In vivo imaging of membrane-associated glycans in developing zebrafish

Abstract

Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chemistry, enabling the visualization of glycans in vivo at subcellular resolution during development. At 60 hours after fertilization, we observed an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal analysis of glycan expression and trafficking and identified patterns that would be undetectable with conventional molecular imaging approaches.

Fig. 1

Ac₄GalNAz is metabolically incorporated into zebrafish glycans. (A) Schematic depicting the use of metabolic labeling with Ac₄GalNAz and copper-free click chemistry using DIFO probes for the noninvasive imaging of glycans during zebrafish development. (B) Flow cytometry analysis of ZF4 cells metabolically labeled with Ac₄GalNAz. ZF4 cells were incubated with Ac₄GalNAz (0 to 100 μM, 3 days) and subsequently reacted with DIFO-488 (10 μM, 1 hour). Error bars represent the standard deviation from three replicate samples. (C) Immuno-blot analysis of lysates from zebrafish embryos at 120 hpf incubated with Ac₄GalNAc (Ac) or Ac₄GalNAz (Az), probed with horseradish peroxidase–conjugated antibody to Flag (top panel) or antibody to β-tubulin (bottom panel).

Fig. 2

In vivo imaging of glycans during zebrafish development. (A and B) Zebrafish embryos were metabolically labeled with Ac₄GalNAz (Az) or Ac₄GalNAc (Ac) starting at 3 hpf. (A) Embryos were reacted at 72 hpf with DIFO-647 for 1 hour. The right panel indicates an exposure time that is 20 times longer than that in the other two panels. (B) Embryos were reacted at 72 hpf with DIFO-647 for 1 to 60 min. Asterisks denote autofluorescence. (C) Zebrafish embryos incu- bated with Ac₄GalNAz or Ac₄GalNAc (fig. S6) starting at 3 hpf were reacted with DIFO-647 at 24 hpf and subsequently at 12-hour intervals, viewed laterally and ventrally (alternating panels). (D and E) Zebrafish from (C) imaged at higher magnification at 60 hpf (D) or 72 hpf (E), viewed laterally (left panels) and ventrally (right panels). Solid arrowhead, olfactory organ; open arrowhead, pectoral fin. Dotted line indicates the pharyngeal epidermis in the jaw region. Scale bars in (A) and (C), 500 μm; in (B), (D), and (E), 200 μm.

Fig. 3

Identification of temporally distinct glycan populations during zebrafish development using two-color labeling. Zebrafish embryos metabolically labeled with Ac₄GalNAz from 3 to 60 hpf were reacted with DIFO-647 between 60 and 61 hpf and then reacted with DIFO-488 either between 61 and 62 hpf [(A) to (D)] or, after an additional 1 hour of metabolic labeling with Ac₄GalNAz, between 62 and 63 hpf [(E) to (I)]. Control embryos incubated with Ac₄GalNAc and otherwise reacted with the same DIFO-fluorophore probes are shown in figs. S9 and S10. (A) Brightfield image of a frontal view. (B) z-projection (left panel) and x-projection (right panel) fluorescence images of the mouth region. (C) Brightfield image of a lateral view. (D) Single z-plane fluorescence image of the pectoral fin region. (E) Brightfield image of a ventral view of an embryo at 63 hpf. (F) Single z-plane fluorescence image of (E) displaying intense DIFO-488 fluorescence but not DIFO-647 fluorescence. (G) Left panel, single z-plane fluorescence image of the jaw region; middle and right panels, z-projection (middle panel) and x-projection (right panel) fluorescence images of the region highlighted in the left panel. (H) z-projection (left panel) and y-projection (right panel) fluorescence images of the mouth. (I) z-projection fluorescence image of the olfactory organ. Highlighted are the olfactory epithelium (oe) and olfactory pit (op) regions. In (B), (D), and (F) to (I), red is DIFO-647 (60 to 61 hpf) and green is DIFO-488 [61 to 62 hpf in (B) and (D) and 62 to 63 hpf in (F) to (I)]. Scale bars in (A), (C), (E), and (F), 100 μm; in (B), (D), (G) (left panel), (H), and (I), 10 μm; in (G) (middle and right panels), 5 μm.

Fig. 4

Spatiotemporal analysis of de novo glycan biosynthesis during zebra-fish development between 60 and 72 hpf. Zebrafish embryos metabolically la- beled with Ac₄GalNAz from 3 to 60 hpf were reacted with DIFO-647 between 60 and 61 hpf, metabolically labeled with Ac₄GalNAz for 1 hour, and reacted with DIFO-488 between 62 and 63 hpf. The embryos were metabolically labeled with Ac₄GalNAz for an additional 9 hours and then reacted with DIFO-555 between 72 and 73 hpf. (A) z-projection fluorescence image of a lateral view. (B) Single z-plane fluorescence images of the region highlighted in (A). (C) Single z-planefluorescenceimage of a ventral view of the jaw region. (D) Left panel, z-projection fluorescence image of cells in the region highlighted in (C); middle and right panels, z-projection (middle panel) and x-projection (right panel) fluorescence images of the cells highlighted in the left panel (white dashed rectangle). (E) z-projection (left panel) and x-projection (right panel) fluorescence images of kinocilia. (F) z-projection fluorescence image of the olfactory organ. Highlighted are the olfactory epithelium (oe) and olfactory pit (op) regions. Blue, DIFO-647 (60 to 61 hpf); green, DIFO-488 (62 to 63 hpf); red, DIFO-555 (72 to 73 hpf). Scale bars in (A), and (C), 100 μm; in (B), 25 μm; in (D) and (F), 10 μm; in (E), 5 μm.