Generating Chimeric Zebrafish Embryos by Transplantation

Abstract

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps. ProtocolA step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage. One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps. To proteolytically dechorionate embryos, incubate at the 1-cell stage for~1-5' in a 0.5 mg/ml pronase solution as described in the Zebrafish Book 1. Moniter dechorionation closely and submerge embryos in Embryo Medium (EM) with Pen/Strep 1 as soon as the chorions begin to visibly collapse. Once released from their chorions, blastula and gastrula stage embryos are fragile and will stick to plastic or disintegrate if exposed to air. Therefore keep dechorionated embryos submerged in Pen/Strep EM, transfer between dishes using a wide-bore fire-polished glass pipet, and maintain in either agar-coated plastic dishes (1.2% agarose in Pen/Strep EM) or autoclaved glass petri dishes. Injecting dechorionated embryos with lineage marker.Prepare an injection dish by inserting a glass slide at a 45 degree angle across the widest part of a Petri dish three-quarters full of 1.2% agarose made in Pen/Strep EM. ...

Cadherin 6 promotes neural crest cell detachment via F-actin regulation and influences active Rho distribution during epithelial-to-mesenchymal transition

“…Cell transplantations were carried out at the blastula stage as described previously (Kemp et al, 2009). Donor embryos [Tg(-4.9sox10:EGFP)] were injected with 1 nl of 3% Texas Red-conjugated Dextran (Life Technologies) at the one-cell stage.…”

Description of Embryonic Development of Spotted Green Pufferfish (Tetraodon nigroviridis)

“…For imaging, approximately 100 embryos were moved into an agarose-coated Ø 9 cm Petri dish with approximately 1 mm long, 1 mm wide, and 0.7 mm deep wells. Those wells were generated by a custom-made plastic mold similar to the one used by Kemp et al 14 The embryos for imaging in the well plates were replaced at least once a day. Embryos were cultured and imaged at 27.3°C.…”

Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression

“…42,43 Creation of zebrafish chimeras has been used to study the cell-intrinsic functions of cloche and bloodless genes in HSPC proliferation, survival, and differentiation. 26,27,44 Here, we demonstrated that transplanted cdh5-silenced blastula cells differentiate into red cells in WT zebrafish embryos.…”