Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Abstract

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered. Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions. Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes. Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras. We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another. We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures. In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo. These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology. Video LinkThe video component of this article can be found at https://www.jove.com/video/1422/ Protocol Part 1: Alexa 594 Labeled Embryos Microinjection Plate and Needle Preparation Making microinjection plates1. Prepare injection plates comprised of three to five 1-mm troughs within an agarose mold using a technique by Westerfield, 2007 1 . These troughs will snuggly hold the embryos in a line for efficient and systematic microinjections. Prior to making the plates, take three 1-mm, 4-inch long non-filament capillaries and carefully break them in half. Using superglue, glue two of these 2-inch long capillaries side-by-side on a flat surface. Glue the third capillary on top of these two, nestled in the grove, creating a pyramid shape with the three capillaries. Allow to dry and repeat until enough "trough molds" are constructed. 2. Pour 20-25mL (5mm depth) of molten 1.5% agarose in embryo medium (EM) [5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2 , 0.33mM MgSO 4 and 0.00003% methylene blue] into a 100mm petri dish. Immediately place three to five of the previously made pyramidshaped trough molds to the botto...

Protocadherin-18b interacts with Nap1 to control motor axon growth and arborization in zebrafish

“…, 1990 ). The procedure was followed according to a published protocol ( Deschene and Barresi, 2009 ).…”

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

“…Various populations of cells can be observed through the use of genetically encoded fluorescent proteins such as GFP, mCherry, or the photoconvertible dye Kaede (to name a few). Further, fluorescently labeled cells within chimeric embryos can also be viewed with this approach 14 .…”

Characterizing the diverse cells that associate with the developing commissures of the zebrafish forebrain

“…Gastrula-staged cell transplantation was carried out as described in (Deschene and M. J. Barresi, 2009). Briefly, donor transgenic embryos and wild-type host embryos were hand dechorionated at 4 hpf and placed into a 2% agarose transplant well plate filled with antibiotic embryo rearing medium (15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO4, 150 µm KH2PO4, 1.0 mM CaCl2, 0.7 mM NaHCO3, 0.5 mg/L Methylene Blue).…”

“…We analyzed the resulting transplants at 28 hpf by immunocytochemistry to label POC axons (Deschene and M. J. Barresi, 2009). We successfully obtained 73 host embryos with isolated colonies of glial cells associating with the developing POC (Figure 8; Figure 9).…”