High resolution, dynamic imaging of early mouse and human liver bud morphogenesis in three dimensions

Liver organogenesis has thus far served as a paradigm for solid organ formation, and has recently attracted interest due to challenges faced in liver regenerative medicine. Murine genetic studies indicate that early steps in morphogenesis are required, suggesting that three-dimensional imaging of early liver morphogenesis at high resolution can improve our understanding. Unfortunately, existing approaches to image early liver morphogenesis have been unable to achieve high spatial resolution (1-5 um) required. In this study, we focused on imaging, visualization, and analysis of early liver development. We utilized available online databases for both mouse (EMAP) and human (3D Atlas of Human Embryology) liver development. To visualize liver bud morphogenesis at high spatial resolution, we performed 3D reconstructions of stacked, digital tissue sections. We show dynamic 3D hepatic cord formation in the mouse in humans. Interestingly, when we quantified fetal liver growth, we showed that 3D fetal liver growth appears to occur in spurts rather continuously, and 3D images suggest that there could be considerable remodeling during these stages. Further, our analysis of the STM, in both mouse and humans, demonstrates that it increases in size during early fetal liver growth, that is highly interconnecting with liver epithelium, and that it can have strong local effects on growth. Finally, we identify and visualize and identify human hepatic cord formation followed by rapid sheet-like growth, which we propose could be an under-appreciated morphological feature that enables rapid growth of early human fetal liver. These studies will motivate future approaches to employ culture and organoid technology to improve human PSC differentiation, and improve disease modeling, and therapeutic opportunities for liver diseases. In conclusion, compared to 2D sectioning, high spatial resolution imaging of the mouse and human 3D liver bud morphogenesis enables greatly improved visualization of the hepatic cords, 3D sheet-like liver cell growth, STM-epithelial cell interactions, and quantitative comparisons between mouse and human liver bud morphogenesis.