Lineage-tracing methods and the kidney
Authors: Benjamin D. Humphreys, Derek P DiRocco
Journal: Kidney International (2013)

The kidney is a complex organ with over 30 different cell types, and understanding the lineage relationships between these cells is challenging. During nephrogenesis, a central question is how the coordinated morphogenesis, growth, and differentiation of distinct cell types leads to development of a functional organ. In mature kidney, understanding cell division and fate during injury, regeneration and aging are critical topics for understanding disease. Genetic lineage tracing offers a powerful tool to decipher cellular hierarchies in both development and disease because it allows the progeny of a single cell, or group of cells, to be tracked unambiguously. Recent advances in this field include the use of inducible recombinases, multicolor reporters, and mosaic analysis. In this review, we discuss lineage-tracing methods focusing on the mouse model system and consider the impact of these methods on our understanding of kidney biology and prospects for future application. The kidney is a complex organ with over 30 different cell types, and understanding the lineage relationships between these cells is challenging. During nephrogenesis, a central question is how the coordinated morphogenesis, growth, and differentiation of distinct cell types leads to development of a functional organ. In mature kidney, understanding cell division and fate during injury, regeneration and aging are critical topics for understanding disease. Genetic lineage tracing offers a powerful tool to decipher cellular hierarchies in both development and disease because it allows the progeny of a single cell, or group of cells, to be tracked unambiguously. Recent advances in this field include the use of inducible recombinases, multicolor reporters, and mosaic analysis. In this review, we discuss lineage-tracing methods focusing on the mouse model system and consider the impact of these methods on our understanding of kidney biology and prospects for future application. In a lineage-tracing experiment, a cell is identified by expression of a reporter gene. If that cell divides, expression of the reporter is passed on to all progeny, and migration of these marked cells can be easily measured. Typical reporter genes include β-galactosidase or a fluorescent protein. Historically, lineage tracing was used most in the field of developmental biology. In its earliest application, lineage tracing was accomplished not by the expression of a fluorescent reporter, but through direct observation of embryos by light microscopy. This allowed the generation of 'fate maps' for cells from the one-cell stage until generation of germ layers.1.Conklin E.G. The organization and cell lineage of the ascidian egg.J Acad Nat Sci Phil. 1905; 12: 1-119Google Scholar Subsequent advances in the field included application of dyes to the surface of an embryo in order to track the movement of groups of cells.2.Vogt W. Gestaltungsanalyse am Amphibienkeim mit ortlicher Vitalfarbung. II. Teil Gastrulation und Mesodermbildung bie Urodelen und Anuren.Wilhelm Roux Arch Entwicklungsmech Org. 1929; 120: 384-706Crossref Scopus (225) Google Scholar However, direct observation is limited to the study of small, transparent embryos, and as a result lipid-soluble dyes were injected into the embryo and later sectioned.3.Axelrod D. Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization.Biophys J. 1979; 26: 557-573Abstract Full Text PDF PubMed Scopus (401) Google Scholar Ultimately, the introduction of genetic markers superseded dye experiments because of their advantages: they do not leak into neighboring cells and they are typically inherited by progeny where they are expressed at levels equal to that of the parental cell. These genetic markers were originally introduced by viral infection, direct injection, or transfection. Over time, techniques for lineage tracing have evolved to the point that they now possess remarkable sensitivity and offer sufficient resolution to track single cells, in real time. The subject of this review is the most commonly used lineage-tracing approach—genetic recombination. The concept of genetic lineage tracing involves the expression of a recombinase enzyme in a cell-specific manner in order to activate the expression of a reporter gene. Cre recombinase (causes recombination of the bacteriophage P1 genome) has become an indispensible tool in the mouse model system, and it has recently been adapted for use in zebrafish.4.Nagy A. Cre recombinase: the universal reagent for genome tailoring.Genesis. 2000; 26: 99-109Crossref PubMed Scopus (960) Google Scholar,5.Hans S. Freudenreich D. Geffarth M. et al.Generation of a non-leaky heat shock-inducible Cre line for conditional Cre/lox strategies in zebrafish.Dev Dyn. 2011; 240: 108-115Crossref PubMed Scopus (76) Google Scholar Flippase is an alternate recombinase that has been used mostly in Drosophila and will not be discussed here. Cre recognizes a 34-base-pair nucleotide sequence called loxP that is not present in the mouse genome. When two loxP sites are oriented in a head-to-tail manner, Cre recombinase will excise the intervening DNA sequence while rejoining the ends together. This is very useful in turning genes on for lineage tracing. Normally, a reporter gene is located downstream of a ubiquitous promoter, such as the Rosa26 locus.6.Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat Genet. 1999; 21: 70-71Crossref PubMed Scopus (4157) Google Scholar If a strong transcriptional stop sequence, such as a triple polyadenylation sequence, is placed between two loxP sequences and located upstream of this reporter gene, this reporter gene will always be turned OFF. However, expression of Cre recombinase will cause deletion of the polyadenylation sequences, turning the reporter ON in that cell and all of its descendants. By choosing cell-specific promoters for expression of Cre and combining it with a reporter, one can mark any cell population when that promoter becomes activated. An ever-growing variety of Cre driver mouse lines are available, and they have been created by several approaches: for example, by random integration of short promoter sequences or by much larger bacterial artificial chromosome transgenes. Of the two, bacterial artificial chromosome transgenics are more likely to faithfully recapitulate the endogenous expression pattern of the gene of interest, as unknown regulatory sequences are more likely to be included on the bacterial artificial chromosome. Knock-in approaches are also useful, and these are also highly likely to recapitulate endogenous expression patterns, as all nearby DNA sequences are intact at the locus of interest. In many cases, it is useful to be able to inducibly mark a cell, and this is accomplished using a Cre enzyme fused to a modified form of the estrogen receptor (CreERt2), which causes Cre to be sequestered in the cytoplasm until it binds to its ligand tamoxifen, triggering nuclear translocation.7.Littlewood T.D. Hancock D.C. Danielian P.S. et al.A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins.Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (702) Google Scholar, 8.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: 305-318Crossref PubMed Scopus (1010) Google Scholar, 9.Feil R. Wagner J. Metzger D. et al.Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.Biochem Biophys Res Commun. 1997; 237: 752-757Crossref PubMed Scopus (733) Google Scholar Both a codon-optimized version (iCreERt2) and a codon-optimized version fused to two ERt2 domains (iERCreER) at the N and C termini are now available and offer improved expression and recombination frequency.10.Shimshek D.R. Kim J. Hubner M.R. et al.Codon-improved Cre recombinase (iCre) expression in the mouse.Genesis. 2002; 32: 19-26Crossref PubMed Scopus (290) Google Scholar,11.Casanova E. Fehsenfeld S. Lemberger T. et al.ER-based double iCre fusion protein allows partial recombination in forebrain.Genesis. 2002; 34: 208-214Crossref PubMed Scopus (57) Google Scholar Alternatively, a constitutively active Cre may be regulated by a drug-controlled promoter such as tetracycline. Tetracycline-regulated systems in particular are quite robust in mouse but are generally used for protein overexpression experiments.12.Saunders T.L. Inducible transgenic mouse models.Methods Mol Biol. 2011; 693: 103-115Crossref PubMed Scopus (44) Google Scholar They are less commonly used for lineage analysis because they may require three separate alleles in this application rather than two, making for a cumbersome (and expensive) breeding strategy. However this approach has been used with success along mouse nephron epithelia, for example, in which a Pax8-rtTA allele, which drives expression of the reverse tetracycline transactivator (rtTA) is crossed to a mouse line with an allele consisting of a tetracycline-response element driving Cre expression as well as a reporter mouse.13.Traykova-Brauch M. Schonig K. Greiner O. et al.An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.Nat Med. 2008; 14: 979-984Crossref PubMed Scopus (198) Google Scholar In this case, administration of doxycycline binds to rtTA present in Pax8-positive renal epithelial cells only, and the doxycycline-rtTA complex drives Cre expression and subsequently activation of the reporter. Among the important issues to consider before choosing an appropriate reporter is the anticipated method of detection. The reporter chosen must possess sufficient signal-to-noise ratio to enable unambiguous detection (Figure 1). The Escherichia coli lacZ gene, β-galactosidase, has been used extensively and produces an intense blue color when incubated with the substrate analog X-gal. Yet β-galactosidase does not provide cellular resolution in thin cells, because the reaction product does not entirely fill long processes but is enriched in the cell body.14.Snyder E.Y. Deitcher D.L. Walsh C. et al.Multipotent neural cell lines can engraft and participate in development of mouse cerebellum.Cell. 1992; 68: 33-51Abstract Full Text PDF PubMed Scopus (757) Google Scholar In addition, there is a lack of specific antibodies that would allow routine indirect immunofluorescence detection in all systems. Therefore, LacZ is not the best reporter to detect interstitial cells. Fluorescent reporters are now the norm, and in this case detection by epifluorescence is far superior to antibody-enhanced methods, which are subject to nonspecific binding of primary and secondary antibodies. Therefore, an adequate expression level of the reporter is critical. Although the Rosa26 locus drives strong expression of reporters in developing kidney, the expression level in adult kidney is reduced and may be inadequate (Humphreys, unpublished observations). Thus, straight Rosa26 reporters may not be ideal for experiments in adults. In such cases, reporters that have an additional CAG promoter knocked into the locus provide much stronger expression levels. Older reporters exist in which viral promoters such as CAG were generated as random integration transgenics, rather than knocked into the Rosa26 locus.15.Novak A. Guo C. Yang W. et al.Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.Genesis. 2000; 28: 147-155Crossref PubMed Scopus (725) Google Scholar These may suffer from mosaic expression as the integration locus may be inactive in certain cells, and this is an important limitation. The mT/mG reporter addresses this limitation because cells that have not undergone recombination express membrane-targeted tdTomato (mT), but after recombination they express membrane-targeted enhanced green fluorescent protein (eGFP) (mG, Figure 1a). In this way, mT expression verifies that cells of interest are capable of expressing the reporter allele.16.Muzumdar M.D. Tasic B. Miyamichi K. et al.A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2265) Google Scholar Fluorophores with strong epifluorescence also improve signal-to-noise ratio. Although eGFP and its variants continue to be quite useful, a new generation of fluorophores offer much brighter fluorescence. The most highly fluorescent reporter currently available is a tandem dimmer of Tomato (tdTomato), which is itself a mutagenized version of DsRed. tdTomato is nearly three times brighter than eGFP, is nontoxic to cells, and is photostable.17.Shaner N.C. Lin M.Z. McKeown M.R. et al.Improving the photostability of bright monomeric orange and red fluorescent proteins.Nat Methods. 2008; 5: 545-551Crossref PubMed Scopus (752) Google Scholar A Rosa26 knock-in reporter mouse expressing tdTomato is available, in which a strong CAG promoter has been inserted to increase expression levels18.Niwa H. Yamamura K. Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4590) Google Scholar and a woodchuck hepatitis virus post-transcriptional regulatory element is present in order to further enhance mRNA stability (Figure 2).19.Madisen L. Zwingman T.A. Sunkin S.M. et al.A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (3866) Google Scholar These mice can be maintained as homozygotes. The primary weakness of this line is that tdTomato fluorescence is so strong that it can easily bleed into the eGFP channel. Nearly any kidney cell type can now be targeted through widely available Cre drivers and reporter strains, but it remains critical to characterize each system in order to interpret results accurately. For example, mosaic expression of Cre in target tissues can lead to incomplete recombination and false-negative results. This may either be due to silencing of the transgene in a subset of cells or due to low absolute expression levels. An even more serious problem is unfaithful expression outside of the cell type that should express Cre, causing false-positive results. Many Cre drivers are created with short transgenic promoters, and these may have unexpected expression patterns ('promiscuous expression') owing to local effects of the integration site, which is random. In an extreme example of this phenomenon, 24 separate founders for one individual transgene consisting of regulatory elements for the thy1 gene had 24 different expression patterns.20.Feng G. Mellor R.H. Bernstein M. et al.Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.Neuron. 2000; 28: 41-51Abstract Full Text Full Text PDF PubMed Scopus (2421) Google Scholar Fortunately, online databases that document Cre driver characterization are becoming available, and these can be of great assistance in planning an experiment (Table 1).21.Murray S.A. Eppig J.T. Smedley D. et al.Beyond knockouts: cre resources for conditional mutagenesis.Mamm Genome. 2012; 23: 587-599Crossref PubMed Scopus (47) Google Scholar,22.Heffner C.S. Herbert Pratt C. Babiuk R.P. et al.Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource.Nat Commun. 2012; 3: 1218Crossref PubMed Scopus (151) Google ScholarTable 1Cre resourcesaAs of May 2013.21.Murray S.A. Eppig J.T. Smedley D. et al.Beyond knockouts: cre resources for conditional mutagenesis.Mamm Genome. 2012; 23: 587-599Crossref PubMed Scopus (47) Google Scholar,22.Heffner C.S. Herbert Pratt C. Babiuk R.P. et al.Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource.Nat Commun. 2012; 3: 1218Crossref PubMed Scopus (151) Google ScholarResource nameNumber of Cre lines characterizedWeb addressMouse Genome Informatics CrePortal1920http://www.creportal.orgGene Expression Nervous System Atlas Cre project258http://www.gensat.org/CrePipeline.jspAllen Brain Atlas215http://connectivity.brain-map.orgNIH Neuroscience Blueprint Cre Driver Network310http://www.credrivermice.orgInternational Mouse Strain Resource920http://www.findmice.orga As of May 2013. Open table in a new tab An important limitation of inducible Cre experiments is leakiness of the CreERt2. Some lines will have a basal rate of recombination even in the absence of tamoxifen. To control for this, tissues must be examined before and after the experimental manipulation from mice that have not been exposed to tamoxifen. An added complication is the potential toxicity of tamoxifen itself. For example, tamoxifen is toxic to the gastric epithelium and liver at high doses.23.Huh W.J. Khurana S.S. Geahlen J.H. et al.Tamoxifen induces rapid, reversible atrophy, and metaplasia in mouse stomach.Gastroenterology. 2012; 142: e27Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar,24.Lee M.H. Kim J.W. Kim J.H. et al.Gene expression profiling of murine hepatic steatosis induced by tamoxifen.Toxicol Lett. 2010; 199: 416-424Crossref PubMed Scopus (32) Google Scholar When administered during pregnancy, tamoxifen acts as a mixed estrogen agonist, and this can induce late-term abortions. One workaround is to administer half the dosage of progesterone along with tamoxifen in this setting.25.Nakamura E. Nguyen M.T. Mackem S. Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton.Dev Dyn. 2006; 235: 2603-2612Crossref PubMed Scopus (201) Google Scholar Genetic lineage analysis has allowed fundamental discoveries in kidney development, homeostasis, and disease. With regard to kidney development, a fate-marking study by Mugford et al.26.Mugford J.W. Sipila P. McMahon J.A. et al.Osr1 expression demarcates a multi-potent population of intermediate mesoderm that undergoes progressive restriction to an Osr1-dependent nephron progenitor compartment within the mammalian kidney.Dev Biol. 2008; 324: 88-98Crossref PubMed Scopus (237) Google Scholar used an OSR1-CreERt2 allele to show that intermediate mesoderm gives rise to the great majority of cell types in the metanephric kidney, including ureteric and metanephric epithelium, interstitial stromal cells, vasculature, and smooth muscle. Anatomic fate-mapping techniques in chick also demonstrate that kidney stromal lineage, which later becomes FoxD1+, is also specified before the formation of the metanephric kidney.27.Guillaume R. Bressan M. Herzlinger D. Paraxial mesoderm contributes stromal cells to the developing kidney.Dev Biol. 2009; 329: 169-175Crossref PubMed Scopus (48) Google Scholar In another experiment from the McMahon group, clonal analysis of Six2+ cap mesenchyme cells provided direct proof that these are multipotent, self-renewing kidney stem cells. Kobayashi et al.28.Kobayashi A. Valerius M.T. Mugford J.W. et al.Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.Cell Stem Cell. 2008; 3: 169-181Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar created a Six2-CreERt2 driver and administered submaximal tamoxifen in order to label single-cap mesenchyme cells. After kidney development was completed, labeled progeny from this single cell could be observed not only in cap mesenchyme but also in the podocyte, proximal, and distal tubule lineages. Similar conclusions were drawn using a Cited1-CreERt2-based lineage tracing strategy.29.Boyle S. Misfeldt A. Chandler K.J. et al.Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia.Dev Biol. 2008; 313: 234-245Crossref PubMed Scopus (219) Google Scholar More recently, Barker et al.30.Barker N. Rookmaaker M.B. Kujala P. et al.Lgr5(+ve) stem/progenitor cells contribute to nephron formation during kidney development.Cell Rep. 2012; 2: 540-552Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar used an inducible genetic strategy to show that Lgr5+ cells within the S-shaped body are an intratubular progenitor population that gives rise to the thick ascending limb and distal convoluted tubule. Genetic recombination is particularly useful in defining cellular hierarchies in adult kidney after injury or stress, because the influx of inflammatory cells and transient state changes (i.e., dedifferentiation) complicate accurate cellular identification by antigen- or marker-based approaches in these settings. An early example of this was provided by Moeller et al.31.Moeller M.J. Soofi A. Hartmann I. et al.Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.J Am Soc Nephrol. 2004; 15: 61-67Crossref PubMed Scopus (158) Google Scholar, who genetically labeled podocytes and induced crescentic glomerulonephritis. The results indicated that cellular crescents are in part populated by podocytes themselves. More recently, lineage analysis showed that parietal cells also contribute to crescent formation, as well as to the formation of focal and segmental glomerulosclerosis lesions.32.Smeets B. Uhlig S. Fuss A. et al.Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells.J Am Soc Nephrol. 2009; 20: 2604-2615Crossref PubMed Scopus (203) Google Scholar,33.Smeets B. Kuppe C. Sicking E.M. et al.Parietal epithelial cells participate in the formation of sclerotic lesions in focal segmental glomerulosclerosis.J Am Soc Nephrol. 2011; 22: 1262-1274Crossref PubMed Scopus (176) Google Scholar Finally, recent studies implicate glomerular parietal cells as a niche for podocyte progenitors. Genetically labeled parietal cells at postnatal day 5 were shown to migrate onto the glomerular tuft and become podocytes in adult mice, providing direct evidence for a new cellular source of podocytes.34.Appel D. Kershaw D.B. Smeets B. et al.Recruitment of podocytes from glomerular parietal epithelial cells.J Am Soc Nephrol. 2009; 20: 333-343Crossref PubMed Scopus (388) Google Scholar A growing body of work has documented epithelial hierarchies in acute kidney disease and chronic kidney fibrosis models using lineage analysis. The approach demonstrated that bone marrow cells do not meaningfully contribute to the epithelium after acute injury, for example, although low levels of cell fusion can be detected.35.Lin F. Moran A. Igarashi P. Intrarenal cells, not bone marrow-derived cells, are the major source for regeneration in postischemic kidney.J Clin Invest. 2005; 115: 1756-1764Crossref PubMed Scopus (386) Google Scholar,36.Duffield J.S. Park K.M. Hsiao L.L. et al.Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells.J Clin Invest. 2005; 115: 1743-1755Crossref PubMed Scopus (528) Google Scholar We have used fate mapping to establish that the cells responsible for repair of damaged nephron epithelial originate from within the tubule and not from any extratubular compartment.37.Humphreys B.D. Valerius M.T. Kobayashi A. et al.Intrinsic epithelial cells repair the kidney after injury.Cell Stem Cell. 2008; 2: 284-291Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar In kidney fibrosis, our fate-mapping approach showed that interstitial myofibroblasts did not originate from epithelial cells through epithelial to mesenchymal transition, but rather they originate from endogenous kidney pericytes and interstitial fibroblasts.38.Humphreys B.D. Lin S.L. Kobayashi A. et al.Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.Am J Pathol. 2010; 176: 85-97Abstract Full Text Full Text PDF PubMed Scopus (1093) Google Scholar These results have been substantiated using separate genetic fate-mapping approaches by other groups.39.Koesters R. Kaissling B. Lehir M. et al.Tubular overexpression of transforming growth factor-beta1 induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells.Am J Pathol. 2010; 177: 632-643Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar Lineage tracing has also provided insight concerning the identification and plasticity of erythropoietin-producing cells in kidney interstitium and the origins of renal vasculature. Gomez used a renin-Cre line to show that renin-expressing cells give rise to multiple lineages during development, and those cells retain the capacity to synthesize renin during stress.40.Sequeira Lopez M.L. Pentz E.S. Nomasa T. et al.Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar More recently, Asada et al. provided lineage-tracing evidence that erythropoietin-producing cells originate from the neural crest, and upon injury differentiate into myofibroblasts while they lose the capacity for erythropoietin production. Notably, erythropoietin production could be rescued by the administration of dexamethasone or neuroprotective agents, suggesting a novel strategy to treat the anemia of chronic kidney disease.41.Asada N. Takase M. Nakamura J. et al.Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.J Clin Invest. 2011; 121: 3981-3990Crossref PubMed Scopus (268) Google Scholar With sparse genetic recombination, visualization of cellular substructures is made possible. We recently showed that tertiary podocyte foot processes can be easily detected by light microscopy using such a strategy.42.Grgic I. Brooks C.R. Hofmeister A.F. et al.Imaging of podocyte foot processes by fluorescence microscopy.J Am Soc Nephrol. 2012; 23: 785-791Crossref PubMed Scopus (28) Google Scholar The increasing sensitivity and fidelity of genetic reporters has made genetic recombination well suited for live-cell imaging. For example, Shan et al.43.Shan J. Jokela T. Skovorodkin I. et al.Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development.Differentiation. 2010; 79: 57-64Crossref PubMed Scopus (36) Google Scholar used kidney organ culture from a Wnt4-GFPCre; Rosa26YFP reporter cross to show differentiation and expansion of Wnt4-positive renal vesicle and tubules. Similar strategies can be used to target most populations in developing kidney, and the ability of kidney to develop in culture makes it uniquely suited for these analyses.44.Costantini F. Watanabe T. Lu B. et al.Imaging kidney development.Cold Spring Harbor Protocols. 2011; 2011: 468-474Google Scholar Live imaging of podocytes from isolated glomeruli, in which a podocin-Cre driver activated the expression of the mT/mG reporter, has been used to demonstrate rhythmic glomerular contractions ex vivo.45.Hohne M. Ising C. Hagmann H. et al.Light microscopic visualization of podocyte ultrastructure demonstrates oscillating glomerular contractions.Am J Pathol. 2013; 182: 332-338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The Barasch group coupled the expression of a gene that is induced by kidney injury (Ngal) to a luciferase reporter and generated a powerful mouse model in which kidney injury activates luciferase expression. This was not a Cre/lox approach; however, luciferase reporters exist and provide a powerful means to assess gene expression and cell localization noninvasively.46.Woolfenden S. Zhu H. Charest A.A. Cre/LoxP conditional luciferase reporter transgenic mouse for bioluminescence monitoring of tumorigenesis.Genesis. 2009; 47: 659-666Crossref PubMed Scopus (15) Google Scholar In other tissues, the dynamic analysis of cell fate in living tissues has also been facilitated by multiphoton microscopy, which allows greater depth of penetration and reporter detection without phototoxicity.47.Livet J. Weissman T.A. Kang H. et al.Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.Nature. 2007; 450: 56-62Crossref PubMed Scopus (1355) Google Scholar Peti-Peterdi and colleagues have reported the first such study in the kidney,48.Hackl M.J. Lam L. Burford J. et al.Cell fate tracking with serial multiphoton imaging of a new podocyte confetti mouse in vivo reveals podocyte proliferation and migration.J Am Soc Nephrol. 2012; 23: SA-OR082Google Scholar and it is very likely that many more will follow. The advent of multicolor reporter alleles has had a profound impact on the stem cell field, and they are sure to have a similar effect in the kidney. The concept is straightforward: by combining multiple different fluorescent reporters with alternating lox sites, mutually exclusive excision possibilities are created and Cre is forced to 'choose' which reporters to delete. This strategy is made possible by the discovery that variant lox sequences will not recombine with the canonical loxP site, but will recombine with an identical copy of the variant. Cre recombinase will also invert a DNA segment between two inward-facing loxP sequences.49.Hoess R.H. Wierzbicki A. Abremski K. The role of the loxP spacer region in P1 site-specific recombination.Nucleic Acids Res. 1986; 14: 2287-2300Crossref PubMed Scopus (232) Google Scholar This property can be exploited by linking two reporters in tandem with the opposite DNA orientation in between loxP sites that are facing each other. As long as Cre is present, this segment will invert the reporter DNA segment repeatedly. Once Cre activity is gone (i.e., after tamoxifen is degraded in a CreERt2 approach), the DNA segment will randomly stabilize in only one orientation. An important consideration here is that in the case of a straight Cre driver whose expression is maintained at the time of analysis, more than one reporter will be expressed because the inversion will continue as long as Cre is present (Figure 3). The original demonstration of this elegant approach was by Livet et al.47.Livet J. Weissman T.A. Kang H. et al.Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.Nature. 2007; 450: 56-62Crossref PubMed Scopus (1355) Google Scholar, who developed the Brainbow2.1 reporter and used it to color-code individual neurons and their axons with up to 90 different colors. Multicolor reporters have allowed the assessment of contributions from multiple cell types, such as with mouse digit tip regeneration. Clevers and colleagues have modified the Brainbow2.1 by simplifying to only four possible colors and transferring the cassette to the Rosa26 locus downstream of the strong CAG promoter, the 'Confetti' mouse. This approach enabled examination of the individual behavior of multiple stem cells in a single niche. In the case of Lgr5-positive intestinal crypt cells, the Confetti reporter revealed that crypt stem cells do not undergo asymmetrical division, but rather symmetrical division to create two identical daughter stem cells that subsequently compete for residency in the niche.50.Snippert H.J. van der Flier L.G. Sato T. et al.Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.Cell. 2010; 143: 134-144Abstract Full Text Full Text PDF PubMed Scopus (1330) Google Scholar This paradigm-shifting finding demonstrated that intestinal stem cell fate is not hierarchical (i.e., stem cell → transit amplifying cell → differentiated cell) but stochastic. Each daughter cell has equivalent potential to assume either fate, and the fate assumed is determined by neutral competition for residency within the niche. Several possible applications for multicolor reporters can be envisioned in the kidney. These include evaluation of the clonality of various proposed progenitor pools during nephrogenesis, such as Six2+ metanephric mesenchyme, FoxD1+ mesenchyme, and HoxB7+ ureteric bud lineages. In adult, several progenitor niches are known and one can imagine multicolor lineage tracing of parietal epithelium which may hold podocyte progenitors,34.Appel D. Kershaw D.B. Smeets B. et al.Recruitment of podocytes from glomerular parietal epithelial cells.J Am Soc Nephrol. 2009; 20: 333-343Crossref PubMed Scopus (388) Google Scholar,51.Ronconi E. Sagrinati C. Angelotti M.L. et al.Regeneration of glomerular podocytes by human renal progenitors.J Am Soc Nephrol. 2009; 20: 322-332Crossref PubMed Scopus (431) Google Scholar evaluation of proliferative potential of proximal tubule after injury,52.Humphreys B.D. Czerniak S. Dirocco D.P. et al.Repair of injured proximal tubule does not involve specialized progenitors.Proc Natl Acad Sci USA. 2011; 108: 9226-9231Crossref PubMed Scopus (278) Google Scholar and interstitial mesenchymal cells some of which may be multipotent.53.Pelekanos R.A. Li J. Gongora M. et al.Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities.Stem Cell Res. 2012; 8: 58-73Crossref PubMed Scopus (47) Google Scholar A final Cre-based approach for lineage tracing involves interchromosomal recombination called mosaic analysis with double markers.54.Zong H. Espinosa J.S. Su H.H. et al.Mosaic analysis with double markers in mice.Cell. 2005; 121: 479-492Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar This technique was pioneered in mice by Liquin Luo. Fluorescent reporters in this approach are artificially split by a loxP-containing artificial intron, and reciprocally chimeric genes that each encode the opposite half of the reporter are placed at identical positions in a pair of homologous chromosomes. Neither reporter is expressed until a Cre-mediated interchromosomal recombination event occurs, which reconstitutes the functional reporters. After the cell subsequently undergoes mitosis, each allele segregates to separate daughter cells and therefore express distinct colors. A powerful advantage of this approach is that many different mosaic analysis with double marker alleles on different chromosomes now exist, and they can be coupled to a mutation of interest such that homozygous mutant cells express GFP and daughter cells that are homozygous wild type express red fluorescent protein.55.Muzumdar M.D. Luo L. Zong H. From the cover: modeling sporadic loss of heterozygosity in mice by using mosaic analysis with double markers (MADM).Proc Natl Acad Sci USA. 2007; 104: 4495-4500Crossref PubMed Scopus (58) Google Scholar Generating the appropriate crosses to execute mosaic analysis with double marker is time-consuming, and although no kidney study has yet been reported there is no other technique that allows single-cell labeling with mutant analysis. Lineage analysis is a powerful tool that is now accessible to nearly all scientists. With a multitude of new Cre driver lines available, the time when any cell type in the kidney can be fate-mapped is on the immediate horizon. These techniques promise to help identify progenitor populations for regenerative medicine and to disambiguate complex cellular relationships in the kidney. Exciting new methods including multicolor reporters and single-cell mosaic analysis will answer mechanistic questions. However, a basic understanding of the limitations of these systems remains mandatory for the proper design and interpretation of a lineage-tracing experiment.