Cell grafting and labeling in postimplantation mouse embryos.

Abstract

Fate mapping experiments provide direct information on the differentiation pathways normally taken by cells or tissues during embryogenesis. Systematic analyses of the developmental fate of cell populations localized in different parts of the embryo enables the construction of fate maps. A comparison of the expression pattern of lineage-specific genes and the fate map allows the identification of precursor tissue for cell lineages well before definitive histogenesis takes place. The ability to trace the early lineage history of cells greatly facilitates the elucidation of the forces and processes that lead to the specification of cell lineages and the determination (or commitment) of cell fate. The knowledge of cell fate may also assist the interpretation of the phenotype of mutant embryos produced by either spontaneous mutation or gene knockout experiments. This chapter describes the technical aspect of fate mapping the mouse embryo during gastrulation (6.5 days post coitum = 6.5 d) (1–3) and organogenesis (8.5 d) (4–8). Two experimental strategies are used to study the developmental fate of cells. First, a specific population of cells can be marked by labeling with vital carbocyanine dyes in situ or by introducing genetic markers by electroporation (9), and second, the same population of cells can be isolated from a transgenic embryo followed by transplantation (grafting) to a host embryo. The pattern of tissue colonization and differentiation of the descendants of these marked or transplanted cells is then analyzed after a period of in vitro development to assay their development fate. This can be done by fluorescence imaging of live whole embryos under the dissecting microscope. Cell labeling, electroporation, and grafting procedures have their special advantages. When grafting a genetically identifiable population of cells or electroporating a gene-expression construct, there is no dilution of the label owing to cell proliferation, so the contribution of transgenic cells to every available lineage can be assessed. Cell transplantation techniques can also be applied to the study of the developmental potency of a population of cells, by confronting the cells with novel tissue environmental or inductive signals (10). The usefulness of the cell grafting approach depends critically on the ability to isolate a defined cell population for transplantation and to place these cells at the appropriate site in the host. In contrast to cell transplantations, in situ labeling experiments do not require tedious dissection of tissue fragments. Fate mapping studies can be carried out directly after in situ marking of the cells with minimal disruption of the existing tissue architecture. However, the label must be noncytotoxic and should remain only among the descendants of the labeled cells. Similar to in situ labeling, embryo electroporation does not require dissection. By a series of electrical pulses that permeate the cell membrane, an expression plasmid encoding a genetic marker can be introduced to cells. A major advantage is that the marker is expressed by all the descendants that inherit the electroporated gene, thereby allowing the complete lineage to be tracked over a substantial length of time or number of cell divisions with no significant diminution of signal intensity. Genetic markers that have been used successfully include green fluorescent protein (GFP) and its spectral variants, β-galactosidase, and alkaline phophatase. Electroporation works most effectively on tissue with an epithelial architecture (such as the endoderm and the ectoderm of the embryo), since the basal lamina can prevent DNA from spreading to other tissue layers during labeling. Furthermore, electroporation enables marking of cells in a thin epithelium (such as the gut endoderm, surface ectoderm, or endothelium) when the conventional technique of transplantation of cells for fate mapping is not feasible.