Embryonic Phase Transition/Separation on Hepatic Liquid Crystal Droplets Is Essential for Liver Development

Phase transition or phase separation occurs in cells and tissues in many physiological events, such as protein granules (P granules) in early C. elegans development. Inappropriate phase transition is often associated with pathologic processes such as RNA-protein complex (RNP) and FUS-mutation-associated diseases. Given its ubiquity, phase transition has been considered a new frontier for comprehending physiological processes and pathological diseases. However, molecular and cellular mechanisms of phase transition in situ remain poorly described. Combining histochemistry with polarization analysis and biochemical thin-layer chromatography, we identified a massive phase transition change during the development of Taihe fowl (Gallus gallus domesticus Brisson). During embryonic day 12 (E12), the livers of these Silkie chicken demonstrated a sudden massive transition from hepatic lipid droplets (HLD) into hepatic liquid crystal droplets (HLCDs). We identified these changes by characterizing the sudden appearance of birefringent Maltese crosses (MCs) typical to liquid crystals (LC) where non-birefringent lipid droplets used to reside within hepatic cells. LC status was confirmed by fluidity with shape-changing and in vitro thermal phase transition tests. These HLCDs were present consistently until the early postnatal days after hatching. Using thin-layer chromatography combined with X-ray diffraction analysis, we determined that these HLCDs were composed of cholesterol, cholesterol ester and lecithin, which are the same as the components of cytoplasmic membrane. There was no change in the quantity of lipid components during liver development to suggest a critical mass of components triggering these changes. However, expressions of membrane-associated autophagy markers LC3A and Beclin 1 increased dramatically during this HLD to HLCD transition. Increases in membrane-associated LC3A and Beclin 1 are localized with massive increases in membrane lipid components of HLCDs. Areas with enhanced LC3A and Beclin 1 signaling have been associated with liquid crystal MCs to the thickness of 69 Å (Bragg d value). These associations indicate the possible regulatory role autophagy plays during liquid crystal phase transition in embryonic liver development. Reactivation of this autophagy pathway may be a possible mechanism behind the development of non-alcoholic fatty liver disease in adulthood.