No abstract

No abstract

Difficulty in imaging the vertebrate intestine in vivo has hindered our ability to model nutrient and protein trafficking from both the lumenal and basolateral aspects of enterocytes. Our goal was to use live confocal imaging to increase understanding of intestinal trafficking of dietary cholesterol and apolipoprotein A-I (APOA-I), the main structural component of high-density lipoproteins. We developed a novel assay to visualize live dietary cholesterol trafficking in the zebrafish intestine by feeding TopFluor-cholesterol (TF-cholesterol), a fluorescent cholesterol analog, in a lipid-rich, chicken egg yolk feed. Quantitative microscopy of transgenic zebrafish expressing fluorescently tagged protein markers of early, recycling, and late endosomes/lysosomes provided the first evidence, to our knowledge, of cholesterol transport in the intestinal endosomal-lysosomal trafficking system. To study APOA-I dynamics, transgenic zebrafish expressing an APOA-I fluorescent fusion protein (APOA-I-mCherry) from tissue-specific promoters were created. These zebrafish demonstrated that APOA-I-mCherry derived from the intestine accumulated in the liver and vice versa. Additionally, intracellular APOA-I-mCherry localized to endosomes and lysosomes in the intestine and liver. Moreover, live imaging demonstrated that APOA-I-mCherry colocalized with dietary TF-cholesterol in enterocytes, and this colocalization increased with feeding time. This study provides a new set of tools for the study of cellular lipid biology and elucidates a key role for endosomal-lysosomal trafficking of intestinal cholesterol and APOA-I. NEW & NOTEWORTHY A fluorescent cholesterol analog was fed to live, translucent larval zebrafish to visualize intracellular cholesterol and apolipoprotein A-I (APOA-I) trafficking. With this model intestinal endosomal-lysosomal cholesterol trafficking was observed for the first time. A new APOA-I fusion protein (APOA-I-mCherry) expressed from tissue-specific promoters was secreted into the circulation and revealed that liver-derived APOA-I-mCherry accumulates in the intestine and vice versa. Intestinal, intracellular APOA-I-mCherry was observed in endosomes and lysosomes and colocalized with dietary cholesterol.

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V).Tet1-/-,Tet1V/V, andTet1HxD/HxDsperm methylomes show that TET1Vand TET1HxDrescue mostTet1-/-hypermethylated regions, demonstrating the importance of TET1&#39s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.

The mammalian genome undergoes extensive epigenetic reprogramming twice during development, once during gestation when primordial germ cells (PGCs) are specified from somatic cells and a second time after fertilization in the preimplantation embryo. PGC differentiation into germ cells involves DNA demethylation and subsequent remethylation. DNA demethylation takes place in two waves in the mouse germline, an early phase where most of the genome is demethylated by replication coupled passive demethylation, and a second phase predominated by active DNA demethylation. Imprinted genes, CpG islands on the inactive X chromosome of females, and germline‐specific genes are among those loci that are demethylated late. The Ten‐Eleven Translocation (TET) family of 5 mC dioxygenases has emerged as active demethylating enzymes that are critical to achieving a DNA hypomethylated state, with TET1 being the most important for imprinted genes. Here, we discuss DNA methylation dynamics in the mammalian genome, with a particular emphasis on DNA demethylation in the germline and the requirement for TET1 in imprinted gene reprogramming.

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.

No abstract

No abstract