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

Background: Liver organogenesis has thus far served as a paradigm for solid organ formation. The developing liver bud is a well established model of organogenesis, and murine genetic studies demonstrate key molecules involved in key morphogenetic changes. However, the analysis of the liver bud is typically limited to 2D tissue sections, which precludes extensive visualization, quantitation, and analysis. Further, the lack of human liver bud data has further hindered our understanding of human liver organogenesis. Therefore, new analytical and visualization approaches are needed to elicit further morphogenetic details of liver organogenesis and to elucidate differences between mouse and human liver bud growth. Results: To address this need, we focused on high resolution imaging, visualization, and analysis of early liver growth by using available online databases for both mouse (EMAP, Toronto Phenogenomics center) and human (3D Atlas of Human Embryology), noninvasive multimodality imaging studies of the murine embryo, and mouse/human liver weight data. First, we performed three-dimensional (3D reconstructions) of stacked, digital tissue sections that had been initially segmented for the liver epithelium and the septum transversum mesenchyme (STM). 3D reconstruction of both mouse and human data sets enabled visualization and analysis of the dynamics of liver bud morphogenesis, including hepatic cord formation and remodeling, mechanisms of growth, and liver-epithelial STM interactions. These studies demonstrated potentially under-appreciated mechanisms of growth, including rapid exponential growth that is matched at the earliest stages by STM growth, and unique differences between mouse and human liver bud growth. To gain further insight into the exponential liver bud growth that was observed, we plotted volumetric data from 3D reconstruction together with fetal liver growth data from multimodality (optical projection tomography, magnetic resonance imaging, micro-CT) and liver weight data to compose complete growth curves during mouse (E8.5-E18) and human (day 25-300) liver development. For further analysis, we performed curve fitting and parameter estimation, using Gompertzian models, which enables the comparison between mouse and human liver bud growth, as well as comparisons to processes like liver regeneration. To demonstrate the importance of mesenchyme in rapid liver bud growth and morphogenesis in the human liver bud, we performed functional analysis in which human pluripotent stem cell (hPSC)-derived hepatic organoids were used to model collective migration that occurs in vivo, demonstrating that migration is strongly dependent upon mesenchyme. Discussion: Our data demonstrates improved visualization with 3D images, under-appreciated and potentially new mechanisms of growth, complete liver growth curves with quantitative analysis through embryonic and fetal stages, and a new functional human stem cell-derived liver organoid assay demonstrating mesenchyme- driven collective migration. These data enhance our understanding of liver organogenesis.