Fig. 1

Visualization of TF-cholesterol absorption in live, larval zebrafish indicates fatty acids are necessary for dietary cholesterol absorption. TF-cholesterol provided in a lipid-rich meal (5% chicken egg yolk) is absorbed by enterocytes and accumulates in distinct subcellular punctae (A). Representative images show that dietary TF-cholesterol absorption is not observed when it is fed with a lipid-poor meal (5% chicken egg white) (B). Approximately sixfold more TF-cholesterol fluorescence is observed in enterocytes when it is provided with egg yolk relative to egg white (C) (means ± SE, student’s t-test, *P < 0.05). n = 3, with 6–9 fish per n. Arrows indicate colocalization; L, intestinal lumen; N, nucleus; TF-cholesterol, TopFluor-cholesterol.

 
Fig. 2

Dietary TF-cholesterol localizes to the endosomal-lysosomal trafficking network in enterocytes. Representative images show that 3 h following the onset of feeding, dietary TF-cholesterol colocalizes to mCherry-Rab5c (early endosomes) (A), mCherry-Rab11a (recycling endosomes) (B), and mCherry-Rab7 (late endosomes) (C). n = 3, with 3–9 fish per n; all fish 6-dpf. TF-cholesterol colocalizes with BODIPY TR ceramide, which marks the trans-Golgi network, in enterocytes after 4 h of feeding (D). n = 3, with 3–6 fish per n. Arrows indicate colocalization; dpf, days postfertilization; L, intestinal lumen; N, nucleus; TF-cholesterol, TopFluor-cholesterol.

 
Fig. 4

Transgenic zebrafish allows for visualization of human APOA-I in vivo. Live tg(lfabp10:hAPOA-I-mCherry) (A–C) and tg(ifabp:hAPOA-I-mCherry) (E–F) larvae show hAPOA-I-mCherry accumulation in the liver and intestine. hAPOA-I-mCherry derived from the liver (outlined by dotted line) accumulates in the intestine outlined by wavy line) and hAPOA-I-mCherry derived from the intestine accumulates in the liver (B and C). Tissue specificity of promoters used in APOA-I-mCherry transgenic fish revealed using whole mount in situ hybridization with antisense riboprobes to mCherry mRNA (D and G). No expression was detected with the sense probes (n = 10–12 fish); all larvae 6-dpf. ApoA-I-mCherry fusion protein is made in transgenic zebrafish and partially degraded (H). ApoA-I-mCherry fusion protein (arrow head) is made in transgenic zebrafish as expected size (50 kDa) (representative image from three experiments). mCherry antisera detected two different bands from Tg(lfabp:hApoA-I-mcherry) and Tg(ifabp:hApoA-I-mcherry) (I). The higher molecular weight bands (arrow head) represent ApoA-I-mCherry full-length protein. The lower molecular weight bands (arrow) is possibly a degradation product of similar size as mCherry protein made from Tg(ef1a:mcherry-CVLL) (representative image from two experiments). mCherry flourscence on native gel (representative image from four experiments) (J). Immunofluorescence for endogenous zApoA-Ia and zApoA-Ib in wild-type fish show punctae within the intestine similar to hAPOA-I-mCherry accumulation (representative image from three experiments) (K). APOA-I, apolipoprotein A-I; dpf, days postfertilization; E, Tg(ef1a:mcherry-CVLL); hAPOA-I, human APOA-I; I, Tg(ifabp:hApoA-I-mcherry); L, Tg(lfabp:hApoA-I-mcherry); W, wild-type; zAPOA-1, zebrafish APOA-1.
Fig. 5

Transgenic zebrafish allows for visualization of zebrafish APOA-Ia in vivo. Live tg(lfabp10:zApoA-Ia-mCherry) (left) and tg(ifabp:zApoA-Ia-mCherry) larvae show APOA-I-mCherry accumulation in the liver and intestine (right). Scale bar = 20 µm, all larvae 6-dpf. APOA-I, apolipoprotein A-I; dpf, days postfertilization.
Fig. 6

hAPOA-I-mCherry localizes to the endosomal-lysosomal trafficking system in enterocytes of tg(ifabp:hAPOA-I-mCherry) larvae. Representative images demonstrate that hAPOA-I-mCherry colocalizes to early endosomes (A), recycling endosomes (B), and late endosomes/lysosomes, as marked by eGFP-Rab7 (C) and Lysotracker (D), in enterocytes. Arrowheads: colocalization; n = 3–4, with 3–12 fish per n, all fish 6-dpf. APOA-I, apolipoprotein A-I; dpf, days postfertilization; hAPOA-I, human APOA-I; L, intestinal lumen; N, nucleus.
Fig. 7

hAPOA-I-mCherry localizes to the endosomal-lysosomal trafficking system in enterocytes of tg(lfabp10:hAPOA-I-mCherry) larvae. Representative images showing hAPOA-I-mCherry colocalized to early endosomes (eGFP-Rab5c) (A), recycling endosomes (eGFP-Rab11a) (B), late endosomes/lysosomes (eGFP-Rab7) (C), and lysosomes (eGFP-Lamp1) (D) in enterocytes. Arrowheads: colocalization; n = 3–4, with 3–12 fish per n. APOA-I, apolipoprotein A-I; hAPOA-I, human APOA-I; L, intestinal lumen; N, nucleus.
Fig. 9

hAPOA-I-mCherry colocalizes with dietary TF-cholesterol in enterocytes of tg(lfabp10:hAPOA-I-mCherry) larvae. Representative images showing hAPOA-I-mCherry and TF-cholesterol colocalization in enterocytes after 3 h (A), 4 h (B), and 6 h (C) of 5% egg yolk feed. Arrowheads: colocalization; n = 3–4, with 3–9 fish per n. A greater percent of TF-cholesterol colocalizes with hAPOA-I-mCherry at 6 h into feeding than at 3 h (means ± SE, 1-way ANOVA of tg(lfabp10:hAPOA-I-mCherry) groups, *P < 0.05) (D). APOA-I, apolipoprotein A-I; hAPOA-I, human APOA-I; L, intestinal lumen; N, nucleus; TF-cholesterol, TopFluor-cholesterol.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Am. J. Physiol. Gastrointest. Liver Physiol.
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