Lineage Marking

Lineage Marking

Chapter 32 Lineage Marking Andras Nagy Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Professor, Department of Medical Genetics and Micro...

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Chapter 32

Lineage Marking Andras Nagy Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Professor, Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada

Chapter Outline Definitions Questions to Ask Markers and Lineage Marking Marking with Endogenous Identifiers Marking with Dyes or Enzymes Marking with Exogenous Genetic Identifiers “Passive” Exogenous Genetic Markers

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DEFINITIONS Mammalian development starts from a singe cell, the zygote, which is the ancestor to all the somatic and germ cells of the entire later organism. This type of developmental capacity is called totipotency. In the first few divisions of preimplantation development, the cells (blastomeres) retain this totipotency, but shortly before implantation, at the blastocyst stage, cells become committed to certain tissues of the later conceptus. Three cell types are clearly distinguishable at this stage: the trophectoderm, committed to the trophoblast cells of the placenta; the primitive endoderm, which derivatives remain extraembryonic and will form the parietal and visceral endoderm; and the primitive ectoderm responsible for all cells in the embryo proper and for some internal extraembryonic membranes, such as allantois, amnion, and yolk sac mesoderm (the inner layer of this membrane). A given cell of the blastocyst is only capable of differentiating into a subset of cells of the conceptus; therefore, these cells are no longer totipotent. Instead they are referred to as pluripotent. After implantation, further subdivisions of developmental commitments occur in the embryo proper, as the embryonic ectoderm, mesoderm, and definitive endoderm form. The following organogenesis produces the final cellular diversity of an individual by differentiating highly specialized cell types (differentiated cells) for organ functions. The cellular diversity consists of approximately 260 distinguishable cell types in mammals, which make up

“Active” Exogenous Genetic Markers Chimera e A Tool of Fate Mapping Cell and Tissue Grafting Using Genetic Switches Future Directions Acknowledgments References

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an estimated total cell number of 1 trillion (1012) in an adult human or 10 billion (1010) in a mouse. The progress of cell commitment to an ever-decreasing range of cell types can be viewed from the opposite direction: every cell among the 260 cell types has its own “history” that can be traced. This trace is often referred to as the lineage (Figure 32.1). If the traces are looked at from a given progenitor or a stem cell (the origin) in the forward direction, the set of traces is the fate of the cell. If the fate remains within a single type of differentiated cell or results in two or more kinds, the origin is called uni-, bi-, or multipotential, respectively. With these definitions, cell lineage and fate are practically the same phenomenon, a set of developmental traces viewed either from the differentiated product or the origin. In Figure 32.1, therefore, the fate of p1 is d1 and d2, and the fate of p2 is d1. The progenitor cell, p1, is bipotential, and p2 is unipotential. For the differentiated cell, the lineage of d2 is simple: it goes back to p1 only. The d1 lineage could be traced back to either the p1 or the p2 progenitor. The development of neurons and oligodendrocytes is a good example for the paths depicted in Figure 32.1. Analyses of cerebral cortical cultures (He et al., 2001) and developing chicken spinal cord (Leber and Sanes, 1991; Leber et al., 1990) indicate that these two cell types share a common precursor. On the other hand, oligodendrocytes originate from several cell types, such as the small foci of cells in the floor of the third ventricle that generated the oligodendrocytes for the optic nerve (Miller and Ono,

Handbook of Stem Cells, Two-Volume Set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00032-9 Copyright Ó 2013 Elsevier Inc. All rights reserved.

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FIGURE 32.1 Relationship between cell lineage and cell fate. See text.

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1998; Ono et al., 1997) and the oligodendrocyte precursors from the motor neuron pool (Zhou et al., 2001; Zhou and Anderson, 2002).

a unique identifier characteristic of the progenitor under study and retained in the derivatives. Providing this identifier is lineage marking, the subject of this chapter.

QUESTIONS TO ASK

Marking with Endogenous Identifiers

There are two basic questions to be asked about cell lineages or cell fates:

It was pointed out earlier that existence of a unique marker gene or genes covering an entire lineage is not common. Fortunately, though, there are useful genetic differences among individuals within or among species that can be used as identifiers in celletissue transplantation or chimera studies. These types of lineage marking were important in early studies of fate mapping. A great deal of knowledge was acquired using embryo transplantation chimeras between quail and chicken (Teillet et al., 1999). The nuclei of quail cells contain heterochromatin condensed into one (or sometimes two) large mass (Le Douarin and Barq, 1969). This easily recognizable phenomenon was used to distinguish quail from chicken cells. In the mouse, in situ hybridization for a specific genomic sequence of Mus caroli can also discriminate between these and Mus musculus cells in chimeric tissues (Rossant et al., 1983; Rossant, 1985). Haplotype cell surface markers have been frequently used to study hematopoietic lineages (Jackson et al., 1999). In situ hybridization for the Y-chromosome can be used as an identifier when grafting male cells into female recipients (review by Jackson et al., 2002). The visualization is relatively straightforward with Y-chromosome-specific probes on histological section (Hutchinson et al., 1989; Mezey et al., 2000). The unique advantage of the endogenous identifiers is that they are “built in”; therefore, they do not require genetic or other modifications of cells. Grafting, chimeramaking, and frequent requirements for bridging between genders or even species, however, limits the use of such endogenous markers. Modern transgenic methods have in the recent years replaced these traditional approaches. Before detailing these, I should mention another historically important approach that provides a short-term, transient marking of a cell and its progenitors.

1. The lineage question: What are the traces and progenitors that lead to a certain differentiated cell type, for example, d1? 2. The fate question: What are the possible differentiated products of a certain progenitor cell, such as p1?

MARKERS AND LINEAGE MARKING As cells go through the diversification process, they correspondingly change their gene expression profile. This profile determines the properties of the cells and therefore their identities. Cells of the same type express similar genes. Within this pool are some genes specific to one particular cell type. These genes are referred to as cell typespecific markers. For example, hepatocytes, astrocytes, neurons, and cardiac myocytes can be identified by the expression of albumin, GFAP, Neu-N, and a-myosin heavy chain, respectively. To identify traces during development or adult regeneration or renewal is not a simple task. There is often no specific marker identifying a lineage along its complete trace. Instead, there is a set of genes expressed at certain segments of the lineage. For example, oligodendrocytes go through several developmental stages, such as proliferative and differentiative phases. The immature oligodendrocytes can first be identified by a surface antigen expression recognized by an antibody, A2B5. As the cells mature, they acquire another specific surface marker recognized by the antibody called O4. Then, as they further differentiate, they start expressing myelin-specific genes such as MBP and PLP (Miller, 2002). Typically, a temporal series of specific markers or the lack of certain gene expression represents a particular lineage. If these markers are known and easy to detect, they can be used to characterize the fate of a progenitor or a stem cell. However, to follow all the descendants of a given progenitor, there is a need for

Marking with Dyes or Enzymes Vital fluorescence dyes or an enzyme with a long half-life can mark cells in a temporary manner. Among these dyes,

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the most popular are DiI (a carbocyanine) and its derivatives because of their high photostability and low toxicity. These dyes label cell membranes by inserting two long hydrocarbon chains into the lipid bilayers, resulting in an orange-red (565 nm) fluorescence emission. DiI can be used with DiO, which gives a green fluorescent color. These dyes are usually applied to cells either from an ethanol solution or directly from the dye crystal. This simple labeling technique has frequently been used in nonmammalian model systems to successfully address lineage, potentiality, and fate questions (Downs et al., 2001; Selleck, and Stern, 1991; Collazo et al., 1993). Horseradish peroxidase (Lawson and Pedersen, 1992; Kadokawa et al., 1987) and Rhodamine-conjugated dextran (Cruz and Pedersen, 1985; Winkel and Pedersen, 1988) has also extensively been used for cell marking; however, these techniques are more invasive, as they have to be microinjected into the cells. The obvious limitations to all these methods are the temporary nature of marking and the need for free access to cells for labeling. In the mouse, the usefulness of these approaches is further limited by the requirement for in vitro culturing of postimplantation-stage embryos.

Marking with Exogenous Genetic Identifiers Rapidly evolving transgene-based approaches are currently the most versatile methods of lineage marking. This fact, however, does not mean that these approaches have replaced more traditional techniques. They all have their optimal applications, and they are still valid members of the arsenal of options. There are two categories of exogenous genetic identifiers: passive and active markers.

“Passive” Exogenous Genetic Markers Passive exogenous markers are identified on the basis of genomic insertion of a known DNA sequence into the genome. Their detection requires techniques such as DNA in situ hybridization or Southern blotting. A typical example of this category is retroviral insertion. Probing with a retroviral sequence in a genomic Southern blot detects an integration site-specific band pattern. In the case of multiple integrations, the Southern blotting can serve as a fingerprint for a group of cells derived from a single precursor or stem cell. Such an identifier can be used as a tool to measure the turnover of active stem cells that generate an entire hematopoietic compartment (Lemischka et al., 1986). Unfortunately, such an approach is not practical at a single-cell level, which restricts its application in lineage studies. A useful passive genetic identifier for many lineage studies and for fate mapping has been created by direct transgene insertion into the mouse genome (Clarke

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et al., 1988; Nagy et al., 1990). In this transgenic mouse line, a b-globin transgene was inserted into chromosome 3 in a tandem repeat of approximately 1000 copies, which created a long enough unique and known sequence to visualize with DNA in situ hybridization on histological sections (Lo, 1986) (Figure 32.2A).

“Active” Exogenous Genetic Markers Active exogenous genetic markers are based on reporter gene expression from a transgene. There are several reporters available and proven to be useful in lineage studies and fate mapping. Depending on the application, one may be better than the other. The most common reporters fall into two groups: enzymes and fluorescent proteins. Enzyme Activity-Based Reporters The b-galactosidase (lacZ gene of the E. coli) has had a very successful “career” as a reporter for all sorts of eukaryotic cells. The expression of the enzyme by a heterologous promoter seems to be neutral for the functioning of cells. A variety of substrates can be used for detecting b-galactosidase. The most common is an indole derivative, 5-bromoe4-chloroe3-indolyleb-Degalactoside (X-gal; Holt and Sadler, 1958), which produces a blue color as it breaks down. This simple histochemical staining works on small tissues as whole mount and on histological cryostat sections. Many useful transgenic mouse lines have been made with different specificity for LacZ expression. Of those, the most known is a gene trap insertion of lacZ into an endogenous locus named Rosa-26 (Friedrich and Soriano, 1991). This locus provides expression throughout the entire embryo and in most of the adult tissues. Figure 32.2B shows an example for use of Rosa-26 transgenic and N-myc-deficient embryonic stem (ES) cells in a chimera study. The derivatives of these cells (LacZ stained blue) are not able to contribute to the chondrocyte lineage of the embryo. Since the aim of most gene trap programs is to generate lacZ insertions randomly in all the genes of the mouse, cell type- or lineage-specific LacZ tagging is now created in huge numbers. Figure 32.3I shows an example of a LacZ-stained embryo in which the trap vector “landed” in the Mef-2c gene (see the Centre for Modeling Human Disease gene trap database at http:// cmhd.mshri.on.ca/ sub/genetrap.asp). In addition to gene trap lines, many targeted alleles contain a lacZ gene placed into the target vector in such a way that the enzyme is expressed under the regulation of the endogenous gene. Figure 32.3B shows an example in which the lacZ gene was knocked in to the endothelial-specific flk-1 locus. Figure 32.2D shows a yolk sac section from a chimeric embryo created by diploidetetraploid embryo aggregation. In the diploid component, LacZ was expressed from both alleles of the vascular endothelial growth factor (VEGF)

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FIGURE 32.2 Lineage marking at the cellular level. (A) DNA in situ hybridization in trophoblast cells transgenic to the multicopy globin gene insertion (Nagy et al., 1990). (B) Chimeric tissue where one of the components is N-myc oncogene deficient and LacZ tagged with the Rosa-26 gene trap. (C) Immunohistochemical staining for podocyte-specific expression of green fluorescent protein (GFP). (D) LacZ staining of a chimeric yolk sac in which one of the components is expressing the reporter from the vascular endothelial growth factor locus (Damert et al., 2002). (E and F) LacZ and human alkaline phosphatase double staining on mosaic intestine and pancreas (Lobe et al., 1999). (F and H) LacZ staining and GFP visualization of an adjacent section of skin of a Z/EG, K14-Cre recombinase double transgenic animal (Novak et al., 2000). Part C provided by S. Quaggin.

FIGURE 32.3 Whole mount embryos and organs with lineagemarked cells. (A) ES cell-derived embryo in the uterus. The trophoblast of the placenta and the yolk sac endoderm is GFP transgenic tetraploid embryo-derived. (B) Whole mount LacZ stained embryo in which the lacZ gene is inserted into the flk-1, endothelial cell specifically expressed receptor kinase (Shalaby et al., 1997). (C) Z/AP and Cre recombinase double transgenic embryo with sporadic human placental alkaline phosphatase activation (Lobe et al., 1999). (D) Heart of the chimeric embryo between Cyan Fluorescent Protein expressor ES cells and GFP transgenic embryo (Hadjantonakis et al., 2002). Z/EG and Cre recombinase double transgenic embryos with (E) GFP podocytespecific (Eremina et al., 2003), (F) complete, (G) differentiated neuronspecific, and (H) chondrocyte-specific activation. (I) LacZ-stained embryo derived from a Mef-2c gene trap ES cell line. Part H provided by J. Haigh, and part I provided by K. Vintersten and B. Stanford.

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gene (Damert et al., 2002), and the tetraploid cells were wild type for VEGF. In the resulting chimera, the yolk sac mesoderm e derived completely from the diploid cells e shows a blue staining. The yolk sac endoderm, however, shows a mosaic LacZ pattern since it is derived from both diploid and tetraploid cells. The LacZ protein is rather tolerant to N- and C-terminal modifications. Heterologous functional domains may be added, which changes the property of the enzyme, such as the intracellular localization. There are variants that translocate the protein to the nucleus or anchor it to the cell membrane (Skarnes et al., 1995). Alkaline phosphatase is an essential, constitutively expressed enzyme in cells. Similar to LacZ, a simple histochemical or whole-mount staining procedure can visualize enzyme activity in cells. Endogenous alkaline phosphatases can easily be inactivated by a brief heat treatment (at 70 C for 15 minutes). However, the alkaline phosphatase expressed by the human placenta (hPLAP) is unique in that it is not affected by such a heat treatment (DePrimo et al., 1996). This unique property has been utilized for detection of hPLAP activity when expressed from a transgene. Since heat treatment can be applied to both whole mount and cryostat sections, hPLAP has become a convenient histological marker, which allows simple detectionerecognition of enzyme-containing cells. Since hPLAP is efficiently transported into axons, it is an ideal reporter if nerve visualization is required. Figure 32.3C shows sporadic hPLAPþ cells in a 10.5-day postcoitus (dpc) embryo, and Figure 32.3E and F show double staining for LacZ and hPLAP on a cryostat section of intestine and pancreas of a mosaic newborn (for more details, see the next sections of this chapter). However, compared to LacZ, hPLAP stain penetration to whole mount tissue is slightly more limited, and the quality of histological sections falls short of excellence because of the heat treatment of the tissue. Green Fluorescent Proteins The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria has joined the group of reporters used to trace lineages and determine cell fate in mouse and other model organisms (Hadjantonakis et al., 1998; Okabe et al., 1997; Zernicka-Goetz and Pines, 2001). Several mutant derivatives have been developed providing different levels of stability and intensity and a spectrum of light emission (Hadjantonakis et al., 2002). The gene that encodes for GFP can be applied in heterologous-transient or -stable integrant transgenic expression settings or its mRNA for transient production of the protein. In the latter method, the mRNA is injected into the cells (ZernickaGoetz et al., 199). This reporter is definitely unique since its visualization does not require cell fixation. Instead, live specimens can be observed, and cells expressing GFP (or its derivatives) can be followed in their dynamic behavior.

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However, not even GFP could overcome the main obstacle in studying mammalian development, that the embryo needs an intra-utero environment. Ex vivo organ culture systems are gaining importance to overcome this limitation; however, the very early postimplantation time is not yet possible to follow in vitro. Establishing Overall-Expressor Transgenic Lines in the Mouse There are several considerations in expressing reporters for lineage marking from stable integrant transgenes. When cell grafting is the approach to studying cell fate, derivatives of the graft should be identifiable. For this purpose, the reporter has to be expressed ubiquitously in the donor. Interestingly, a reliable ubiquitous expression is difficult to achieve. For more than 10 years, the Rosa-26 gene trap line has been the overall LacZ expressor reporter in the mouse (Downs et al., 2001). Second on the “popularity list” is a LacZ line that has the transgene integration on the X-chromosome. This line has been used successfully in several studies (Tam et al., 1994; Stone et al., 2002; Kinder et al., 2001) to address cell fate and lineage determination questions during gastrulation. Establishing a new overall expressor, a ubiquitous reporter transgenic mouse line, is a challenging task. Even the most promising, endogenously ubiquitous promoter gives unstable, mosaic, or restricted expression from most insertion sites in the genome. Generally, a transgenic mouse line is produced by injecting the desired transgene into the pronucleus of a zygote. This approach requires far too many founder animals to be established and tested for expression. For this reason, ES cell-mediated transgenesis may be a better alternative. If the same constructs are introduced into ES cells, hundreds of transgenic clones can be isolated in a short time. These clones, which all represent a unique genomic integration site of the transgene, can be screened for overall expression of the reporter, first in undifferentiated ES cells and then in in vitro differentiation assays. If only the best overall expressor ES cell line is used to generate mice, the chance of obtaining satisfying reporter expression is high (Lobe et al., 1999). ES cells may also provide control on transgene insertion. The most common method is targeted integration of a transgene (reporter) into well-characterized loci, for example, Rosa-26 (Soriano, 1999; Srinivas et al., 2001). Recombinasemediated cassette exchange is an alternative that would eliminate the uncertainty of transgene expression because of random genomic insertion. Three recombinaseeintegrase systems are available for such an approach; the Cre, Flip (Flp), and PhiC31 (Kolb, 2001; Araki et al., 1997; Belteki et al., 2003). Using any of these, a “docking” site can to be prepared and characterized for expression permissiveness. The transgene is then equipped with a recognition site or

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sites of the recombinase and introduced into the cells with the enzyme. Site-specific genomic integration of the transgene can thus be achieved at a high efficiency.

Chimera e A Tool of Fate Mapping The possibility of combining two or more embryos in one chimera contributed tremendously to the understanding of lineage determination and cell fate in mouse development (for a review, see Nagy and Rossant, 2001). Identifiers described previously are essential to detecting and characterizing the contribution of a chimeric component to a lineage, cell type, or organ. Injection of cells isolated from the preimplantation embryo into a blastocyst and characterization of the allocation of derivatives of each compartment in later stages revealed the first specification events (Gardner, and Rossant, 1979). Chimeras (Figure 32.3D) have also been very informative in addressing basic organization questions, for example whether complex structures are derived from single progenitors (clonality in development) (Schmidt et al., 1985; Rossant et al., 1986). The use of ES cells and tetraploid embryos expanded the horizon of questions that could be addressed. ES cells are not able to contribute to the trophectoderm and primitive endoderm lineages of the developing chimeras (Nagy et al., 1990). On the other hand, cells derived from tetraploid embryos are excluded from the primitive ectoderm lineage when they have to compete with diploid embryo or ES cell derivatives (Nagy et al., 1990, 1993). For chimera production, one can choose any two of the following three sources as components: diploid embryo, tetraploid embryo, and ES cells. In addition, two diploid embryos can be chosen, increasing the number of possible combinations to four.

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Depending on the actual combination, different lineage allocation can be obtained for the selected components (Nagy and Rossant, 2001, 2000) (Figure 32.4). The most extreme separation occurs when tetraploid embryos are aggregated with ES cells; practically no chimeric lineages are generated. The cells in the resulting embryo are either tetraploid derived (trophoblast lineage of the placenta, visceral, and parietal endoderm) or ES cell derived (amnion, allantois, embryonic mesoderm component of the placenta and yolk sac, umbilical cord, and embryo proper) (Figure 32.3A). In a broader sense, the lineage restrictions in chimeras could be considered lineage marking.

Cell and Tissue Grafting The classical chimera production restricts the components to the sources discussed in the previous paragraph and the timing to the preimplantation stages. Later stage cell mixing is also possible, but it is more demanding and the possible applications are more limited. Small tissue transplantation can be performed between two postimplantation stage embryos. If the graft is tagged with a unique identifier, the fate of these cells can be followed during development of the recipient. This technique has been in practice for almost 20 years. Developmental trajectories, as they are depicted in Figure 32.1, are dynamic processes not only at the level of gene expression but also at the level of cell allocations in the developing embryo. These two levels interact: allocation can induce gene expression changes, and gene expression changes can influence cell allocations. Knowing the complexity of mammalian gastrulation, one can easily imagine the heroic effort of creating the map of cell movements in the early

FIGURE 32.4 Diagram of lineage contributions in different kinds of aggregation chimeras (Nagy and Rossant, 2000). Solid blacks and grays indicate nonchimeric tissues, and stripes indicate chimeric tissues.

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postimplantation embryo using mostly LacZ-marked tissue grafting and still pictures showing the allocation of the graft derivatives (Davidson et al., 1999). Further limitations associated with this approach come from our inability to successfully place the postimplantation-stage embryo back into the uterus environment where it could develop further. In vitro postimplantation embryo culture sets a limitation on the “developmental stage window” of isolation from 6.5 dpc to 10.5 dpc and on survival for a period of only 2e3 days.

Using Genetic Switches Grafting identifier-tagged tissues from one embryo to another creates a small population of progenitor cells whose derivatives can be easily recognized at later stages. The experimental achievement of such a scenario is rather invasive, and it is full of limitations, as it was pointed out in the previous paragraph. State-of-the-art tools, however, can be used to achieve a similar situation with much less invasiveness. The key element is the Cre site-specific recombinase, which recombines DNA between two consensus loxP sites. If two loxP sites are similarly oriented in the same DNA strand, the result of such a recombination is the excision of the intervening sequence. This property allows the design of the Cre excision-conditional reporter Rosa26R, a knock-in transgenic mouse line that uses the widespread expression from the Rosa26 locus, where now the LacZ expression is Cre excision conditional (Soriano, 1999; Mao et al., 1999). A similar reporter line has been developed for the other popular recombinase system: the Flp (Awatramani et al., 2001). The features of a sophisticated “switch reporter,” the Z/EG transgene (Novak et al., 2000), are the following: a strong overall expressor promoter (CMV enhancer þ Chicken b-actin promoter; Niwa et al., 1991) is driving a lacZ-neomycin resistance fusion gene (bgeo) followed by three polyadenilation (pA) sites. The bgeo and the 3xpAs are flanked by loxP sites, and a coding region of an enhanced green fluorescent protein (EGFP) with its own pA follows this flanked region. This transgene was inserted as a single copy into a single site of the mouse genome leading to the expression of the bgeo only. However, EGFP expression can be activated by Cre recombinase; Cre activity removes bgeoþ3xpA and moves the EGFP coding region under the transcriptional control of the promoter (Figure 32.3F). The EGFP activation is, therefore, dependent on the presence and action of the Cre recombinase. Z/AP, another reporter line (Lobe et al., 1999; Soriano, 1999; Mao et al., 1999; Novak et al., 2000), can be used in the same way as Z/EG, except that the second reporter is hPLAP instead of EGFP. Cre recombinase has had a great career in mouse genetics (Nagy, 2000). Dozens of transgenic mouse lines have been produced, expressing this phage enzyme with

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different spatial and temporal control (http://www.mshri. on.ca/nagy/Cre-pub.html). Double transgenic combination of a Cre transgene and the Z/EG (or Z/AP) reporter turns on EGFP (hPLAP) expression not only in the actual Cre expressor cells but also in any cell that had an ancestor express the recombinase during the course of development. Therefore, the EGFPþ cells in these double transgenic embryos or animals are the union of all the cells expressing the recombinase and those that had a Cre recombinase expressor ancestor (Figure 32.3EeH). It is easy to recognize that an efficient Cre recombinase excision is not always advantageous in this situation, since the resulting set of EGFPþ (hPLAPþ) cells could be too large, obscuring the recognition of fates derived from a single Creþ progenitor. A less efficient Cre recombinase is preferable here. If the excision (activation of the second reporter) only occurs sporadically, the allocation of the descendent cells may still reflect the clonality (Figure 32.3C) and reveal the potential of the progenitor. Low-frequency activation of the Cre recombinase can be achieved by the use of either the tamoxifen- (Hayashi and McMahon, 2002; Metzger and Chambon, 2001; Metzger et al., 1995) or the tetracyclineinducible (St. Onge et al., 1996) Cre recombinase systems. Proper titering of the inducer could create the most informative excisioneactivation frequency of progenitors. In addition, withdrawal of the inducer may be used to create an upper developmental limit of the excisioneactivation of the reporter (Perl et al., 2002; Lindeberg et al., 2002). The induction of low-frequency or controlled activation of the conditional reporter is also possible by direct injection of the recombinase (Joshi et al., 2002; Jo et al., 2001) or its mRNA into cells of developing embryos.

FUTURE DIRECTIONS It is difficult to predict what the distant future will bring into this exciting area. In the near future, however, efforts will certainly lead to novel features that will provide means to follow the behavior of cells. New imaging techniques will allow the recording of the dynamic property of cells, such as movements, speed, direction, interactions with other cells in in vitro cultures, or formation of specific embryonic structures in ex vivo cultures of early postimplantation embryos (Jones et al., 2002). The study of later development may require slice cultures for filming cellular events, such as the birth of neurons from radial glia cells (Miyata et al., 2001). Another technical development, ultrasound-guided embryonic transplantation, is expected to have an effect on lineage studies and fate mapping. It is becoming possible to graft reporter-tagged cells into early postimplantation-stage embryos (Liu et al., 1998) while in utero and to recover the embryos at a later stage for studying the derivatives of the graft.

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Remembering the enormous effort behind generating the complete map of lineage development in C. elegans e which consists of less than 1000 cells e the task of untangling the same system in the mammalian embryo may seem impossible. This conclusion comes not only from the dramatic increase in cell number but also from the increased plasticity of higher organisms. Plasticity creates a stochastic component in differentiation and cell determination processes. Therefore, the lineage studies and fate mapping have to take this uncertainty component into account. Nevertheless, the increasing understanding of lineage determination and differentiation will place us in a good position to control these processes both in vivo and in vitro. ES cells have been playing an important role in this process. The acquired knowledge, in return, promotes the development of technologies aiming controlled in vitro differentiation of ES cells into therapeutically useful cell types.

ACKNOWLEDGMENTS I am grateful to Patrick Tam for useful discussion and to Kristina Vintersten for very valuable comments and help to finalize this chapter.

REFERENCES Araki, K., Imaizumi, T., Okuyama, K., Oike, Y., Yamamura, K., 1997. Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. J. Biochem. (Tokyo) 122, 977e982. Awatramani, R., Soriano, P., Mai, J.J., Dymecki, S., 2001. An Flp indicator mouse expressing alkaline phosphatase from the ROSA26 locus. Nat. Genet. 29, 257e259. Belteki, G., Gertsenstein, M., Ow, D.W., Nagy, A., 2003. Site-specific cassette exchange and germ line transmission with mouse ES cells expressing phiC31 integrase. Nat. Biotechnol. 21, 321e324. Clarke, H.J., Varmuza, S., Prideaux, V.R., Rossant, J., 1988. The development potential of parthenogenetically derived cells in chimeric mouse embryos: implications for action of imprinted genes. Development 104, 175e182. Collazo, A., Bronner-Fraser, M., Fraser, S.E., 1993. Vital dye labeling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration. Development 118, 363e376. Cruz, Y.P., Pedersen, R.A., 1985. Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers. Dev. Biol. 112, 73e83. Damert, A., Miquerol, L., Gertsenstein, M., Risau, W., Nagy, A., 2002. Insufficient VEGFA activity in yolk sac endoderm compromises hematopoietic and endothelial differentiation. Development 129, 1881e1892. Davidson, B.P., Camus, A., Tam, P.P.L., 1999. In: Moody, S.A. (Ed.), Cell Lineage and Fate Determination. Academic Press, San Diego, pp. 491e504.

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Lineage Marking

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