Differential Lectin Binding Patterns Identify Distinct Heart Regions in Giant Danio (Devario aequipinnatus) and Zebrafish (Danio rerio) Hearts

Abstract

SummaryLectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish (Danio rerio) and giant danio (Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins-wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)-in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins' ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts. (J Histochem Cytochem 64:687-714, 2016)

Visualization of Mouse Choroidal and Retinal Vasculature Using Fluorescent Tomato Lectin Perfusion

“…20,[23][24][25] Although Ulex europaeus agglutinin I strongly labels blood vessels in human tissues, rodents do not express ligands for Ulex europaeus agglutinin I on their blood vessel walls, 26,27 so this lectin is not suitable for animal models. Tomato lectin (TL; Lycopersicon esculentum agglutinin) recognizes poly-N-acetyl lactosamine-type oligosaccharide moieties 28,29 that is relatively specific for microvessels [30][31][32][33] in mice. 34,35 We are not aware of any published reports of TL perfusion for the assessment of normal choroidal vasculature in flat mounts, but it has been described for hyaloidal vessel analysis and choroidal neovascularization detection in the subretinal space.…”

Heart Development, Coronary Vascularization and Ventricular Maturation in a Giant Danio (Devario malabaricus)

“…The hearts were washed and stained immediately. Fluorescein-conjugated and rhodamine-conjugated BS lectin, Con A, or wheat germ agglutinin (WGA) (Vector Laboratories, Burlingame, CA, USA) were used as previously described [ 37 ]. Hearts and larvae were incubated wholemount in Cellstar 24 well cell culture plates (Santa Cruz, Biotech, Santa Cruz, CA, USA) overnight at 4 °C using in 400 μL of lectin-containing Tris buffer saline (50-mM Tris, 150-mM NaCl, 1-mM CaCl2, 1-mM MgCl 2 , pH 7.6) at the following final lectin concentrations: BS lectin (1:400, 5 μg/mL), Con A (1:1000, 2 μg/mL), WGA (1:1000, 2 μg/mL).…”

“…Studies in our lab have shown that the heart of an adult giant danio ( D. cf aequipinnatus ) possesses robust regenerative capacities similar to those seen in the zebrafish [ 36 ]. Recently, we reported on the use of lectins as histochemical tools to study cardiac biology in giant danio and zebrafish [ 37 ]. However, little is known regarding the cardiac development, growth and maturation of Devario species.…”

Structure, development, and functional morphology of the cement gland of the giant danio, Devario malabaricus

“…We first serendipitously observed an area on the superior aspect of 3-day postfertilization D. malabaricus larval head, highly reactive to wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I), (not shown), while carrying experiments for comparative cardiac development using lectin histochemistry. 36 Closer inspection under brightfield microscopy revealed the profile of an oval shaped and markedly raised protuberance over the dorsal epithelial surface of the larval head ( Figure 4A-E). We identified this structure to be the mucus producing cement gland of the species and studied its development from hatching up to 3 weeks post fertilization (wpf).…”

“…For whole-mount embryos and larvae imaging, sample were fixed in 4% paraformaldehyde, washed in 1X PBS, and permeabilized with 0.5% Triton X-100 in 1X PBS overnight at 4 C. Fluorescein-conjugated and rhodamine-conjugated lectins (Vector Laboratories, Burlingame, California) were used. Larvae were incubated in Cellstar 24-well cell culture plates (Santa Cruz, Biotech, Santa Cruz, California) overnight at 4 C, as previously described 36 using in 400 μL of lectincontaining Tris buffer saline (50-mM Tris, 150-mM NaCl, 1-mM CaCl2, 1-mM MgCl2, pH 7.6) at final concentrations between 2 and 5 μg/mL. Next, larvae were washed in PBS, and cell nuclei were stained with Hoechst stain (Invitrogen, Eugene, Oregon), wash in 1X PBS.…”