This study is led by Prof. Lusheng Huang (National Key Laboratory for Swine Genetic Improvement and Germplasm Innovation, Jiangxi Agricultural University).
Liver executes essential metabolic and immunological functions throughout the postnatal periods, while cellular basis underlying postnatal liver development remains limited. Single cell technology facilitates the study of development at a higher resolution. As the mini pigs Bamaxiang are widely applied as human diseases model animal and are also considered as donors for human organ transplantations, they aimed to dissect the cellular heterogeneity, define cell type-specific transcription factors, and reveal intercellular interactions between hepatocytes and non-parenchymal cells in four important developing Bamaxiang mini pig time points: Day 30 (the suckling period), Day 42 (the postweaning period), Day 150 (the rapid growth period), and Day 730 (the adult period).
Lin Rao et al. first generated an up-to-date most comprehensive single cell atlas for postnatal liver development in all mammals, in terms of single cell number and its detecting approaches. They applied both scRNA-seq and snRNA-seq technologies and generated 84,824 single cells at four developmental stages in the healthy pig livers, with male and female being investigated at each stage.
Subsequently, in the total of 84,824 liver single cells and nuclei from four age states, they uncovered 23 cell types, including three rare cell types in liver: plasmacytoid dendritic cells (pDCs), CAVIN3+IGF2+ endothelial cells, and EBF1+ fibroblasts. The pDCs cluster displayed significant male and female cell proportion and function heterogeneity in all studied ages, with the tolerance induction genes expression significantly enriched in males in days 30 and 42, but vice versa in day 730.
By further exploring the highly heterogeneous hepatocytes over four ages and constructing the developmental trajectory, the team found that 5693 genes exhibited significant expression change in the hepatocytes across the three ages. They revealed 33 developmental stage-specific transcription factors (TFs), among which D30 enriched TF EZH2 was involved in regulating postnatal hepatocyte maturation, D42 enriched CLOCK in circadian rhythms regulation, D730 enriched NFIL3 in regulating bile and xenobiotic metabolism.
Furthermore, they investigated the spatiotemporal heterogeneity of liver sinusoidal endothelial cells (LSECs), revealed their age-specific function changes, especially the scavenger functions. They discovered and validated LUZP2 as an adult LSEC-specific TF by multiplex immunohistochemistry assays and single cell multi-omics (snRNA-seq and snATAC-seq).
They discovered that it is the lymphoid cells (NK and T cells) that govern the immune systems in the liver since day 30, which can be subclustered into nine various immune subtypes, among which tissue resident NK cells (trNKs) display virus defense function and retain the proliferating feature at the age of D730. The cross-species comparison analyses showed the trNKs in pig liver presented higher conservative TF expression pattern with humans compared to mouse liver.
In conclusion, this study presented the dynamic changes in liver metabolism and immune function during the postnatal period. A further dissection of the adult LSEC-specific TF LUZP2 is required to better understand the concordant postnatal liver development.