An important aspect of developmental biology is investigating where particular cells of interest originate in the early embryo, and how and when they arrive at their final locations. A classic approach to studying cell and tissue origins is to generate a fate map, which outlines the developmental history of specific cells or regions of the early embryo. Lineage analysis traces a progenitor cell through its divisions to form all of its daughter cells and tracks their contributions to the embryo to map the fate of that progenitor cell. It is a powerful tool for deciphering which and how many cells form the various structures of the developing embryo and how the structures evolve their shape. To produce a fate map, a single cell or a few cells are labeled in a specified location, and the label is transmitted to the progeny of the initial cell(s) independently of where the cells migrate, generating lineages of labeled progeny. The types of cells that derive from the original progenitor and their locations and lifespans can then be determined by assessing the positions, phenotypes, and behaviors of the labeled cells at successive time points following the initial labeling (Bronner-Fraser and Fraser,1989; Fox et al.,2008; Kulesa et al.,2009; Sato et al.,2010; Wacker et al.,2007).
A critical element of constructing a fate map is selecting a suitable lineage label or cell marking. Over the past 100 years, numerous different labeling methods have been attempted (Rodriguez-Gallardo et al.,2005). Researchers can label single cells using many different techniques and reagents, including but not limited to vital dyes, radioactive tracers, genetic markers, and retroviruses.
Radiographic Labeling Approach
The need for a generalizable method of labeling cells led to radiographic labeling. The autoradiographic mapping approach was first considered by Hughes et al. (1958) and tested in vitro by Trinkaus and Gross (1961) in cultured chicken melanocytes to determine the specificity of the labeling technique. Several groups then applied radioactive labeling to study cell migration in vivo in chick embryos (Orts-Llorca and Collado,1968; Rosenquist,1966,1970; Stalsberg and DeHaan,1969; Weston,1963). Lineage analysis by radioactive labeling involves removing grafts of tissue from a donor, immersing the cells or tissue in tritiated thymidine for its incorporation into DNA, and grafting the tissue back into the host. Tissue must be sectioned, and the approximate positions of nuclei in two-dimensions (2D) are detected by the beta emissions released. Centers of beta emission are captured on autoradiographic paper at specified time points after grafting (Weston,1963). No differences in cell behavior were detected between radioactively labeled cells versus unlabeled cells at the dosage used (Trinkaus and Gross,1961); however, subtle effects on cell survival or behavior could not be assayed.
Excess unlabeled thymidine was transferred with grafts to the recipient embryo to prevent unintended labeling of cells in the recipient embryo and ensure that only grafted cells were labeled. While this provided considerable specificity of cell labeling, label detection could not be resolved to single cells within dense tissue, emissions could only be detected from the surface of sections, and label was diluted with cell division (Hughes et al.,1958). Cell death could also result in uptake of labeled DNA by migratory macrophages, which would confound interpretations of labeled cell migration (Trinkaus and Gross,1961). Furthermore, developing the autoradiographs took days or weeks and required fixation and sectioning, resulting in snapshots of the general locations of some of the labeled nuclei at different time points. A more sensitive method for detecting label, and labels that could be detected in living tissue, were required for precise fate mapping and study of the dynamics of morphogenesis.
Chick-Quail Genetic Chimeras
In 1969, Nicole Le Douarin advanced the field of lineage analysis by establishing the use of chick-quail chimeras for fate mapping (Le Douarin,1969). Le Douarin recognized that the similarity between quail and chicken embryonic development would enable grafting between them without obstructing development. She capitalized upon the ability to distinguish quail and chicken cells from one another by staining DNA with Schiff's reagent. Quail interphase nuclei contain dense heterochromatin in the nucleoli, which stain brightly with Schiff's reagent, while nucleoli in the chick do not label distinctly (Le Douarin,1973,1996).
Thus, Le Douarin exploited an intrinsic nuclear marker that enabled her to identify grafted cells in a chimaeric embryo. Her approach involves transplanting cells or tissues between embryos of matched developmental stage in ovo, generally from the quail embryo into the corresponding region of the chick embryo after removing the identical chick tissue (Couly et al.,1992; Le Douarin,1973,1996; Le Douarin and Barq,1969). The eggs are reincubated for a desired period to enable the transplanted quail cells to assimilate into the host chicken tissue as normal development ensues. At later stages of development, the grafted cells are identified with microscopic analysis of stained tissue sections. The approach avoids the aforementioned problem of leaky dyes disseminating to neighboring cells and permits high cellular resolution for lineage analysis studies (Le Douarin,1986; Le Douarin et al.,1974).
The strengths of the chick-quail chimera technique are that the label is integral to the cell, and it does not spread to adjacent cells or dilute with cell proliferation (Balaban et al.,1988; Bortier and Vakaet,1992; Le Douarin,2008). Furthermore, the locations of grafted cells can be more precisely determined compared with the radiographic approach. The traditional drawbacks are that chick-quail chimeras are analyzed statically, the tissue contributions are attributed to groups of cells rather than individual cells, the requirement for precise staging between quail and chick complicates the method, the technique is not broadly applicable to all species, and the transplant scar can induce developmental problems.
Dye Labeling of Individual Cells
While tissue grafting techniques can map the embryonic contributions of groups of cells, the technique can disrupt development, and grafting is generally restricted to superficial sites on the embryo. Tracking the ancestry of tissues deep within an embryo requires a more generalizable means of labeling and visualizing individual progenitors.
Fifty years after Vogt's pioneering experiments using vital dyes to trace cell fate, breakthroughs were made to label living cells in a way that confined the label to the injected cells and circumvented the ambiguities resulting from dye leaking to adjacent cells. In 1978, Weisblat et al. fate mapped single cells (Weisblat et al.,1978). By injecting horseradish peroxidase into single leech blastomeres, they identified derivative tissues following a histochemical reaction with benzidine. They improved this approach by injecting dyes and dye-conjugated dextran, which can be directly visualized in the cytoplasm of cells (Gimlich and Braun,1985; Heasman et al.,1984; Weisblat et al.,1978,1980).
The development of vital dyes that are confined to labeled cells improved label resolution and permitted dynamic in vivo observation of individual cell movements. Dextran-dye conjugates were transmitted to daughter cells and remained traceable in the cytoplasm of cells for a week or more (Gimlich and Braun,1985). Conjugates of different colored dyes enabled multicolor tracing experiments, and fixable dextran-amines enabled immunostaining of dextran-labeled tissue sections (Gimlich and Braun,1985). Fluorescently labeled dextrans were injected into individual cells for fate mapping studies, for instance to trace the lineages of avian neural crest cells (Bronner-Fraser and Fraser,1989,1988,1991), Hensen's node in the chick (Selleck and Stern,1991), and zebrafish blastomeres (Kimmel and Law,1985), among others.
Carbocyanine Vital Dyes
The vital carbocyanine dyes DiA, DiI, and DiO simplifed fluorescent dye use as cell labels for lineage tracing (Schlessinger et al.,1977; Wu et al.,1977). These hydrophobic dyes fluoresce at distinct wavelengths and insert into lipid cell membranes (Axelrod,1979). Because the dyes integrate into the plasma membrane, they are distributed among the progeny of labeled cells during division, but the dyes usually do not flip into the membranes of neighboring cells (Honig and Hume,1989). Thus, they faithfully trace the lineages of the progenitor cells that incorporated the label. Vital carbocyanine dyes fluoresce with strong intensity, and the diverse colors that different dyes emit enable distinction of different populations of adjacent cells using multispectral techniques (Carette and Ferguson,1992; Clarke and Tickle,1999; Gan et al.,1999; Holland and Holland,2007; Honig and Hume,1989; Scherson et al.,1993; Serbedzija et al.,1989).
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).
Viral Delivery of Genetic Markers
In the 1980s, Joshua Sanes and Constance Cepko and their coworkers engineered replication-defective retroviruses to label cells for lineage analysis (Price et al.,1987; Sanes et al.,1986). Upon retroviral infection of a cell, the marker gene [i.e., horse radish peroxidase, alkaline phosphatase, β-galactosidase, or fluorescent protein (FP)] becomes integrated into the cell genome, thereby stably labeling the cell and its descendants for tracking over time (Price et al.,1987; Sanes et al.,1986; Turner and Cepko,1987). The retrovirus is rendered replication-incompetent by elimination of its gag, pol, and env genes, which results in expression of the marker genes in a cell-autonomous manner in clonally derived cells (Cepko et al.,1998; Sanes1989). Because retroviral vectors require disassembly of the nuclear envelope to access the genome of an infected cell, they are only able to integrate into the genomes of actively dividing cells (Cepko et al.,1995). However, lentiviral vectors, which belong to the Retroviridae family, can stably deliver gene cargo into the DNA of both proliferating and quiescent cells (Naldini et al.,1996b).
Marking cells by viral infection has several advantages as the viruses themselves are relatively harmless to the infected cell, no infective particles are produced by infected cells so there is no transmission to neighboring cells from the infected cell, and the integrated retroviral genome is dependably inherited by all offspring of an infected progenitor cell (Cepko et al.,1998; Mikawa et al.,1992; Sanes1989; Turner and Cepko,1987; Turner et al.,1990). However, implementation of the technique can sometimes be complicated by the silencing of integrated proviruses, and infection of too many progenitors may confound interpretation of lineage relationships (Cepko et al.,1998; Poynter and Lansford,2008; Sanes1989).
When labeling cells by viral gene transfer, it can be difficult to identify all the cells derived from a single progenitor, at the exclusion of those derived from other labeled progenitors. Even injecting a very low titer of retrovirus typically labels several individual cells (Cepko et al.,1998; Sanes1989; Turner and Cepko,1987). If it is known that minimal migration and intercalation of cells occurs, then it can be presumed that groupings of cells marked with the reporter are clonally related, but if widespread intermixing of cells is suspected, then a more stringent evaluation of clonality is needed (Austin and Cepko,1990; Turner and Cepko,1987). In a laudable effort to verify lineage clonality, Cepko and coworkers devised a complex library of retroviral constructs in which a unique DNA oligomer tags each individual virus and can be identified using polymerase chain reaction (PCR) and DNA sequencing (Golden et al.,1995). Cells that are clonally related contain identical DNA tags. The drawback of this approach is that it is extremely arduous to characterize individual cells, and extracting and PCR-amplifying the contents of single cells are nontrivial.
Although the approach of recombinant DNA oligomer tagging would be too laborious to trace the lineages of thousands of cells during embryonic morphogenesis, the notion of marking individual cells and their descendants with a stable genetic label was an integral advancement for in vivo dynamic lineage mapping. The discovery of green FP (GFP) in jellyfish (Shimomura et al.,1962) and its cloning (Prasher et al.,1992) and expression in cells of other species (Chalfie et al.,1994; Gervaix et al.,1997; Okada et al.,1999; Tsien,1998) opened the door for fate mapping by dynamic imaging. Introducing FPs to cells by infection with lentiviral vectors stably marks the infected cells, and they reliably transmit the fluorescent marker to their progeny. Labeled cells proceed to divide, migrate, and differentiate as usual (Carleton et al.,2003; LaRue et al.,2003; Okada et al.,1999), and the stable genetic marking permits tracking of labeled progenitors and their descendants in living embryos in real time using time-lapse videography, confocal, and two-photon (2P) microscopies (Dickinson et al.,2002; Okada et al.,1999; Sato et al.,2010).
Lentiviruses are efficient gene delivery vehicles that can bestow enduring gene transduction to a range of cell types in vivo (Blomer et al.,1997; Leber et al.,1996; Lois et al.,2002; Naldini et al.,1996a,b; Watson et al.,2005). As described below, lentiviral vectors are particularly useful because they are able to stably express the delivered genes in a tissue-specific manner in somatic cells and in the germ line of transgenic animals (Kootstra and Verma,2003; Lois et al.,2002). Lentiviral vectors have been used to efficiently produce transgenic mice, rats, quail, chickens, monkeys, and other organisms with high expression of the fluorescent marker (Chapman et al.,2005; Lois et al.,2002; McGrew et al.,2004; Michalkiewicz et al.,2007; Palfi et al.,2002; Remy et al.,2010; Sasaki et al.,2009; Sato et al.,2010; Scott and Lois,2005; Sosa et al.,2010; Tarantal et al.,2001; Yang et al.,2008). Combining stable labeling of cells via lentiviral gene transduction with FP expression provides the means for decoding the complex developmental morphogenesis and lineage structures in vertebrates using dynamic multispectral imaging.