Fate-Mapping Technique: Using Carbocyanine Dyes for Vital Labeling of Cells in Gastrula-Stage Mouse Embryos Cultured In Vitro

Abstract

INTRODUCTIONThe allocation of different progenitor populations to embryonic structures can be visualized by tracking the distribution of cells to specific tissues in the live embryo. A critical prerequisite for cell tracking is to identify unambiguously the progenitors and their descendants during morphogenesis. This can be achieved by using molecular markers that are expressed from transgenes integrated into the genome or as episomal DNA constructs, or by tagging the cells with exogenous markers that are incorporated into the cell membrane or cytoplasmic components of the cells. These labels can be introduced by dye-labeling the membrane, injecting marker enzyme into the cytoplasm, or integrating reporter constructs by transfection or electroporation. This protocol describes how to label cells in the endoderm (which, at this stage of development, is the superficial tissue layer) of live mouse embryos at 7.0-7.5 days post-coitum (dpc), using two carbocyanine dyes (DiI and DiO).

Dynamic lineage analysis of embryonic morphogenesis using transgenic quail and 4D multispectral imaging

“…Depending on the desired application, fluorescent labels can be delivered by administering dye to cell membranes, injecting, electroporating, or transfecting DNA, RNA, or marker enzymes into the cytoplasm, or integrating reporters into the genome by viral infection or transgenesis (Bildsoe et al .,2007; Bronner‐Fraser,1996a,b; Dickinson et al .,2002; Itasaki et al .,1999; Sato et al .,2010).…”

“…Carbocyanine dyes can also be used for time‐lapse studies in living embryos (Ezin et al .,2009; Hatada and Stern,1994; Kulesa and Fraser,1998; Kulesa et al .,2000). The vital dyes can be focally applied to small numbers of desired cells of (i.e., 1–100 cells) or to larger areas of tissue in living embryos and then followed using fluorescence microscopy (Bildsoe et al .,2007; Wetts and Fraser,1988). Drawbacks with all of these direct cell‐labeling methods are that labeling can be nonuniform, and the topically applied label is diluted upon cell division and is thus unsuitable for lineage analysis studies of extended duration (Bildsoe et al .,2007; Clarke and Tickle,1999; Dickinson et al .,2002; Frank and Sanes,1991; Honig and Hume,1989).…”

The mouse fetal‐placental arterial connection: A paradigm involving the primitive streak and visceral endoderm with implications for human development

“…Given that the primitive streak defines antero‐posterior polarity in the embryo, further analysis of the ACD focused on these spatial coordinates. Introducing lipophilic DiI (Bildsoe, Franklin, & Tam, ) into the ACD resulted in labeled cells remaining in situ while others formed an axial file that extended through the allantoic midline (Downs et al, ) (Figure a). This behavior was reminiscent of the node, whose labeled cells both remained in place while at the same time extended an anterior file of cells that became incorporated into the notochord (Beddington, ) (Figure b).…”