Advertisement

Lineage Tracing Mediated by Cre-Recombinase Activity

      Introduction

      Clinical research aims to unravel mechanisms leading to disease. Looking at a disease state, frequently asked questions are when and how did cells change their native behavior and how do these cells circumvent regulation by the healthy environment? Answers to these questions can often be identified through the use of a powerful technique called lineage tracing. A cell lineage describes a single stem or progenitor cell that gives rise to progeny that can adopt differential cell fates. This cellular fate is represented through differential cell properties or migration to specific regions within an organ or the organism. Lineage tracing allows us to study these dynamic processes through visualization of the cell lineage within an organism. Lineage tracing originated in the field of developmental biology. In the early twentieth century, Walter Vogt and his colleagues developed a novel method by injection of “vital dyes” into single cells of an amphibian embryo to trace the cells’ fate during gastrulation using light microscopy (
      • Vogt W.
      Gestaltungsanalyse am Amphibienkeim mit örtlicher Vitalfärbung. II. Teil Gastrulation und Mesodermbildung bei Urodelen und Anuren.
      ). This technique was restricted by dilution of the dye following each cell division, and ultimately it was lost from the cell lineage. Although various ways to trace cells were described in past decades, advances in mouse transgenesis led to genetic lineage tracing techniques, which are now the preferred approaches. We will discuss how the Cre–loxP system can circumvent the problem of label dilution through induction of reporter gene expression in specific cell populations and how these reporter genes can be used to visualize cell lineages and analyze their behavior in vivo.
      Figure thumbnail fx1

      Basics of lineage tracing technology

      Several reporter systems are suitable for the labeling of cell lineages. In general, reporter genes are used for the visualization of a cell without undesired interference with intracellular processes that might affect the cell’s behavior in vivo. To achieve controlled expression of reporter genes, the Cre–loxP system is commonly used, which allows site-specific DNA recombination and can be induced in a space- and time-dependent manner. The two most common Cre-induced reporters are β-galactosidase (β-gal) and fluorescent proteins. The β-gal enzyme, encoded by the lacZ gene, is derived from the Escherichia coli bacterium, where it is involved in the hydrolysis of β-galactosides (
      • Jacob F.
      • Monod J.
      Genetic regulatory mechanisms in the synthesis of proteins.
      ). Cells expressing β-gal can be visualized via staining with the substrate X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside). Here, cleavage of X-gal causes a dark blue precipitate in β-gal-expressing cells and thereby allows their identification (
      • Soriano P.
      Generalized lacZ expression with the ROSA26 Cre reporter strain.
      ). The main disadvantage of the β-gal reporter system is the specific staining method of the induced cells, which requires their fixation and impedes analysis of living cells within the organism. By contrast, the advantage of a fluorescent reporter is its easy visualization. The first fluorescent reporter used for lineage tracing was the green fluorescent protein (GFP) from the jellyfish Aequorea victoria (
      • Mao X.
      • Fujiwara Y.
      • Chapdelaine A.
      • et al.
      Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain.
      ). GFP emits green fluorescent light upon exposure to UV light. Nowadays, many spectral derivates of GFP are available that allow the visualization of cells within the organism via live-cell imaging technologies and multicolor lineage tracing.
      The Cre–loxP system is commonly used to control gene expression and was the topic of a previously published Research Techniques Made Simple article (
      • Scharfenberger L.
      • Hennerici T.
      • Király G.
      • et al.
      Transgenic mouse technology in skin biology: generation of complete or tissue-specific knockout mice.
      ). Briefly, the Cre-recombinase is a site-specific DNA recombinase that mediates the deletion or inversion of a loxP site–flanked DNA region. To induce reporter gene expression, the most common method is to introduce a loxP site–flanked stop codon in front of the reporter gene. Whereas under normal conditions the expression of the reporter is prevented by the stop codon, upon Cre expression this stop codon is excised and the reporter gene is expressed, resulting in labeling of the cell. Importantly, this permanent genetic manipulation circumvents the problem of label dilution due to the constitutive reporter gene expression in the daughter cells after a cell division.

      Regulation of Cre activity

      To achieve cell population or tissue-specific Cre activation, the Cre-recombinase is expressed under the control of a promoter that is only active in the cell population of interest, whereas the reporter gene is expressed under control of a ubiquitous expressed gene such as ROSA26 or β-actin. As an example, if the Cre-recombinase is expressed under control of a hair follicle bulge stem cell–specific promoter, excision of the stop codon, and thereby reporter gene expression, is mediated only in the stem-cell population and its cell progeny (Figure 1).
      Figure thumbnail gr1
      Figure 1Lineage tracing of hair follicle bulge stem cells. Cell population–specific expression of the Cre-recombinase is achieved by placing the Cre gene under the control of a promoter specific for the cell population of interest. In cells in the bulge cell lineage (green cells), the Cre-recombinase mediates the deletion of the loxP site–flanked stop codon in front of the reporter gene and thereby induces reporter gene expression. This permanent genetic modification is passed on to the cell progeny, independent of Cre expression, and facilitates tracing of the cell lineage. In cells that are not in the bulge cell lineage (gray cell), the Cre gene is not expressed and therefore the reporter gene will not be expressed.
      If single cells need to be labeled to follow not only the cell fate decisions of whole cell populations but also single cells within the population and/or at defined developmental time points, an inducible Cre-recombinase is favorable (
      • Günschmann C.
      • Chiticariu E.
      • Garg B.
      • et al.
      Transgenic mouse technology in skin biology: inducible gene knockout in mice.
      ). Here, the Cre activity can be temporally regulated on the transcriptional or posttranscriptional level. The expression of the Cre-recombinase can be transcriptionally controlled by the Tet-On/Off system. Using the Tet-Off system, the gene of interest is expressed under control of the tetracycline response element, consisting of TetO operator sequences, which are placed in front of the gene promoter. In addition, the expression of a tetracycline transactivator (tTA) protein, which is able to bind to the TetO sequence, is controlled by a promoter, which is chosen according to the cell population of interest. If tetracycline is present, it binds the tTA protein, thus impairing the binding to the TetO operator sequence and the transcription of the Cre-recombinase. Using the Tet-On system, binding of tetracycline to the tTA protein enables binding to the TetO operator sequences and induces Cre expression. tTA is responsive not only to tetracycline but also to its derivates, such as doxycycline, which is mostly used in genetic mouse models (
      • Bujard H.
      • Gossen M.
      Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
      ). To induce Cre activity in single cells, the Tet-On system is favorable to the Tet-Off system owing to its faster responsiveness and better control of dosage-dependent induction of the tTA protein in single cells. As an example,
      • Mannik J.
      • Alzayady K.
      • Ghazizadeh S.
      Regeneration of multilineage skin epithelia by differentiated keratinocytes.
      used the Tet-Off system to temporally induce reporter gene expression during keratinocyte differentiation using Cre-recombinase under the control of the involucrin promoter (Figure 2). This promoter is only active in differentiated keratinocytes residing in the spinous and granular layer of the epidermis. Using this technology, the investigators showed that differentiated keratinocytes, which were positive for the yellow fluorescent protein (YFP), can regenerate the complete epidermis, including its appendages, when transplanted onto nude mice (
      • Mannik J.
      • Alzayady K.
      • Ghazizadeh S.
      Regeneration of multilineage skin epithelia by differentiated keratinocytes.
      ).
      Figure thumbnail gr2
      Figure 2Lineage tracing example: inducible Cre-recombination via the Tet-Off system. Mannik et al. (2010) used a lineage tracing approach to demonstrate that differentiated keratinocytes are able to regenerate a fully functional epidermis when transplanted onto nude mice. The investigators induced Cre expression in differentiated keratinocytes, placing the tetracycline transactivator (tTA) protein under control of the involucrin (INV) (keratinocyte differentiation marker) promoter. Using this system, tetracycline administration activates Cre-recombinase expression and thereby the excision of a loxP site–flanked neomycin cassette (a). Through induced expression of the fluorescent reporter YFP, a yellow fluorescent protein, differentiated keratinocytes could be isolated via FACS and transplanted onto nude mice (b).
      Adapted from Mannik et al., 2010. FACS, fluorescence activated cell sorting
      For regulation of Cre activity on the posttranscriptional level, Cre can be fused to a mutated form of the human estrogen receptor (CreER). In the absence of tamoxifen, the ligand of the mutated estrogen receptor, CreER, resides in the cytoplasm as a result of binding to the heat-shock protein HSP90. In the presence of tamoxifen, the ligand binding causes a conformational change of the receptor, its release from HSP90, and the nuclear translocation of CreER, where it can recombine LoxP sites. In this way, CreER can be used to excise a loxP-flanked STOP codon in front of the reporter gene, which induces its expression. The activation of transcriptional and posttranscriptional regulated Cre-recombinases is dose dependent of tetracycline or tamoxifen, which means that low doses of the substances induce fewer cells and the induction can be optimized down to the single-cell level in specific cell populations (
      • Hayashi S.
      • McMahon A.P.
      Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.
      ). The administration of tetracycline and tamoxifen can be achieved via food, water, topical treatment, or injection.

      Advantages and limitations

      Lineage tracing enables us to gain detailed information about the migration and cell fate decisions of cell populations or single cells within their native (patho-)physiological environment, which makes it an advantageous tool. Beside the expression of a reporter gene and thereby the visualization of cell lineages within the living organism, additional gene modifications can be introduced into these lineages. Thereby we can learn whether genetic manipulation of specific genes affects the interaction of cells with their environment, migratory behavior, or intrinsic cell fate decisions during development or under homeostatic conditions. For example, the mutation or deletion of tumor suppressor genes can induce tumor formation; here lineage tracing enables the analysis of the interaction of tumor cells within the healthy tumor environment and during the metastatic processes. But although lineage tracing seems to be a straightforward technique, a detailed characterization of the Cre-recombinase and the reporter genes, in terms of both activity and specificity, is required to prevent system leakage. In addition, the expression of reporter genes and Cre-inducing agents such as tamoxifen may have a direct effect on cell behavior because of the lack of suitable controls.
      Figure thumbnail fx2

      CME ACCREDITATION

      This activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of the Duke University School of Medicine and Society for Investigative Dermatology. The Duke University School of Medicine is accredited by the ACCME to provide continuing medical education for physicians. To participate in the CME activity, follow the link provided. Physicians should only claim credit commensurate with the extent of their participation in the activity.
      To take the online quiz, follow the link below:

      SUPPLEMENTARY MATERIAL

      A PowerPoint slide presentation appropriate for journal club or other teaching exercises is available at http://dx.doi.org/10.1038/jid.2014.472.

      References

        • Bujard H.
        • Gossen M.
        Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
        Proc Natl Acad Sci USA. 1992; 89: 5547-5551
        • Günschmann C.
        • Chiticariu E.
        • Garg B.
        • et al.
        Transgenic mouse technology in skin biology: inducible gene knockout in mice.
        J Invest Dermatol. 2014; 134: e22
        • Hayashi S.
        • McMahon A.P.
        Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.
        Dev Biol. 2002; 244: 305-318
        • Jacob F.
        • Monod J.
        Genetic regulatory mechanisms in the synthesis of proteins.
        J Mol Biol. 1961; 3: 318-356
        • Mannik J.
        • Alzayady K.
        • Ghazizadeh S.
        Regeneration of multilineage skin epithelia by differentiated keratinocytes.
        J Invest Dermatol. 2010; 130: 388-397
        • Mao X.
        • Fujiwara Y.
        • Chapdelaine A.
        • et al.
        Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain.
        Blood. 2001; 97: 324-336
        • Scharfenberger L.
        • Hennerici T.
        • Király G.
        • et al.
        Transgenic mouse technology in skin biology: generation of complete or tissue-specific knockout mice.
        J Invest Dermatol. 2014; 134: e16
        • Soriano P.
        Generalized lacZ expression with the ROSA26 Cre reporter strain.
        Nat Genet. 1999; 21: 70-81
        • Vogt W.
        Gestaltungsanalyse am Amphibienkeim mit örtlicher Vitalfärbung. II. Teil Gastrulation und Mesodermbildung bei Urodelen und Anuren.
        Wilhelm Roux Arch Entwicklungsmech Org. 1929; 120: 384-706