Live imaging of hepatic and intestinal intracellular ApoA‐I in zebrafish
Authors: Otis Jessica P , Caldwell Blake A , Howles Philip N , Farber Steven A 
Published: November 2023

Elevated high‐density lipoprotein (HDL) cholesterol is incontrovertibly correlated with protection from cardiovascular disease in humans. The main structural component of HDL isapolipoprotein A‐I (APOA‐I). Cellular APOA‐I dynamics (e.g., transport, degradation, recycling) directly influence circulating APOA‐I and HDL levels. APOA‐Icell biology has remained largely unexplored, despite the strong link between cellular APOA‐I, circulating HDL, and cardiovascular disease risk. Furthermore, both the liver and the intestine synthesize HDL and APOA‐I, but the biological significance, and whether APOA‐I cell biology differs by tissue, is not currently understood. The optically clear larval zebrafish presents an excellent opportunity to visualize global and cellular APOA‐I dynamics in a live animal. Zebrafish have two ApoA‐I proteins; our insitu hybridization analysis shows ApoA‐Ia is expressed strongly in the intestine and weakly in the liver, and that ApoA‐Ib is expressed in the liver. We created transgenic zebrafish expressing fluorescently labeled human orzebrafish APOA‐I (APOA‐I‐mCherry) driven by liver‐ or intestine‐specific promoters to visualize APOA‐I of hepatic vs. intestinal origin in vivo. Agarose gel electrophoresis of adult zebrafish plasma indicates that the human and zebrafish APOA‐I‐mCherryfusion proteins are incorporated into HDL particles. Confocal microscopy of live larvae reveals that both secreted APOA‐I‐mCherry fusions localize to specific tissues and subcellular domains. We hypothesized that human APOA‐I‐mCherrylocalizes to the endosomal transport system of enterocytes and hepatocytes, and that due to the unique biology of hepatocytes and enterocytes, this trafficking may vary by cell type. To investigate this hypothesis, the human APOA‐I transgenic larvae were studied in genetic backgrounds that express Rab‐GFP fusion proteins, which mark specific endosomal compartments, on the ubiquitous, inducible HSP70 promoter. Using live imaging, we found that human APOA‐I‐mCherry co‐localizes with markers of early(GFP‐Rab5c), recycling (GFP‐Rab11a), and late endosomes/lysosomes (GFP‐Rab7, Lysotracker). This data also suggests that whether APOA‐I‐mCherry co‐localizes with these endosomal markers is influenced by both the tissue APOA‐I‐mCherry isderived from, and the tissue it is taken up by; for example, APOA‐I‐mCherry secreted from the intestine co‐localizes with recycling endosomes in the liver, but APOA‐I‐mCherry secreted from the liver does not co‐localize with recycling endosomes in the intestine. On‐going experiments will examine the route of intracellular APOA‐I transport and how it differs by tissue. In conclusion, we harnessed the larval zebrafish to visualize apolipoprotein dynamics at both the multi‐organ and subcellular levels. To our knowledge, this is the first time tissue‐specific apolipoprotein transport has been visualized in vivo.Support or Funding InformationThis research was supported by the NIH (DK093399 and NIDDK F32DK096786 (JO)), Carnegie Institution of Washington Endowment, and G. Harold and Leila Y. Mathers Charitable Foundation.