Visualizing enveloping layer glycans during zebrafish early embryogenesis

Abstract

Developmental events can be monitored at the cellular and molecular levels by using noninvasive imaging techniques. Among the biomolecules that might be targeted for imaging analysis, glycans occupy a privileged position by virtue of their primary location on the cell surface. We previously described a chemical method to image glycans during zebrafish larval development; however, we were unable to detect glycans during the first 24 hours of embryogenesis, a very dynamic period in development. Here we report an approach to the imaging of glycans that enables their visualization in the enveloping layer during the early stages of zebrafish embryogenesis. We microinjected embryos with azidosugars at the one-cell stage, allowed the zebrafish to develop, and detected the metabolically labeled glycans with copper-free click chemistry. Mucin-type O-glycans could be imaged as early as 7 hours postfertilization, during the gastrula stage of development. Additionally, we used a nonmetabolic approach to label sialylated glycans with an independent chemistry, enabling the simultaneous imaging of these two distinct classes of glycans. Imaging analysis of glycan trafficking revealed dramatic reorganization of glycans on the second time scale, including rapid migration to the cleavage furrow of mitotic cells. These studies yield insight into the biosynthesis and dynamics of glycans in the enveloping layer during embryogenesis and provide a platform for imaging other biomolecular targets by microinjection of appropriately functionalized biosynthetic precursors.

Fig. 1.

In vivo imaging of glycans in early zebrafish embryos using microinjection of azidosugars and detection with copper-free click chemistry. (A) Metabolic labeling of mucin-type O-glycans with azidosugars via the GalNAc salvage pathway. The enzymatic transformations shown are catalyzed by (i) nonspecific esterases, (ii) GalNAc-1-phosphate kinase, (iii) UDP-GalNAc pyrophosphorylase, and (iv) ppGalNAcTs and other glycosyltransferases. (B) Two-step strategy for imaging glycans in vivo. (C and D) Zebrafish embryos were microinjected with UDP-GalNAz (C, top), GalNAz (D, top), UDP-GalNAc (C, bottom), or no sugar (D, bottom), along with the tracer dye rhodamine-dextran, allowed to develop to 7 hpf, reacted with DIFO-488 (100 μM, 1 h), and imaged by confocal microscopy. Shown are maximum intensity z-projection images. Green, DIFO-488; red, rhodamine-dextran. (Scale bar: 200 μm.)

Fig. 2.

Two-color, time-resolved labeling enables visualization of O-glycan trafficking. Zebrafish embryos were microinjected with GalNAz, allowed to develop to 9 hpf, and reacted with DIFO-488 (100 μM, 1 h). The embryos were then allowed to further develop for 2 h (A) or 12 h (B), at which point they were reacted with DIFO-555 (100 μM, 1 h) and then imaged by confocal microscopy. Shown are maximum intensity z-projection images of superficial enveloping layer cells. Green, DIFO-488; red, DIFO-555. (Scale bar: 10 μm.)

Fig. 3.

Imaging of sialylated glycans using a nonmetabolic approach. (A) Schematic for chemical labeling of sialylated glycans by treatment with NaIO₄ to expose aldehydes on sialic acids, followed by detection using aminooxy-fluorophore conjugates. (B and C) Zebrafish embryos (10 hpf) were bathed in NaIO₄ (500 μM, 30 min, top row) or no reagent (bottom row), reacted with aminooxy-Alexa Fluor 488 (AO-488, 100 μM, 1 h, pH 6.7), and imaged by confocal microscopy. (B, left panel) Brightfield; (B, right panel) and (C), maximum intensity z-projection images of AO-488 fluorescence. [Scale bars: 200 μm (B), 20 μm (C).]

Fig. 4.

Simultaneous visualization of O-glycans and sialylated glycans using two independent bioorthogonal chemistries. (A) Schematic depicting dual labeling of O-glycans and sialylated glycans. (B and C) Zebrafish embryos were microinjected with GalNAz or no sugar, allowed to develop to 10 hpf, and then bathed in NaIO₄ (500 μM, 30 min) or no reagent. Embryos were then reacted in a mixture of DIFO-555 (100 μM) and AO-488 (100 μM) in PBS (pH 6.7) for 1 h, rinsed, and imaged by confocal microscopy. Green, AO-488; red, DIFO-555. [Scale bars: 200 μm (B), 20 μm (C).]

Fig. 5.

Time-lapse monitoring of mitotic cells reveals dramatic glycan reorganization during cell division. (A) Wild-type zebrafish embryos were microinjected with GalNAz, allowed to develop to 10 hpf, reacted with DIFO-488 (100 μM, 1 h), and imaged by confocal microscopy. Arrowheads indicate intense staining at the cleavage furrow of dividing cells. Maximum intensity z-projection images are shown. (B) H2A-GFP transgenic zebrafish embryos were microinjected with GalNAz and allowed to develop to 10 hpf. The embryos were reacted with DIFO-647 (100 μM, 1 h) and imaged by confocal microscopy. Shown are single z-plane frames from a time-lapse movie (Movie S1). Indicated in the top right corner of each image is time (h:min:s). Green, H2A-GFP; red, DIFO-647. (C) H2A-GFP zebrafish were microinjected at the one-cell stage with GalNAz and memCherry mRNA and allowed to develop to 10 hpf. The embryos were reacted with DIFO-647 (100 μM, 1 h) and imaged by confocal microscopy. Blue, H2A-GFP; green, memCherry; red, DIFO-647. Arrowheads indicate location of new membrane between daughter cells. Maximum intensity z-projection images are shown. [Scale bars: 100 μm (A), 20 μm (B, C).]