Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP

Abstract

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Developmental Dynamics 232:359 -368, 2005.

Solute carrier family 3 member 2 (Slc3a2) controls yolk syncytial layer (YSL) formation by regulating microtubule networks in the zebrafish embryo

“…Wild-type and Tg[β-actin:GFP] strains of zebrafish (27) were maintained according to standard procedures (1).…”

“…We therefore speculated that abrogation of Slc3a2 in the zebrafish embryo might alter plasma membrane fusion during YSL formation. To examine this possibility, plasma membrane dynamics during YSL formation was analyzed using two-photon microscopy to image membranetagged GFP-labeled cells (27). Acquisition of both two-photon fluorescence (TPF) (for GFP) and coherent anti-Stokes Raman scattering (CARS) (for detecting lipids) enabled visualization of plasma membranes of the blastomeres and the yolk cell throughout cell-division cycles.…”

Ion imaging during axolotl tail regeneration in vivo

“…Of course, penetration of such vital dyes is more effective into thin tissues (up to 0.5 mm) such as tails of axolotl larvae used in this study, zebrafish embryos (Cooper et al, 2005), or mammalian retina (Bianchini et al, 2008). By these variations in thickness a quantification of these values may be difficult.…”

Ubc9 Regulates Mitosis and Cell Survival during Zebrafish Development

“…To be able determine ratios of cells in M-phase, pH3 stainings were performed in transgenic fish expressing membrane-bound GFP under the control of a cytoskeletal actin promoter, thereby visualizing cell boundaries (Cooper et al, 2005). Furthermore, specimens were stained for the endodermal pouch marker Zn5, allowing us to distinguish chondrocyte precursors of the different gill arches.…”