schmidt et al., 1995; Weinstein et al., 2001; Zhao and Duncan, 2005; Tanimizu and Miyajima, 2007). In the liver, hepatoblasts undergoing budding express genes that are characteristic of hepatocytes, including albumin (ALB), transthyretin (TTR), and alpha-fetoprotein (AFP) (Lemaigre and Zaret, 2004; Zaret, 2008). Following invasion of the mesenchyme, these bipotential cells differentiate into hepatocytes (α-fetoprotein⁺/albumin⁺) and cholangiocytes (cytokeratin (CK)-19⁺) (Germain et al., 1988; Jung et al., 1999). The precise equilibrium between hepatocyte and cholangiocyte populations derived from hepatoblasts is maintained through coordinated signaling and transcriptional networks. The Jagged-Notch pathway is crucial in guiding hepatoblast differentiation toward a biliary epithelial phenotype (McCright et al., 2002; Tanimizu and Miyajima, 2004), whereas Hepatocyte Growth Factor (HGF) inhibits biliary differentiation. In conjunction with oncostatin M (OSM), HGF promotes hepatocyte differentiation (Suzuki et al., 2003). After lineage segregation, the proportion of bipotential cells decreases significantly, with most differentiating into either hepatocyte or cholangiocyte lineages. Committed cells undergo progressive morphological and physiological changes, with maturation continuing postnatally for several weeks. This process has been elucidated through various gene array analyses of rodent liver development (Kelley-Loughnane et al., 2002; Jochheim et al., 2003; Petkov et al., 2004).

In this study, human embryonic livers ranging from 2-month-old to 7-month-old were collected for transcriptome sequencing. The study identified differentially expressed genes (DEGs) and biological processes associated with human embryonic liver development at various stages. Our findings establish a foundational basis for investigating liver development in human embryos and identifying diseases caused by abnormalities in embryonic liver function.

The normal development of the liver during embryogenesis is crucial to the proper function of the adult liver. To explore the predominantly expressed genes and associated biological processes at various developmental stages of embryonic liver, we collected human embryonic liver tissue ranging from 2-month-old to 7-month-old. We then employed RNA sequencing analysis to identify differentially expressed genes (DEGs). Our research provides researchers with transcriptomic profiles of normal embryonic liver development, validates the expression of essential genes in this process, and offers a reference for studies on embryonic abortion resulting from abnormal liver development.

Compared to the liver of 2-month-old human embryo, the upregulated genes associated with humoral immune response and B cell mediated immunity were identified in the liver of 3-month-old human embryo (Figure 1C). The genes, such as CFH, PGLYRP1, LTF, CFHR2, BLNK, CTSG, FCER2, HPX, HRG, CFHR3, CFHR4, CXCL9, SERPING1, C1R, S100A12, PF4, CXCL10, RNASE3, LGALS4, AZU1, C1QA, H2BC4, PRTN3, CFI, DEFA1, C4B, DEFA3 were highly expressed in the liver of 3-month-old human embryo, indicating a distinct immune profile at this development stage (Figure 1E). Previous research suggested that complement factor H (CFH) regulated alternative complement activation by inhibiting the cleavage of the central complement component C3 (Kiss et al., 2019). Lactoferrin (LTF), a critical molecule in human first-line defense against infections, is frequently targeted by humoral autoimmune responses (Hu et al., 2017). The B-cell linker protein (BLNK), also known as BASH or SLP-65, encodes an adaptor protein essential for B-cell receptor signaling (Kurata et al., 2021). Heparin interacts with the endogenous tetrameric protein platelet factor 4 (PF4), forming PF4/heparin complexes that can cause a severe immune-mediated adverse reaction called heparin-induced thrombocytopenia (HIT) (Nguyen et al., 2015). The upregulation of these genes in the liver of 3-month-old human embryo underscores the dominance of humoral responses mediated by B cells as primary biological processes during this developmental stage. This suggests an early establishment of immune functions crucial for fetal protection. The prominence of B-cell-mediated responses in the embryonic liver likely plays a crucial role in the maturation of the immune system.

Compared to the liver of 3-month-old human embryo, the upregulated genes related to the biological process of vitamin response and regulation of brown fat cell differentiation, such as PTGS2, BMP7, SPP1, FGF23, EPO, POSTN, ALPL, FNDC5, METRNL were identified in the liver of 4-month-old human embryo (Figure 2E). Previous studies have demonstrated that BMP7 induces the conversion of primary human adipose stem cells from white to brown (Elsen et al., 2014). Bone morphogenetic protein 7 (BMP7) plays a critical role in the differentiation and development of brown adipose tissue (BAT) (Syamsunarno et al., 2021). Additionally, BMP7 contains a vitamin D receptor (VDR) response element within its promoter region and contributes to cellular response to mechanical loading (Santos et al., 2011). Vitamin D exerts its influence on viral hepatitis through non-genomic factors, including matrix metalloproteinase, endothelial vascular growth factor, prostaglandins, PTGS2 (Cyclooxygenase-2), and oxidative stress (Luong and Nguyen, 2012). Cyclooxygenase-2 regulates energy homeostasis in mice by recruiting brown adipocytes (Vegiopoulos et al., 2010). Vitamin C protects retinal ganglion cells by upregulating SPP1 expression in glaucoma (Li and Jakobs, 2023). Fibroblast growth factor 23 (FGF23) is a critical regulator of both phosphate and vitamin D homeostasis. It participates in an FGF23-Vitamin D-PTH (parathyroid hormone) regulatory axis that governs mineral homeostasis (Blau and Collins, 2015). Periostin (POSTN), a member of a newly identified family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells (Coutu et al., 2008). Alkaline phosphatase (ALPL) is an essential enzyme that influences the bioavailability of vitamin B6, functioning as a rate-limiting factor in its metabolism (Spinelli et al., 2023). FNDC5, a membrane protein, undergoes cleavage and is secreted as irisin. Irisin acts on white adipose cells to stimulate the expression of thermogenin (UCP1), inducing a program of brown fat-like development (Bostrom et al., 2012). Meteorin-like (METRNL), a hormone secreted by various tissues, including thermogenically active brown and beige adipose tissues, is associated with brown adipose tissue activity in early infancy (Garcia-Beltran et al., 2023). The upregulation of these genes indicated that the 4-month-old embryonic liver is actively engaged in vitamin response and brown fat cell differentiation regulation. This indicates that the liver in the 4-month-old human embryo is actively involved in essential metabolic processes, including nutrient sensing and thermogenesis. The regulatory mechanisms at this stage may be critical for maintaining proper energy homeostasis and could have enduring effects on the health of the human embryos.

In comparison to the liver of 4-month-old human embryo, the upregulated genes associated with the biological process of T cell differentiation, such as TNFRSF9, SPI1, TBX21, PTPRC, LOXL3, MYB, FOXO3, IL1B, BCL11B, RIPK3, PTPN22, IRF4, VAV1, RORC, IKZF3, NLRP3, SLAMF6, EOMES, SYK, CD3D, RHOH, IL7R, PTGER4, PIK3CD, RASGRP1, LILRB4, CARD11, TOX, CR1 were identified in the liver of 5-month-old human embryo (Figure 3E). CD137, also known as TNFRSF9, plays a critical role in the activation of T cells and NK cells, as well as cytokine production (Vinay et al., 2004). The T cell-specific T-box transcription factor TBX21 is integral to immune system regulation, promoting the differentiation of T helper 1 (T(H)1) cells while inhibiting the commitment of T helper 2 (T(H)2) cells, in conjunction with the homeobox transcription factor HLX1 (Suttner et al., 2009). PTPRC enhances immune responses mediated by CD8⁺ T cells and increases drug sensitivity in breast cancer treatment (Li et al., 2023). FOXO3 regulates the memory of CD8⁺ T cells through intrinsic cellular mechanisms (Sullivan et al., 2012). BCL11B is essential for T-cell development and the preservation of T-cell identity (Liu et al., 2010). RIPK3 and Caspase-1/11 are required for the optimal antigen-specific CD8⁺ T cell response (Rana et al., 2020). PTPN22 associates with end-binding protein 1 (EB1) to modulate T-cell receptor signaling (Zhang et al., 2020). SLAMF6 is crucial for T cell activation, enhancing T cell functionality (Gartshteyn et al., 2023). CD3D is responsible for initiating T cell signal transduction and is associated with the antitumor immune response across various cancer types (Wei et al., 2022). Concurrent deficiencies in PIK3CD and TNFRSF9 result in chronic active Epstein-Barr virus infections in T cells (Rodriguez et al., 2019). LILRB4 signaling in leukemia cells promotes T cell suppression and tumor infiltration (Deng et al., 2018). CARD11 signaling is vital for regulating T cell development and function (Deng et al., 2018). Furthermore, TOX plays a critical role in the differentiation of tumor-specific T cells (Scott et al., 2019). The increased expression of these genes in the liver of 5-month-old human embryo indicates significant biological processes, primarily involving the immune system, are actively occurring. These processes encompass T cell differentiation, which is essential for adaptive immune responses, and myeloid leukocyte activation, crucial for innate defense mechanisms. Additionally, lymphocyte differentiation reflects the maturation of B cells and T cells, which are necessary for antigen-specific immunity. Collectively, these biological processes suggest an advanced developmental stage of the embryonic immune system.

In comparison to the liver of 5-month-old human embryo, the upregulated genes associated with coagulation regulation, hemostasis, hormone transport, hormone secretion, and blood coagulation, such as TFPI, VWF, FGG, FGA, FGB, GAS6, F8, GIPR, CPE, FGF23, VAMP7, AGTR1, ADM, BMP6, KCNB1, RPH3AL, EFNA5, IRS2, EDN1, PROCR, FGL1, SERPINE1, ST3GAL4, INHBB, F2RL1, C1QTNF1, C4BPB, PDGFRA, SERPINA10, SLC7A11, F2RL1 were identified in the liver of 6-month-old human embryo (Figure 4E). The suppression of Tissue Factor Pathway Inhibitor (TFPI) activity can re-establish effective hemostasis via the extrinsic blood coagulation pathway, independently of factors VIII or IX (Peterson et al., 2016). Fibrinogen, a hexameric glycoprotein encoded by three clustered genes including FGA, FGB, and FGG on chromosome 4q, plays a critical role in the final stages of coagulation as a precursor to fibrin monomers (Acharya and Dimichele, 2008). The growth arrest-specific 6 (GAS6) gene and its receptor, AXL, are pivotal in vascular hemostasis and the pathogenesis of atherosclerosis (Lee et al., 2014). The von Willebrand factor (VWF) is a critical glycoprotein that facilitates primary hemostasis (Kanaji et al., 2012). The gastrointestinal peptide hormone receptor GIPR is involved in linking energy availability to the regulation of hematopoiesis (Pujadas et al., 2020). Carboxypeptidase E (CPE) plays a vital role in processing prohormones into mature hormones and is abundantly expressed in various neuroendocrine tissues (Chen et al., 2023). Fibroblast Growth Factor 23 (FGF23) is a phosphaturic hormone that originates from bone and regulates phosphate and vitamin D metabolism (Takashi and Fukumoto, 2018). The effects of Angiotensin II (ANG II) on blood pressure regulation, water-electrolyte balance, and hormone secretion are primarily mediated through AGTR1 (Vervoort et al., 2002). Adrenomedullin (ADM), a peptide hormone with widespread expression across various tissues, is implicated in a diverse range of physiological processes, including the regulation of hormonal secretion, glucose metabolism, and the inflammatory response (Wong et al., 2014). In liver sinusoidal endothelial cells (LSECs), homeostatic adaptation to systemic iron overload involves the transcriptional induction of Bone Morphogenetic Protein 6 (BMP6) (Charlebois et al., 2023). Depletion of IRS2 impairs cell proliferation and reduces hormone secretion in mouse granulosa cells (Lei et al., 2018). EPHRIN-A5 is essential for maintaining optimal fertility and eliciting a full ovulatory response to gonadotropins in female mice (Buensuceso et al., 2016). The Rabphilin-3A-like (RPH3AL) protein plays a pivotal role in regulating hormone exocytosis (Chen et al., 2011). The elevated expression of these genes suggests that the regulation of coagulation, hemostasis, hormone transport, hormone secretion, and blood coagulation are principal biological processes occurring in the liver of 6-month-old human embryo. Notably, this increased gene expression indicates that the liver may effectively regulate blood coagulation and maintain hemostatic balance. It appears that the liver possesses the capacity to regulate blood coagulation and hemostasis through hormone secretion at this stage of development.

In comparison to the liver of 6-month-old human embryo, the upregulated genes associated with the biological process of steroid metabolic process, such as CYP26A1, APOL1, SULT2A1, PLEKHA1, UGT2B10, APOA4, BMP5, PDE8B, LEPR, NR5A2, PROX1, FGF23, EGR1, INSIG2, BMP2, APOL2, CYP2E1, UGT2A3 were identified in the liver of 7-month-old human embryo (Figure 5E). CYP26A1 plays a crucial role in hepatic function by regulating retinoic acid metabolism (Wang et al., 2021). SULT2A1 preferentially acts on hydroxysteroids, including dehydroepiandrosterone, testosterone/dihydrotestosterone, and pregnenolone, as well as on amphipathic sterol bile acids derived from cholesterol (Chatterjee et al., 2005). APOA4 is involved in lipid transport and metabolism by modulating hormonal regulation (Nazzari et al., 2024). PDE8B regulates basal corticosterone synthesis through both acute and chronic mechanisms (Tsai and Beavo, 2011). Polymorphisms in the LEPR gene are associated with type 2 diabetes and related metabolic traits in a Chinese population (Zhang et al., 2018). The increased expression of these genes indicate that the liver of 7-month-old human embryo likely possesses the capability to regulate metabolic processes associated with steroids. This observation suggests a potential functional maturity in the enzymatic systems of the embryonic liver involved in steroid metabolism. Further research into this phenomenon could provide essential insights into the emergence of liver functions during human development and the molecular mechanisms governing metabolic pathways of fetal maturation. Additionally, the biological processes of complement activation, blood coagulation, cellular carbohydrate metabolism, serine family amino acid metabolism, myeloid leukocyte activation, humoral immune response, T cell proliferation, T cell differentiation, response to peptide hormone, cellular response to peptide hormone stimulus, steroid metabolic process and response to steroid hormone were identified along the pseudotime from 3-month-old to 7-month-old (Figure 6A). These findings align with previously reported data, demonstrating that distinct biological processes occur in the liver at various developmental stages. Collectively, these results suggest that the embryonic liver possesses not only hematopoietic functions but also other biological functions that emerge and evolve throughout the embryonic period.

Finally, the genes associated with lymphocyte differentiation, such as TNFSF13B, CD79A, ITK, LEPR, EGR1, PTPN22, IL6, CD8A, FZD7, MS4A1, CD1D, EOMES, NFIL3, GPR183, ONECUT1, PTGER4 exhibited high expression levels in the liver of 7-month-old human embryo (Figure 7B). B cell-activating factor (BAFF), also known as CD257, TNFSF13B, and BLyS, is recognized as a critical regulator of B cell development and differentiation (Tangye et al., 2006). CD79a is expressed on both normal and neoplastic B cells and is considered B-cell-specific (Pilozzi et al., 1998). Interleukin-2 Tyrosine Kinase (ITK) plays a crucial role in the differentiation of T helper 9 (Th9) cells through Interleukin-2 (IL-2) and Interferon Regulatory Factor 4 (IRF4) (Gomez-Rodriguez et al., 2016). EGR1 and NFAT2 have been shown to cooperatively regulate the expression of the expression of the regulatory factor ID3 and the recombinase RAG2, both of which are essential for T-lymphocyte differentiation (Koltsova et al., 2007). PTPN22 encodes lymphoid-specific tyrosine phosphatase LYP, a non-receptor type protein tyrosine phosphatase. LYP is critical for lymphocyte activation and differentiation (Gianchecchi et al., 2013). NFIL3 is essential for the development of all innate lymphoid cell subsets (Seillet et al., 2014). Additionally, the genes associated with the regulation of hemopoiesis, including CYP26B1, TBX21, LAG3, SFRP1, IL4I1, PTN, VNN1, LOX, CD80, TNFAIP6, INHA, PCK1, IRF1, IL2RA, IL10, IRF4, CTLA4, IL15, TLR3, TNFRSF11B, MALT1, TRIB1, GPR171, TCIM, EGR3, CIB1, IRF7, ZFP36L1, ISG15, HLA-DRB1 were identified in the liver of 7-month-old human embryo (Figure 7B). SFRP1 plays a vital role in maintaining hematopoietic stem cell (HSC) homeostasis by externally regulating β-catenin signaling (Renstrom et al., 2009). Loss of IRF1 impairs HSC self-renewal, increases stress-induced cell cycle entry, and confers resistance to apoptosis (Rundberg Nilsson et al., 2023). Tribbles pseudokinases, consisting of TRIB1, TRIB2, and TRIB3, are crucial regulators of both normal and malignant hematopoiesis (Salome et al., 2018). GPR171, identified as a putative P2Y-like receptor, is involved in the negative regulation of myeloid differentiation in murine hematopoietic progenitor cells (Rossi et al., 2013). TCIM (C8orf4) exerts regulatory control over hematopoietic stem and progenitor cells, as well as hematopoiesis (Jung et al., 2014). ZFP36L1 enhances monocyte/macrophage differentiation by repressing CDK6 (Chen et al., 2015). IRF7 inhibits hematopoietic regeneration under stress by targeting CXCR4 (Chen et al., 2021). The genes associated with liver development, such as HGF, CPS1, OTC, PROX1, ASS1, GATA6 were significantly upregulated in the liver of 7-month-old human embryo (Figure 7B). PROX1 is essential for hepatocyte migration during liver development. A deficiency in PROX1 results in a reduced-size liver, characterized by a diminished population of clustered hepatocytes (Sosa-Pineda et al., 2000). Hepatocyte growth factor (HGF) is clinically significant in liver fibrosis, hepatocyte regeneration post-inflammation, and post-transplant liver regeneration (Zhao et al., 2022). GATA6 is involved in various stages of liver development, including endoderm liver-lineage determination, liver specification, hepatic bud outgrowth, and hepatoblast differentiation (Zhang and He, 2018). Building on these findings, Figure 7A illustrates the gene interaction network associated with regulation of hemopoiesis, lymphocyte differentiation and liver development. Analysis of this network reveals complex interactions among genes, underscoring the necessity of their coordinated expression for typical liver maturation. The delineation of this network has provided significant insights into the coordinated molecular activities essential for the appropriate development and functionality of the embryonic liver. Understanding these gene interactions is imperative for elucidating the complexities of embryonic liver development and its diverse physiological roles.

In this study, the gene expression profiles in the liver across various development stages of human embryos were determined. Genes with high expression levels were enriched in the biological processes such as humoral immune response, B cell mediated immunity, response to vitamin and brown fat cell differentiation in the livers of 3-month-old and 4-month-old human embryos. As development progressed, the genes involved in T cell differentiation, coagulation, hormone transport, T cell activation and steroid metabolic process were upregulated in the liver. These results indicate that immune response and hematopoiesis are significant processes in the early embryonic liver. As the embryonic liver matures, it progressively acquires metabolic functions. These results suggest that the liver in embryogenesis not only possesses hematopoietic function but also develops other biological functions that emerge and evolve throughout the embryonic period. Moreover, this study identified not only genes previously associated with liver development but also a plethora of uncharacterized genes that exhibit high expression levels relevant to liver development. These insights will establish a groundwork for investigating fetal liver development and pathologies linked to embryonic liver abnormalities.