Gastroenterology
Volume 138, Issue 5 , Pages 1681-1696, May 2010

Leucine-Rich Repeat-Containing G-Protein-Coupled Receptors as Markers of Adult Stem Cells

  • Nick Barker

      Affiliations

    • Corresponding Author InformationReprint requests Address requests for reprints to: Nick Barker, PhD, Hubrecht Institute, Uppsalalaan 8, 3584CT Utrecht, The Netherlands. fax: +31 30 2121801
    • ,
  • Hans Clevers

Hubrecht Institute, Uppsalalaan, Utrecht, The Netherlands; and University Medical Center Utrecht, Utrecht, The Netherlands

Received 3 February 2010; accepted 5 March 2010.

John P. Lynch and David C. Metz, Section Editors

Article Outline

Molecular markers are used to characterize and track adult stem cells. Colon cancer research has led to the identification of 2 related receptors, leucine-rich repeat–containing, G-protein–coupled receptors (Lgr)5 and Lgr6, that are expressed by small populations of cells in a variety of adult organs. Genetic mouse models have allowed the visualization, isolation, and genetic marking of Lgr5+ve and Lgr6+ve cells and provided evidence that they are stem cells. The Lgr5+ve cells were found to occupy locations not commonly associated with stem cells in the stomach, small intestine, colon, and hair follicles. A multipotent population of skin stem cells express Lgr6. Single Lgr5+ve stem cells from the small intestine and the stomach can be cultured into long-lived organoids. Further studies of these markers might reveal adult stem cell populations in additional tissues. Identification of the ligands for Lgr5 and 6 will help elucidate stem cell functions and modes of intracellular signaling.

Keywords: Lgr5, Lgr6, Stem Cell Marker, GPCR

Abbreviations used in this paper: Ascl2, Achaete scute–like 2, CBC, crypt base columnar, FSH, follicle-stimulating hormone, GFP, green fluorescent protein, Lgr, leucine-rich repeat–containing, G-protein–coupled receptor, LH, leutinizing hormone, 7TM, 7-transmembrane, TSH, thyroid-stimulating hormone

 

The maintenance and repair of adult tissues depends on resident specialized stem cells. As a minimal definition, stem cells are cells that maintain themselves over long periods of time (termed self-renewal), producing all differentiated cell types of that tissue (termed multipotency). Each adult tissue contains its own unique type(s) of dedicated stem cells. Mammals probably have many different types of stem cells; most await discovery. It is unclear if shared molecular and cell biology principles underlie the behavior of different types of adult stem cells. In addition to longevity and multipotency, stem cell biology often tacitly is assumed to involve 2 other phenomena: quiescence and asymmetric cell division. Stem cells in most rapidly renewing tissues such as intestine or stomach therefore have been proposed to divide infrequently, and, when they divide, to do so asymmetrically—division of a single stem cell generates one rapidly cycling cell and another that replaces the parent stem cell. The rapidly cycling daughter cells, also called transit-amplifying cells, are responsible for replacing damaged or exfoliated tissue. Transit-amplifying cells typically undergo a limited number of cell divisions, after which they terminally differentiate (Figure 1A).

  • View full-size image.
  • Figure 1. 

    A scheme of adult stem cell–driven tissue-renewal in organs such as intestine and stomach. (A) Stem cells concomitantly self-renew and generate rapidly dividing transit-amplifying daughter cells via asymmetric cell division. The transit-amplifying (ta) cells undergo several rounds of division before differentiating into the mature, functional cell types of the adult tissue. (B) Adult stem cells potentially can follow 3 modes of cell division: symmetric division to generate 2 stem cells, asymmetric division to generate 1 stem cell and 1 TA cell, or symmetric division to generate 2 TA cells.

Although quiescent stem cells have been shown to exist in locations such as hair follicles1 and bone marrow,2 there is no a priori reason why stem cells should be exclusively quiescent. Also, the size of stem cell populations should be stable over time, yet this does not have to be accomplished at the level of a single stem cell (ie, by obligatory asymmetric cell division). Physically defined stem cell niches could maintain stable stem cell populations and allow individual stem cells to generate 2 stem cells, 2 transit-amplifying cells, or one of each (Figure 1B).

Two experimental strategies allow for a direct demonstration of stem cell function (stemness). Molecular markers such as CD34, CD133, and nestin, alone or in combination, can be used to identify potential rare stem cells among a multitude of other cell types in a given tissue.3 These markers can be used in fluorescence-activated cell sorting of candidate stem cells, which then are cultured in vitro and/or transplanted into animals. Candidate stem cells also can be marked genetically, so that it and its offspring can be visualized over time (lineage tracing). This second approach to studying stem cell activity is particularly appealing because it does not involve any physical manipulation of the candidate cells and they can be studied in an unperturbed environment. Unfortunately, it can be applied only when a single definitive gene marker is available that distinguishes the stem cells from all other cell types.

Technologies are currently not available for the isolation or genetic marking of adult stem cells from most tissues. In such cases, quiescence (visualized by long-term retention of DNA labels) often is used as a surrogate marker of stemness. However, there are many examples of stem cells that are not quiescent. Moreover, long-lived differentiated cells also incorporate and subsequently retain DNA label during early maturation, which can complicate long-term label-retention analyses in candidate stem cells (Barker and Clevers, unpublished data). Progress in stem cell biology research therefore depends on the identification of definitive markers that can discriminate stem cells from all other cells; ideally, gene products that are coupled to the function of the stem cell. If common molecular principles underlie the functions of diverse types of stem cells, single definitive markers might be used to identify stem cells in many different types of tissues. Are leucine-rich repeat–containing, G-protein–coupled receptors (Lgr)5 and Lgr6 among these markers?

Back to Article Outline

Lgr5 Is a Wnt Target Gene in Colon Cancer 

The Wnt signaling pathway regulates the proliferative activity of intestinal crypt cells.4, 5, 6 It is therefore not surprising that mutations that activate the Wnt pathway, most notably through loss of its negative regulator, adenomatous polyposis coli (APC), have been associated with many forms of colon cancer.7, 8 Wnt signaling ultimately induces transcription of genes via transcription factors of the T-cell factor (Tcf) family. The genetic programs that are activated by Wnt/Tcf signaling are tissue-specific. Tcf4/Tcf7l2 represents the Tcf family member that mediates Wnt signaling in normal and malignant intestinal cells. It is the most dominantly expressed Tcf protein in colon tumors7 and its deletion in mice leads to the immediate loss of crypt stem cell compartments.5

The genetic program that is activated inappropriately in APC-mutant human colon cancer cells is the same as that expressed by proliferative cells in healthy crypts.9 This profile consists of a core of about 80 genes that are regulated by Wnt signaling.10 We performed histologic expression studies for each of the 80 Wnt target genes to identify those that might be expressed only in potential crypt stem cells. Although most were expressed either throughout the proliferative crypt compartment or specifically in postmitotic Paneth cells,11 expression of several genes was restricted to crypt bottoms. One of the genes that Tcf4 targets, Lgr5/Gpr49, was expressed in a unique fashion: it appeared to be specifically active in the small cycling cells that are interspersed between the Paneth cells of the small intestine.12 These so-called crypt base columnar cells were identified originally by Leblond et al13 by electron microscopy more than 35 years ago, but have gone largely unstudied since.14 In studies of genetic mouse models, Lgr5+ve cells were found to represent the long-lived stem cells of the small intestine and colon.12, 15

Back to Article Outline

The Lgr4, Lgr5, and Lgr6 Receptors 

In 1998, Hsu et al16 cloned 2 molecules that were related to the hormone receptors for thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and leutinizing hormone (LH). These receptors belong to the large, G-protein–coupled, 7-transmembrane (7TM) family of proteins. They are unique in that they have a large N-terminal extracellular (ecto-) domain that contains a series of leucine-rich repeats. In the LH-, FSH-, and TSH-receptor molecules, the ectodomain is crucial for binding of the glycoprotein hormones. The 2 novel G-protein–coupled receptors were termed leucine-rich repeat–containing, G-protein–coupled receptors -4 and -5 (Lgr4 and Lgr5).16 In 2000, the same investigators identified a third member of this subfamily, Lgr6.17 The ectodomains of Lgr4, Lgr5, and Lgr6 consist of a central array of multiple leucine-rich repeats (18 in Lgr4 and Lgr5, and 13 in Lgr6) that are flanked by N- and C-terminal cysteine-rich sequences. In comparison, only 9 leucine-rich repeats are found in the glycoprotein hormone receptors. The leucine-rich repeats of Lgr4, Lgr5, and Lgr6 each consist of 24 amino acids and show similarity to repeats found in functionally unrelated proteins such as slit, decorin, and Toll. The junctions between the ectodomain and the first transmembrane region, as well as the rhodopsin-like 7TM domains, are highly conserved between Lgr4, Lgr5, and Lgr6 (Figure 2A). These 3 receptors are of ancient evolutionary origin because homologous proteins are found in invertebrates including sea anemone,18 mollusk,19, 20 the nematode Caenorhabditis elegans,21 and Drosophila melanogaster.22, 23

  • View full-size image.
  • Figure 2. 

    Lgr5/GPR49 is an orphan, G-protein–coupled receptor related to the glycoprotein hormone receptors. (A) Predicted structure of Lgr5, comprising a large extracellular domain with multiple leucine-rich repeats that mediate ligand interaction, a 7TM domain, and an intracellular domain for signal transduction. (B) Phylogenetic relationship between Lgr5 and related family members. The ligands for Lgr4, Lgr5, and Lgr6 have not been identified.

In the glycoprotein hormone receptors, ligand-induced recognition and activation steps are performed by separate domains of the proteins. Binding of the cognate hormones involves the leucine-rich N-terminal ectodomain, which induces a conformational change in the receptor that allows the ectodomain to activate the rhodopsin-like 7TM region of the receptor.24 By analogy, the leucine-rich repeat region of Lgr4, Lgr5, and Lgr6 is predicted to adopt a horseshoe shape that provides a binding site for an unknown peptide ligand.24, 25 The 7TM serpentine portion of the Lgr proteins subsequently would translate ligand binding of peptide agonists into the activation of undefined trimeric G proteins. Downstream events likely involve the generation of second messengers such as Ca++ and/or cyclic adenosine monophosphate (cAMP).

Back to Article Outline

Ligands for Lgr4, Lgr5, and Lgr6 

To fully understand the signaling functions of the Lgr4, Lgr5, and Lgr6 orphan G-protein-coupled receptors (GPCRs), it is important to identify their elusive ligands. Some predictions can be made based on the ligands of related receptors. Phylogenetic analysis indicates that there are 3 Lgr subgroups: the LH/FSH/TSH glycoprotein hormone receptors; Lgr4, Lgr5, and Lgr6; and Lgr7 and Lgr8 (Figure 2B). The ligands of the first subgroup (LH, FSH, and TSH) are defined structurally by the presence of a cysteine knot domain that is common to a range of extracellular signaling proteins.26 Ligands for Lgr7 and Lgr8 belong to a different class: small heterodimeric peptides with homology to insulin, including the pregnancy hormone relaxin and insulin-like 3.27, 28

Studies in insects have provided insights into the nature of the Lgr4, Lgr5, and Lgr6 ligand(s). Similar to all arthropods, insects regularly can replace their cuticle (exoskeleton). Immediately after shedding the old cuticle, the neurohormone bursicon causes the hardening and darkening of the new cuticle. Flies with mutations in the gene rickets fail to initiate these processes.28, 29 Cloning of rickets revealed that it encodes the glycoprotein hormone receptor Drosophila Lgr2,29 which appears to be the fly orthologue of mammalian Lgr4, Lgr5, and Lgr6. Although rickets mutants produce the bursicon hormone, they fail to respond to it. Hence, rickets appeared to encode the bursicon receptor.

Bursicon is a neurohormone that can be isolated from insect neural tissue. In an attempt to characterize it molecularly, bursicon protein was purified from homogenates of almost 3000 nerve cords from Periplaneta americana cockroaches and microsequenced.30 Three follow-up studies31, 32, 33 showed that bursicon consists of 2 proteins, encoded by the insect genes burs and pburs (partner of burs), also termed bursicon α and β, respectively. The pburs/burs heterodimer from D melanogaster binds with high affinity and specificity to the G-protein–coupled receptor Drosophila Lgr2, stimulating cAMP signaling in vitro. In vivo, tanning of the exoskeleton could be induced by injection of bursicon in blowflies in which endogenous bursicon action was blocked by tying a lace around the neck of the insect. The burs/pburs subunit genes encode cysteine-knot domain proteins (the same class of proteins to which FSH, LH, and TSH belong).32 These proteins are similar to vertebrate bone morphogenetic protein antagonists of the CAN subfamily, such as gremlin, protein related to DAN and Cerberus (PRDC), and Cerberus.34 The possibility therefore exists that the elusive ligands of mammalian Lgr4, Lgr5, and Lgr6 are present within the large family of bone morphogenetic protein antagonists.

Both the availability of these ligands and specific loss-of-function models for the individual Lgr genes will be needed to elucidate their in vivo function.

Back to Article Outline

Genetic Analysis of Lgr4 Function in Mice 

Lgr4 has been better studied than Lgr5 or 6. Several mutant alleles of mouse Lgr4 have been generated and analyzed and the Lgr4 expression pattern has been well characterized35, 36 (Figure 3). A gene-trapping screen identified a mouse line (Lgr4Gt(pGTOTMpfs)1Wcs) in which the Lgr4 gene was disrupted by a gene trap vector that expressed a bacterial β-galactosidase LacZ fusion protein and placental alkaline phosphatase (Figure 3A).37 Van Schoore et al36 investigated LacZ and placental alkaline phosphatase activity patterns in heterozygous mice at macroscopic and at histologic levels. A broad expression pattern was noted, with particularly strong activity in cartilage, heart, hair follicles, kidneys, reproductive tracts, and the nervous system cells. In reviewing these data (Barker and Clevers, unpublished data), it appears that Lgr4 is expressed broadly within proliferative compartments, but definitely is not restricted to the rare stem cells within such compartments.

  • View full-size image.
  • Figure 3. 

    An overview of the Lgr4/Lgr5/Lgr6 alleles currently used for exploring expression and function, in vivo. Top panel: domain structure of the Lgr proteins. (A) Lgr4 genetrap allele disrupts endogenous Lgr4 expression and marks Lgr4+ve cells in vivo via expression of the β-galactosidase and placental alkaline phosphatase reporter genes. (B) Lgr4 genetrap allele disrupts endogenous Lgr4 expression and marks Lgr4+ve cells in vivo via expression of the β-galactosidase reporter gene. (C) Inducible Lgr4 allele facilitates inducible Lgr4 deletion in tissues expressing Cre enzyme. (D) Lgr5-LacZ allele disrupts endogenous Lgr5 expression and marks Lgr5+ve cells in vivo via expression of the β-galactosidase reporter gene. (E) Lgr5-EGFP-ires-CreERT2 allele disrupts endogenous Lgr5 expression, marks Lgr5+ve cells in vivo via expression of the EGFP reporter gene, and facilitates lineage tracing in combination with inducible reporter mice. (F) Lgr6-LacZ allele disrupts endogenous Lgr6 expression and marks Lgr6+ve cells in vivo via expression of the β-galactosidase reporter gene.

Mazerbourg et al35 observed that only 40% of Lgr4-null mice were born; most of these died within the first 2 days after birth, displaying a pleiotropic phenotype. All Lgr4-null embryos showed intrauterine growth retardation, most notably in the kidney and liver.35 In a follow-up study, Mendive et al38 reported that when the Lgr4 allele was crossed onto a different genetic background (CD1), homozygous mice survived to adulthood. In the adult Lgr4-null mice, Lgr4 regulated the postnatal tissue remodeling required for elongation and convolution of the efferent ducts and epididymis. In Lgr4-knockout male mice, tube elongation did not occur in the male reproductive tract; instead, the mice developed a hypoplastic and poorly convoluted tract with severely decreased proliferation in the affected tissue, subsequent decreases in fluid reabsorption, and severe dilation of rete testis.38 The investigators concluded that these reproductive tract defects resulted from a loss of Lgr4 regulation of c-AMP–dependent estrogen receptor-α activity.39

Another mouse line, with the gene trap allele Lgr4Gt(pU-21)1Kymm, was generated by Hoshii et al40 (Figure 3B). The homozygous mutant mice expressed 10% of normal levels of Lgr4 messenger RNA and 60% survived to adulthood. The homozygous male mice were infertile, displaying morphologic abnormalities in testes and epididymides that were similar to those of the Lgr4-knockout mice but were related to distortion of basal membranes. Lgr4Gt/Gt embryos developed a normal gall bladder bud, but the bladder bud did not undergo elongation beyond midgestation. The Lgr4Gt/Gt mice did not develop a gall bladder or cystic duct, although there were no discernable effects on liver or pancreas development.41

Mice with a similar gene trap Lgr4 allele (Lgr4Gt(LST020)Byg) also were generated by Song et al.42 Mice homozygous for this allele again displayed a pleiotropic phenotype. One study described the effects on the anterior segment structure of the eye, which included microphthalmia, iris hypoplasia, iridocorneal angle malformation, cornea dysgenesis, and cataracts.43 Lgr4 mutant embryos of this strain displayed transient anemia during midgestation and abnormal definitive erythropoiesis.42 Deletion of Lgr4 delayed osteoblast differentiation and mineralization, as well as postnatal bone remodeling, but not in chondrocyte proliferation and maturation.44

Nishimori et al45 generated mice with a conditional knockout allele (Lgr4tm1.2knis) that facilitated inducible deletion of the exon encoding the 7TM domain of Lgr4 when the mice were crossed to strains of mice with tissue-specific expression of Cre (Figure 3C). Embryonic deletion of this exon using mice expressing Cre in all tissues resulted in embryonic/perinatal lethality. Neonatal, Lgr4-null mice had renal hypoplasia that was accompanied by a notable decrease in the total number of glomeruli.45 Jin et al46, 47 also observed that mutant pups were born with opened eyes, an abnormality that might result from a defect in motility of keratinocytes in the skin. They confirmed this eye-open phenotype by conditional deletion of Lgr4 using mice that expressed Cre specifically in skin.46 These Lgr4K5 KO mice survived to adulthood, with sparse head hair and focal alopecia behind their ears. Lgr4−/− mice showed similar abnormalities in hair follicles, leading to the conclusion that Lgr4 has a role in hair follicle development.48

Therefore, Lgr4 appears to be involved in the development of a wide variety of embryonic tissues, of ectodermal, mesodermal, and endodermal origin. However, little is known about Lgr4 signaling pathways.

Back to Article Outline

Lgr5 Function 

Little was known about mammalian Lgr5 before 2007. Lgr5/Gpr49 is a Wnt target gene as well as a cancer gene; it was on the original list of Wnt/Tcf4 targets that are active in colorectal cancers9 and is overexpressed in tumors of the ovary and liver, likely because of the mutational activation of the Wnt pathway in these tumors.49, 50, 51 Lgr5 expression was observed in basal cell carcinomas52 and in healthy cyclic endometrium.53

The phenotype of a mouse knockout for Lgr5 (Lgr5tm1Ah) was published by Morita et al54 in 2004 (Figure 3D). This strain harbors a lacZ reporter gene just N-terminal to the region of Lgr5 that encodes the first transmembrane domain, essentially creating a null allele. Homozygous disruption of Lgr5 resulted in neonatal lethality, characterized by ingestion of air at birth, resulting in gastrointestinal tract dilation and the absence of milk from the stomach. Macroscopic and histologic examination revealed fusion of the tongue to the floor of the oral cavity in newborns, a condition called ankyloglossia. Lgr5 was found to be expressed in the epithelium of the tongue and in the mandible of wild-type embryos. The observed phenotype indicated that Lgr5 is an essential gene, yet the lethal neonatal phenotype precluded the study of the role of Lgr5 in adult tissues. Garcia et al observed that the same Lgr5-null strain also had accelerated maturation of Paneth cells in the developing intestine.55 They also described a potential role for Lgr5 in negatively regulating Wnt signaling during neonatal development of the intestine.

Back to Article Outline

Lgr5 Marks Stem Cells in the Intestine 

The crypts of the small intestine contain functional stem cells. Although researchers agree that every crypt contains 4–6 independent stem cells, there is disagreement about their exact identity.56 The stem cells are believed to reside at position +4 (on average), relative to the crypt bottom, with positions 1–3 occupied by the terminally differentiated Paneth cells. Potten et al57, 58 provided experimental support for the +4 stem cell model, reporting that radiation-sensitive, label-retaining cells reside specifically at this position. Additional support for this model recently was provided by a B lymphoma Mo-MLV insertion region 1 homolog 1 (Bmi-1) lineage tracing approach.59 An alternative model was proposed based on the identification of crypt base columnar (CBC) cells—small cycling cells that are interspersed between the Paneth cells.60, 61 Bjerknes and Cheng62, 63 proposed that the CBC cells represent true stem cells (Figure 4A). However, until recently, neither of these hypotheses was supported by direct evidence for the stemness of these cells, assessed by transplantation or lineage tracing.

  • View full-size image.
  • Figure 4. 

    In vivo lineage tracing reveals Lgr5+ve cells to be cycling intestinal stem cells. (A) Restricted expression of Lgr5 in the CBC cells at the crypt bottom. ta, transit-amplifying. (B) Lgr5-lacZ expression in the CBC cell population of Lgr5-lacZ knock-in mouse intestine. (C) Lgr5-EGFP expression in the CBC cell population of Lgr5-EGFP-ires-CreERT2 knock-in mouse intestine. (D) Upper panel: LacZ reporter gene activity initially is activated stochastically in Lgr5+ve cells at the crypt base after Tamoxifen administration (black arrows). Lower panel: at later time points, entirely Lgr5+ve cell-derived LacZ+ve crypt/villus units are visible throughout the intestine. Lgr5 cells continue generating this lacZ+ve epithelium over the entire lifetime of the mouse. (E) Generation of the major functional cell types of the intestine from the lgr5+ve CBC stem cell population.

Two alleles of Lgr5 have been used to investigate whether its product is a marker of intestinal stem cells.12, 64 Lgr5tm1Ah mice (hereafter referred to as Lgr5-LacZ) express a LacZ reporter gene under the control of the endogenous Lgr5 promoter, facilitating a detailed analysis of Lgr5 expression in vivo.54 Heterozygous Lgr5-LacZ embryos had a dynamic and complex expression pattern of LacZ (Barker and Clevers, unpublished data). By contrast, in adult mice, LacZ expression was restricted to rare scattered cells in the eye, brain, hair follicle, mammary gland, reproductive organs, stomach, and intestinal tract. In the small intestine, we confirmed Lgr5 expression in the diminutive CBC cells between the Paneth cells (Figure 4A–C). By morphology analysis, the slender Lgr5+ve cells could be distinguished from the adjacent, terminally differentiated Paneth cells by their large nuclei and limited cytoplasm.12, 64 The CBC cells expressed the Ki67 cell-cycle marker and 5-bromo-2′-deoxy-uridine labeling studies showed that the average cycling time of CBC cells was in the order of 1 day. However, there is no evidence for the existence of quiescent Lgr5+ve cells at crypt bottoms. In colon, Lgr5 expression was observed in a handful of cells of similar shape, interspersed with cells that had a morphology similar to that of goblet cells, at the bottom of each crypt.12, 64

In a different strain of mice (Lgr5tm1(cre/ESR1)cle), a cassette that encodes green fluorescent protein (GFP) and a tamoxifen-inducible version of the Cre recombinase enzyme (CreERT2) was inserted at exon 1 of Lgr512 (Figure 3E). The GFP pattern observed in adult tissues faithfully recapitulated the Lgr5-LacZ expression pattern. By confocal imaging, GFP+ve cells could be visualized at the bottoms of the crypts of the small intestine (Figure 4C) and colon. Immunoelectron microscopy illustrated the unique ultrastructural anatomy of the GFP+ve cells.12 Typically, the CBC cells were relatively broad at their base and contained a flat, wedge-shaped nucleus and very little cytoplasm or organelles. A slender extension of apical cytoplasm extended between the neighboring Paneth cells to the crypt lumen and carried a few microvilli.

To test the Lgr5+ve cells for stemness by lineage tracing, mice with the knock-in allele were crossed with the Cre-activatable R26R-LacZ reporter strain.65 Injection of tamoxifen should activate CreERT2, resulting in Cre-mediated excision of the roadblock sequence in the R26R-LacZ reporter uniquely in the Lgr5+ve cells. This marked these cells irreversibly. Although potential progeny of the Lgr5+ve cells did not express GFP, the activated LacZ reporter served as a genetic marker. Adult mice injected with a low dose of tamoxifen activated the R26R-LacZ reporter stochastically in a limited number of Lgr5+ve cells. On day 1, some Lgr5+ve cells in the crypts of small intestine and colon expressed LacZ12 (Figure 4D, upper panel). At later time points, parallel ribbons of blue cells emanated from these marked CBC cells and moved up the flanks of adjacent villi. As predicted, the ribbons reached the villus tips 5 days after inducton. The Lgr5+ve cells were capable of long-term maintenance of the self-renewing epithelium. Even after 2 years, the frequency of blue crypts and ribbons essentially was identical to that observed in the first few weeks after induction64 (Figure 4D, lower panel).

The blue ribbons of cells observed in mouse intestines 60 days after induction included normal percentages of enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. M cells in Peyer's patches (de Lau and Clevers, unpublished data) and Tuft cells (van Es and Clevers, unpublished data) also were derived from Lgr5+ve cells (Figure 4E). By using mutational marking, Bjerknes and Cheng61 reported the existence of different types of long-lived epithelial clones, namely columnar (enterocyte) clones, mucous (goblet) clones, and mixed clones. The clones observed in our study were exclusively of the mixed variety.

We observed identical results in the colons of the Lgr5 transgenic mice. Blue clones emanated from the crypt bottom and contained colonocytes as well as goblet cells, and did not change over 2 years.64 Thus, the Lgr5+ve cells in small intestine and colon fulfill the requirements for designation as stem cells in that they self-renew and are capable of generating all cell types of the respective epithelium.

Back to Article Outline

Lgr5 Marks Stem Cells in the Stomach 

The stomach is similar to the intestine; it is of common endodermal origin and has a constantly renewing epithelium. The equivalent of the intestinal crypt is called the gastric gland (or glandular unit) in the stomach epithelium. Several of these glands feed into a pit that opens out onto the surface epithelium. The structure and composition of these gastric units varies in different anatomic regions of the stomach.66 In the pyloric region (antrum), each gastric unit is composed of several short glands that feed into a single long pit. The epithelium of these pyloric gastric units is composed mainly of mucous cells, gastrin-secreting enteroendocrine cells, and some parietal cells (which secrete hydrochloric acid and various growth factors).67, 68 In the corpus of the stomach, the gastric units are composed of several longer glands that feed into short pits. The corpus epithelium contains many parietal cells and chief cells (secreting zymogen and pepsinogen), as well as mucous cells and various types of enteroendocrine cells.69

The existence of multipotent stem cells within the glandular stomach has been shown by clonal marking studies.70, 71, 72 However, the unavailability of specific endogenous markers has hampered the definitive identification of these cells. Gastric stem cells are believed to reside in a region between the gland and the pit termed the isthmus, because immature cells and cellular proliferation are most predominant at this location.73, 74, 75, 76, 77 Gastric units in the pyloric and corpus region are considered to be functionally monoclonal (ie, all cellular progeny are derived from a single stem cell).71, 78, 79

We observed restricted expression of Lgr5 in the stomach epithelium of both Lgr5tm1Ah and Lgr5tm1(cre/ESR1)cle mice. Is Lgr5 a bona fide marker for adult stem cells in the stomach? In neonatal mice, Lgr5 expression occurred within shallow indentations, the prospective glands, throughout the epithelium of the developing stomach. In adult mice, Lgr5 expression was confined strictly to the base of the glands in the pyloric region and throughout the lesser curvature of the distal stomach, toward the gastric–esophageal junction and along the border of the corpus and squamous forestomach (Figure 5A). Lgr5 expression was essentially absent from the corpus region after the first few weeks of life.

  • View full-size image.
  • Figure 5. 

    In vivo lineage tracing reveals Lgr5+ve cells to be cycling stomach stem cells. (A) Restricted expression of Lgr5 in 3–4 cells at the base of the pyloric glands. (B) Left panel: LacZ reporter gene activity initially is activated stochastically in Lgr5+ve cells at the gland base after Tamoxifen administration (black arrows). Right panel: at later time points, entirely Lgr5+ve cell-derived LacZ+ve glandular units are visible throughout the pyloric region. Lgr5 cells continue generating this LacZ+ve epithelium over the entire lifetime of the mouse.

By lineage tracing, it was observed that the Lgr5+ve cells represent actively dividing, multipotent stem cells that contribute to the long-term renewal of the entire gastric epithelium80 (Figure 5B and C). Several well-known Wnt-regulated genes, such as Axin2, were highly expressed in the sorted Lgr5+ve cells; this indicates active Wnt signaling in the stem cell zone at the gland base. In accordance with this observation, mutational inactivation of APC in this Lgr5+ve stem cell population rapidly initiated tumor formation in the pyloric epithelium. These observations identified the Lgr5+ve cells as self-renewing, multipotent stem cells that are responsible for the long-term renewal of the gastric epithelium. Moreover, the similarity between stomach and intestine extends the involvement of Wnt signaling as a regulator of epithelial stem cells.

The location of the Lgr5+ve stem cells at the gland base contradicts current models that favor a stem cell zone within the isthmus. Without specific markers to assess the stem cell characteristics of isthmus populations independently it is difficult to reconcile the 2 models. We speculate that the immediate progeny of the Lgr5+ve stem cells at the crypt base rapidly migrate to the isthmus, where they undergo transit amplification (similar to the transit-amplifying cells of the intestine), before undergoing terminal differentiation during their bidirectional migration toward the pit or gland.

Back to Article Outline

Lgr5 Marks Stem Cells in the Hair Follicle 

The control of self-renewal in cells of the intestinal crypts is similar to that of hair follicles; both processes are regulated by Wnt signaling.81 Although the interfollicular epidermis undergoes constant self-renewal, the hair follicles go through cyclic phases of growth, involution, and rest.82 In mice, hair follicle stem cells reside in the follicle bulge and are characterized by expression of the cell-surface marker CD34 and cytokeratin 15.83, 84, 85, 86 These stem cells are believed to be quiescent, based on their retention of chromatin labels over long periods of time.1, 87 The gene expression profiles of telogen bulge stem cells have been reported; Lgr5 was among the most highly enriched genes.85 These findings prompted studies on Lgr5 as a potential stem cell marker in skin.88

Lgr5 was expressed in limited numbers of cells in hair follicles throughout life in both the Lgr5tm1Ah and Lgr5tm1(cre/ESR1)cle mice (Figure 6A). Unexpectedly, the Lgr5-expressing cells were actively cycling, at least during the growth phase of the hair follicle (anagen). The Lgr5+ve cells resided in the lower bulge and secondary germ of hair follicles in the resting phase (telogen). Lineage tracing revealed that the Lgr5+ve cells constituted a multipotent stem cell population that was able to give rise to new hair follicles and maintain all cell lineages of the hair follicle over long periods of time (Figure 6B). Sorted Lgr5+ve hair follicle stem cells express high levels of prototypic hair follicle markers such as CD34.89

  • View full-size image.
  • Figure 6. 

    In vivo lineage tracing reveals adult Lgr5+ve and Lgr6+ve cells to be cycling skin stem cells. (A) Various stem cell populations responsible for maintaining hair follicle/epidermal self-renewal in adult skin. (B) Left panel: LacZ reporter gene activity initially is activated stochastically in Lgr5+ve cells at the base of the bulge after Tamoxifen administration in Lgr5-EGFP-ires-CreERT2/Rosa-lacZ mice (black arrow). Right panel: at later time-points, Lgr5+ve cell-derived LacZ+ve hair follicles are visible throughout the skin. (C) Left panel: LacZ reporter gene activity initially is activated stochastically in Lgr6+ve cells above the bulge after Tamoxifen administration in Lgr6-EGFP-ires-CreERT2/Rosa-lacZ mice (black arrow). Right panel: at later time points, Lgr6+ve cell-derived LacZ+ve sebaceous glands (black arrow) and epidermis (red arrows) are visible throughout the skin. Both the Lgr5 and Lgr6 populations continue generating the lacZ+ve epithelium over the entire lifetime of the mouse.

In an independent strategy, Quigley et al90 used crosses of Mus spretus and Mus musculus strains to map locations of genetic variants.90 Combined with gene expression studies in normal skin, they generated a network view of the gene expression architecture of mouse skin. This approach identifies expression motifs that contribute to tissue organization and functions such as cell-cycle control and tumor susceptibility. This study confirmed that the stem cell marker Lgr5 is a candidate master regulator of the hair follicle.

Back to Article Outline

Lgr6 as a Marker of Adult Stem Cells in Skin 

Because Lgr5 appeared to be expressed by a variety of unrelated adult stem cells, we obtained mice with LacZ- or GFP-ires-CreERT2 inserted at Lgr4 and Lgr6, to identify cells that express these genes and to assess their stem cell potential via lineage tracing. Lgr4 does not seem to be a marker of stem cells—it is expressed more broadly and in many proliferative compartments36 (and Haegebarth et al, unpublished data). In contrast, Lgr6 expression in adult Lgr6-LacZLacZ knock-in mice (Figure 3F) was restricted to rare cells in the brain, the mammary gland, the airways of the lungs, and the hair follicle. Expression was absent throughout the entire gastrointestinal tract. Lgr6 expression in embryos was most prominent in the skin, corroborating earlier gene expression profiling studies that showed that both Lgr6 and 5 are highly enriched in late embryonic (embryonic day 17.5) hair follicle stem cells.85

In the skin, Lgr6 is first expressed in embryonic day 14.5 hair placodes. Lgr6+ve cells remain restricted to hair pegs until the growing hair penetrates the epidermis, when some Lgr6+ve cells exit into the basal layer of surrounding neonatal epidermis. In adult hair follicles, Lgr6+ve cells are located in a previously unrecognized band directly above the bulge. Immunofluorescence, DNA label retention, and microarray analyses confirmed that Lgr6+ve cells are distinct from CD34+ve and Lgr5+ve bulge stem cells. Lineage tracing studies showed that prenatal Lgr6+ve cells establish the hair follicle, sebaceous gland, and interfollicular epidermis.89 Postnatal Lgr6+ve cells primarily generate the sebaceous gland and epidermis, and their contributions to the bulge and hair diminish progressively with age. Lgr6 therefore marks a novel population of stem cells located above the bulge at the central isthmus, which gives rise to all lineages of the skin (Figure 6A and C). Unlike Lgr5, Lgr6 does not appear to be controlled by Wnt signaling. This is in agreement with the concept that the active hair lineage in the lower bulge requires Wnt signaling whereas the sebaceous and epidermal lineages are Wnt-independent (reviewed by Fuchs and Horsley91). A Wnt-independent, Lgr6+ve population of stem cells appears to renew sebaceous cells and seed the epidermis throughout life, whereas a Wnt-dependent, Lgr5+ve population is derived from the Lgr6+ve pool early in life, but then becomes relatively independent.

The Lgr6+ve skin stem cells appear to mediate wound repair. Lineage tracing of Lgr6+ve cells for 3 months after excision of back skin revealed that bulge stem cells were labeled;92 convergent bands of blue cells emanated from the border of the wound and migrated toward its center. The bands that arise from bulge stem cells previously were reported to be of a transient nature and essentially disappear by 20 days after the wound was created.92 However, the Lgr6-derived cells remained at the repaired wound, within the newly formed epidermis, for more than 3 months. Ito et al93 reported that hair follicles can form de novo within the wound epithelium. Hair follicles arose in the wound epithelium and some were derived from Lgr6+ve stem cells. Therefore, adult Lgr6+ve cells appear to make important contributions to long-term wound healing, including hair follicle regeneration within the newly formed epidermis.

Back to Article Outline

Refining Experimentation on Adult Stem Cells 

Lgr5 and 6 are unique markers in that they can be used to discriminate stem cells from their immediate, transit-amplifying daughters. Other candidate markers of intestinal stem cells, such as Musashi94 or CD133,95, 96, 97 typically are expressed in a shallower gradient along the crypt axis and a consequently broader pattern. Similarly, doublecortin and calcium/calmodulin-dependent kinase-like 1 (DCAMKL-1) is expressed on a rare population of terminally differentiated cells, called Tuft cells, distributed throughout the intestinal epithelium.98

Differential gene profiling of fluorescence-activated cell sorted Lgr5-GFPhi stem cells and their Lgr5-EGFPlo immediate daughter cells from adult Lgr5tm1(cre/ESR1)cle mouse intestines identified a limited set of stem cell–specific genes. In addition to Lgr5, this stem cell signature contained the Wnt target Achaete scute–like 2 (Ascl2).99 Transgenic expression of this transcription factor throughout the intestinal epithelium induced crypt hyperplasia and ectopic crypts on villi. Deletion of Ascl2 specifically in the adult small intestine led to disappearance of the Lgr5+ve stem cells within days. The combined results from these gain-of-function and loss-of-function experiments indicate that Ascl2 controls intestinal stem cell fate.99

There are many other interesting genes that appear in the intestinal stem cell signature. OlfM4 is expressed robustly by Lgr5+ve stem cells in mouse and human intestinal tracts, so it might be used in immunohistochemical analyses to visualize these stem cells in healthy tissues and in colon tumors.100

Single, live, stem cells can be sorted from Lgr5tm1(cre/ESR1)cle mice based on expression of GFP.101 Although a variety of culture systems have been described,102, 103, 104, 105 no long-term culture system was able to maintain the basic crypt–villus physiology.106 We developed such an intestinal culture system by combining previous in vitro–defined growth requirements, with informed assumptions based on our knowledge of intestinal epithelial renewal in vivo. Of note, we took into account the likely strict requirement for active Wnt signaling in maintaining crypt proliferation based on various in vivo observations,3, 4 and the observed crypt hyperplasia caused by overexpression of the Wnt agonist R-spondin1.107 In addition, epidermal growth factor signaling also induces intestinal cell proliferation108 and transgenic expression of Noggin increases the number of crypts.109 Finally, isolated intestinal cells undergo anoikis outside the normal tissue context, dictating the inclusion of an inhibitor.110 Laminin-rich Matrigel (BD Bioscience) was chosen as the support matrix because Laminin (a1 and a2) are enriched at the crypt base.111 By using this information, we established long-term culture conditions under which single stem cells generate crypt–villus organoids that included all differentiated cell types and maintain the basic epithelial architecture.101 In these culture conditions, intestinal crypt–villus units appear as self-organizing structures that are generated from a single stem cell, in the absence of a nonepithelial cellular niche. This technology was adapted to grow long-term stomach epithelial organoids from Lgr5+ve stomach stem cells.80 Another Wnt-dependent culture system was later described by Ootani et al112 that facilitated sustained intestinal epithelial culture from neonate intestinal fragments.

The Lgr5tm1(cre/ESR1)cle mice express a tamoxifen-inducible Cre enzyme that facilitates selective genetic modification of the Lgr5 stem cells using standard Cre-LoxP technology. This approach recently was used to identify the Lgr5+ve stem cells as the cell-of-origin of intestinal cancer.113 Cre-mediated deletion of Apc in Lgr5+ve stem cells resulted in rapid formation of growing microadenomas at crypt bottoms. These microadenomas showed unimpeded growth and developed into macroscopic adenomas within a month. The presence of Lgr5+ve cells within stem-cell–derived adenomas indicated that a stem cell/progenitor cell hierarchy was maintained in these early neoplastic lesions. By contrast, when Apc was deleted in short-lived transit-amplifying cells using a different Cre mouse, the growth of the induced microadenomas stalled rapidly.113

Back to Article Outline

Future Directions 

In all tissues in which Lgr5- and Lgr6-expressing cells have been studied by lineage tracing, these cells have been found to represent stem cells. Most attention has been restricted to tissues that display a spontaneous, high level of self-renewal activity. In many organs, expression of the 2 markers is observed during development and/or upon injury, but does not occur in the adult under normal conditions. These tissues typically have very low rates of spontaneous self-renewal (eg, pancreas, liver, kidney, ovary, and brain). By using the Lgr5 and Lgr6 mouse models, more types of stem cells likely will be discovered, in spontaneously self-renewing tissues and in tissues that regenerate after damage.

The specific expression pattern of Lgr5 and Lgr6 has allowed precise experimentation on stem cells in mouse models. Unfortunately, they are expressed at very low absolute levels in mice and human beings, which has hampered their use in fluorescence-activated cell sorting or immunohistochemical studies. Anti-Lgr5 or anti-Lgr6 antibodies have not been isolated that are specific for mouse intestine and that do not react with tissues of knockout mice; until specific antibodies are available, studies cannot be performed with human tissue samples. Additional markers might be identified in the mouse models that have the same specific expression patterns but are expressed at higher levels. Such markers, such as OlfM4, would simplify clinical studies of Lgr5+ve stem cells. It is expressed specifically in Lgr5+ve cells in human small intestine and colon (cancer), but is not in nonintestinal stem cell populations.99, 100

Lgr5+ve stem cells likely will be found in tissues outside the intestinal tract and skin. Comparing Lgr5+ve and Lgr6+ve stem cells from different sources might lead to the identification of a common, minimal gene expression pattern that defines stemness. Reagents to identify Lgr5 and Lgr6 expression in healthy and diseased human tissues are needed. Identification of the ligands of Lgr5 and Lgr6 is another important goal of stem cell biology research; this would allow comparison of their activities with factors that regulate bone marrow stem cells, such as erythropoietin or granulocyte colony stimulating factor. Lgr5+ve and Lgr6+ve stem cells also might be developed for regenerative medicine strategies.

Back to Article Outline

Acknowledgments 

The authors thank Hugo Snippert for Lgr6 tracing figures; and Gilbert Vassart, Aaron Hsueh, and laboratory members for critical review of the manuscript.

Both authors contributed equally to the design and writing of this review.

Back to Article Outline

References 

  1. Tumbar T, Guasch G, Greco V, et al. Defining the epithelial stem cell niche in skin. Science. 2004;303:359–363
  2. Kiel MJ, He S, Ashkenazi R, et al. Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU. Nature. 2007;449:238–242
  3. Alison MR, Islam S. Attributes of adult stem cells. J Pathol. 2009;217:144–160
  4. Pinto D, Gregorieff A, Begthel H, et al. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 2003;17:1709–1713
  5. Korinek V, Barker N, Moerer P, et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet. 1998;19:379–383
  6. Hoffman J, Kuhnert F, Davis CR, et al. Wnts as essential growth factors for the adult small intestine and colon. Cell Cycle. 2004;3:554–557
  7. Korinek V, Barker N, Morin PJ, et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma. Science. 1997;275:1784–1787
  8. Morin PJ, Sparks AB, Korinek V, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science. 1997;275:1787–1790
  9. van de Wetering M, Sancho E, Verweij C, et al. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell. 2002;111:241–250
  10. Van der Flier LG, Sabates-Bellver J, Oving I, et al. The intestinal Wnt/TCF signature. Gastroenterology. 2007;132:628–632
  11. van Es JH, Jay P, Gregorieff A, et al. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nat Cell Biol. 2005;7:381–386
  12. Barker N, van Es JH, Kuipers J, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449:1003–1007
  13. Cheng H, Leblond CP. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine (V. Unitarian theory of the origin of the four epithelial cell types). Am J Anat. 1974;141:537–561
  14. Giannakis M, Stappenbeck TS, Mills JC, et al. Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches. J Biol Chem. 2006;281:11292–11300
  15. Barker N, Clevers H. Tracking down the stem cells of the intestine: strategies to identify adult stem cells. Gastroenterology. 2007;133:1755–1760
  16. Hsu SY, Liang SG, Hsueh AJ. Characterization of two Lgr genes homologous to gonadotropin and thyrotropin receptors with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol. 1998;12:1830–1845
  17. Hsu SY, Kudo M, Chen T, et al. The three subfamilies of leucine-rich repeat-containing G protein-coupled receptors (Lgr): identification of Lgr6 and Lgr7 and the signaling mechanism for Lgr7. Mol Endocrinol. 2000;14:1257–1271
  18. Nothacker HP, Grimmelikhuijzen CJ. Molecular cloning of a novel, putative G protein-coupled receptor from sea anemones structurally related to members of the FSH, TSH, LH/CG receptor family from mammals. Biochem Biophys Res Commun. 1993;197:1062–1069
  19. Herpin A, Badariotti F, Rodet F, et al. Molecular characterization of a new leucine-rich repeat-containing G protein-coupled receptor from a bivalve mollusc: evolutionary implications. Biochim Biophys Acta. 2004;1680:137–144
  20. Tensen CP, Van Kesteren ER, Planta RJ, et al. A G protein-coupled receptor with low density lipoprotein-binding motifs suggests a role for lipoproteins in G-linked signal transduction. Proc Natl Acad Sci U S A. 1994;91:4816–4820
  21. Kudo M, Chen T, Nakabayashi K, et al. The nematode leucine-rich repeat-containing, G protein-coupled receptor (Lgr) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells. Mol Endocrinol. 2000;14:272–284
  22. Hauser F, Nothacker HP, Grimmelikhuijzen CJ. Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals. J Biol Chem. 1997;272:1002–1010
  23. Nishi S, Hsu SY, Zell K, et al. Characterization of two fly Lgr (leucine-rich repeat-containing, G protein-coupled receptor) proteins homologous to vertebrate glycoprotein hormone receptors: constitutive activation of wild-type fly Lgr1 but not Lgr2 in transfected mammalian cells. Endocrinology. 2000;141:4081–4090
  24. Vassart G, Pardo L, Costagliola S. A molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci. 2004;29:119–126
  25. Kajava AV. Structural diversity of leucine-rich repeat proteins. J Mol Biol. 1998;277:519–527
  26. Vitt UA, Hsu SY, Hsueh AJ. Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules. Mol Endocrinol. 2001;15:681–694
  27. Hsu SY, Nakabayashi K, Nishi S, et al. Activation of orphan receptors by the hormone relaxin. Science. 2002;295:671–674
  28. Kumagai J, Hsu SY, Matsumi H, et al. INSL3/Leydig insulin-like peptide activates the Lgr8 receptor important in testis descent. J Biol Chem. 2002;277:31283–31286
  29. Baker JD, Truman JW. Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program. J Exp Biol. 2002;205:2555–2565
  30. Honegger HW, Market D, Pierce LA, et al. Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle-sclerotizing neurohormone. J Comp Neurol. 2002;452:163–177
  31. Dewey EM, McNabb SL, Ewer J, et al. Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading. Curr Biol. 2004;14:1208–1213
  32. Luo CW, Dewey EM, Sudo S, et al. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor Lgr2. Proc Natl Acad Sci U S A. 2005;102:2820–2825
  33. Mendive FM, Van Loy T, Claeysen S, et al. Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLgr2. FEBS Lett. 2005;579:2171–2176
  34. Avsian-Kretchmer O, Hsueh AJ. Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists. Mol Endocrinol. 2004;18:1–12
  35. Mazerbourg S, Bouley DM, Sudo S, et al. Leucine-rich repeat-containing, G protein-coupled receptor 4 null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality. Mol Endocrinol. 2004;18:2241–2254
  36. Van Schoore G, Mendive F, Pochet R, et al. Expression pattern of the orphan receptor Lgr4/GPR48 gene in the mouse. Histochem Cell Biol. 2005;124:35–50
  37. Leighton PA, Mitchell KJ, Goodrich LV, et al. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature. 2001;410:174–179
  38. Mendive F, Laurent P, Van Schoore G, et al. Defective postnatal development of the male reproductive tract in Lgr4 knockout mice. Dev Biol. 2006;290:421–434
  39. Li XY, Lu Y, Sun HY, et al. G protein-coupled receptor 48 upregulates estrogen receptor alpha expression via cAMP/PKA signaling in the male reproductive tract. Development. 2010;137:151–157
  40. Hoshii T, Takeo T, Nakagata N, et al. Lgr4 regulates the postnatal development and integrity of male reproductive tracts in mice. Biol Reprod. 2007;76:303–313
  41. Yamashita R, Takegawa Y, Sakumoto M, et al. Defective development of the gall bladder and cystic duct in Lgr4- hypomorphic mice. Dev Dyn. 2009;238:993–1000
  42. Song H, Luo J, Luo W, et al. Inactivation of G-protein-coupled receptor 48 (Gpr48/Lgr4) impairs definitive erythropoiesis at midgestation through down-regulation of the ATF4 signaling pathway. J Biol Chem. 2008;283:36687–36697
  43. Weng J, Luo J, Cheng X, et al. Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2. Proc Natl Acad Sci U S A. 2008;105:6081–6086
  44. Luo J, Zhou W, Zhou X, et al. Regulation of bone formation and remodeling by G-protein-coupled receptor 48. Development. 2009;136:2747–2756
  45. Kato S, Matsubara M, Matsuo T, et al. Leucine-rich repeat-containing G protein-coupled receptor-4 (Lgr4, Gpr48) is essential for renal development in mice. Nephron Exp Nephrol. 2006;104:e63–e75
  46. Kato S, Mohri Y, Matsuo T, et al. Eye-open at birth phenotype with reduced keratinocyte motility in Lgr4 null mice. FEBS Lett. 2007;581:4685–4690
  47. Jin C, Yin F, Lin M, et al. GPR48 regulates epithelial cell proliferation and migration by activating EGFR during eyelid development. Invest Ophthalmol Vis Sci. 2008;49:4245–4253
  48. Mohri Y, Kato S, Umezawa A, et al. Impaired hair placode formation with reduced expression of hair follicle-related genes in mice lacking Lgr4. Dev Dyn. 2008;237:2235–2242
  49. McClanahan T, Koseoglu S, Smith K, et al. Identification of overexpression of orphan G protein-coupled receptor GPR49 in human colon and ovarian primary tumors. Cancer Biol Ther. 2006;5:419–426
  50. Yamamoto Y, Sakamoto M, Fujii G, et al. Overexpression of orphan G-protein-coupled receptor, Gpr49, in human hepatocellular carcinomas with beta-catenin mutations. Hepatology. 2003;37:528–533
  51. Zucman-Rossi J, Benhamouche S, Godard C, et al. Differential effects of inactivated Axin1 and activated beta-catenin mutations in human hepatocellular carcinomas. Oncogene. 2007;26:774–780
  52. Tanese K, Fukuma M, Yamada T, et al. G-protein-coupled receptor GPR49 is up-regulated in basal cell carcinoma and promotes cell proliferation and tumor formation. Am J Pathol. 2008;173:835–843
  53. Krusche CA, Kroll T, Beier HM, et al. Expression of leucine-rich repeat-containing G-protein-coupled receptors in the human cyclic endometrium. Fertil Steril. 2007;87:1428–1437
  54. Morita H, Mazerbourg S, Bouley DM, et al. Neonatal lethality of Lgr5 null mice is associated with ankyloglossia and gastrointestinal distension. Mol Cell Biol. 2004;24:9736–9743
  55. Garcia MI, Ghiani M, Lefort A, et al. Lgr5 deficiency deregulates Wnt signaling and leads to precocious Paneth cell differentiation in the fetal intestine. Dev Biol. 2009;331:58–67
  56. Barker N, van de Wetering M, Clevers H. The intestinal stem cell. Genes Dev. 2008;22:1856–1864
  57. Potten CS. Extreme sensitivity of some intestinal crypt cells to X and gamma irradiation. Nature. 1977;269:518–521
  58. Potten CS, Kovacs L, Hamilton E. Continuous labelling studies on mouse skin and intestine. Cell Tissue Kinet. 1974;7:271–283
  59. Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet. 2008;40:915–920
  60. Cheng H, Leblond CP. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine (I. Columnar cell). Am J Anat. 1974;141:461–479
  61. Bjerknes M, Cheng H. Clonal analysis of mouse intestinal epithelial progenitors. Gastroenterology. 1999;116:7–14
  62. Bjerknes M, Cheng H. The stem-cell zone of the small intestinal epithelium (I. Evidence from Paneth cells in the adult mouse). Am J Anat. 1981;160:51–63
  63. Bjerknes M, Cheng H. The stem-cell zone of the small intestinal epithelium (III. Evidence from columnar, enteroendocrine, and mucous cells in the adult mouse). Am J Anat. 1981;160:77–91
  64. Barker N, van Es JH, Jaks V, et al. Very long-term self-renewal of small intestine, colon, and hair follicles from cycling Lgr5+ve stem cells. Cold Spring Harb Symp Quant Biol. 2008;73:351–356
  65. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–71
  66. Lee ER, Trasler J, Dwivedi S, et al. Division of the mouse gastric mucosa into zymogenic and mucous regions on the basis of gland features. Am J Anat. 1982;164:187–207
  67. Lee ER, Leblond CP. Dynamic histology of the antral epithelium in the mouse stomach: IV. Ultrastructure and renewal of gland cells. Am J Anat. 1985;172:241–259
  68. Lee ER, Leblond CP. Dynamic histology of the antral epithelium in the mouse stomach: II. Ultrastructure and renewal of isthmal cells. Am J Anat. 1985;172:205–224
  69. Karam SM, Leblond CP. Identifying and counting epithelial cell types in the “corpus” of the mouse stomach. Anat Rec. 1992;232:231–246
  70. Bjerknes M, Cheng H. Multipotential stem cells in adult mouse gastric epithelium. Am J Physiol Gastrointest Liver Physiol. 2002;283:G767–G777
  71. McDonald SA, Greaves LC, Gutierrez-Gonzalez L, et al. Mechanisms of field cancerization in the human stomach: the expansion and spread of mutated gastric stem cells. Gastroenterology. 2008;134:500–510
  72. Qiao XT, Ziel JW, McKimpson W, et al. Prospective identification of a multilineage progenitor in murine stomach epithelium. Gastroenterology. 2007;133:1989–1998
  73. Hattori T, Fujita S. Tritiated thymidine autoradiographic study on cellular migration in the gastric gland of the golden hamster. Cell Tissue Res. 1976;172:171–184
  74. Karam SM, Leblond CP. Dynamics of epithelial cells in the corpus of the mouse stomach (V. Behavior of entero-endocrine and caveolated cells: general conclusions on cell kinetics in the oxyntic epithelium). Anat Rec. 1993;236:333–340
  75. Karam SM, Leblond CP. Dynamics of epithelial cells in the corpus of the mouse stomach (III. Inward migration of neck cells followed by progressive transformation into zymogenic cells). Anat Rec. 1993;236:297–313
  76. Karam SM, Leblond CP. Dynamics of epithelial cells in the corpus of the mouse stomach (II. Outward migration of pit cells). Anat Rec. 1993;236:280–296
  77. Karam SM, Leblond CP. Dynamics of epithelial cells in the corpus of the mouse stomach (I. Identification of proliferative cell types and pinpointing of the stem cell). Anat Rec. 1993;236:259–279
  78. Nomura S, Esumi H, Job C, et al. Lineage and clonal development of gastric glands. Dev Biol. 1998;204:124–135
  79. Tatematsu M, Fukami H, Yamamoto M, et al. Clonal analysis of glandular stomach carcinogenesis in C3H/HeN<==>BALB/c chimeric mice treated with N-methyl-N-nitrosourea. Cancer Lett. 1994;83:37–42
  80. Barker N, Huch M, Kujala P, et al. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell. 2010;6:25–36
  81. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature. 2005;434:843–850
  82. Alonso L, Fuchs E. The hair cycle. J Cell Sci. 2006;119:391–393
  83. Blanpain C, Lowry WE, Geoghegan A, et al. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell. 2004;118:635–648
  84. Cotsarelis G, Sun TT, Lavker RM. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell. 1990;61:1329–1337
  85. Morris RJ, Liu Y, Marles L, et al. Capturing and profiling adult hair follicle stem cells. Nat Biotechnol. 2004;22:411–417
  86. Trempus CS, Morris RJ, Bortner CD, et al. Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol. 2003;120:501–511
  87. Braun KM, Niemann C, Jensen UB, et al. Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in whole mounts of mouse epidermis. Development. 2003;130:5241–5255
  88. Jaks V, Barker N, Kasper M, et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet. 2008;40:1291–1299
  89. Snippert HJ, Kasper M, Jaks V, et al. Lgr6 stem cells generate all cell lineages of the skin. Science. 2010;327:1385–1389
  90. Quigley DA, To MD, Perez-Losada J, et al. Genetic architecture of mouse skin inflammation and tumour susceptibility. Nature. 2009;458:505–508
  91. Fuchs E, Horsley V. More than one way to skin. Genes Dev. 2008;22:976–985
  92. Ito M, Liu Y, Yang Z, et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med. 2005;11:1351–1354
  93. Ito M, Yang Z, Andl T, et al. Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding. Nature. 2007;447:316–320
  94. Potten CS, Booth C, Tudor GL, et al. Identification of a putative intestinal stem cell and early lineage marker; musashi-1. Differentiation. 2003;71:28–41
  95. Zhu L, Gibson P, Currle DS, et al. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature. 2009;457:603–607
  96. Snippert HJ, van Es JH, van den Born M, et al. Prominin-1/CD133 marks stem cells and early progenitors in mouse small intestine. Gastroenterology. 2009;136:2187e1–2194e1
  97. Montgomery RK, Shivdasani RA. Prominin1 (CD133) as an intestinal stem cell marker: promise and nuance. Gastroenterology. 2009;136:2051–2054
  98. Gerbe F, Brulin B, Makrini L, et al. DCAMKL-1 expression identifies Tuft cells rather than stem cells in the adult mouse intestinal epithelium. Gastroenterology. 2009;137:2179–2181
  99. Van der Flier LG, van Gijn ME, Hatzis P, et al. Transcription factor Achaete scute-like 2 (Ascl2) controls intestinal stem cell fate. Cell. 2009;136:903–912
  100. van der Flier LG, Haegebarth A, Stange DE, et al. OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells. Gastroenterology. 2009;137:15–17
  101. Sato T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459:262–265
  102. Evans GS, Flint N, Somers AS, et al. The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J Cell Sci. 1992;101:219–231
  103. Fukamachi H. Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture. J Cell Sci. 1992;103:511–519
  104. Perreault N, Jean-Francois B. Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures. Exp Cell Res. 1996;224:354–364
  105. Whitehead RH, Demmler K, Rockman SP, et al. Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans. Gastroenterology. 1999;117:858–865
  106. Bjerknes M, Cheng H. Intestinal epithelial stem cells and progenitors. Methods Enzymol. 2006;419:337–383
  107. Kim KA, Kakitani M, Zhao J, et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science. 2005;309:1256–1259
  108. Dignass AU, Sturm A. Peptide growth factors in the intestine. Eur J Gastroenterol Hepatol. 2001;13:763–770
  109. Haramis AP, Begthel H, van den Born M, et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science. 2004;303:1684–1686
  110. Hofmann C, Obermeier F, Artinger M, et al. Cell-cell contacts prevent anoikis in primary human colonic epithelial cells. Gastroenterology. 2007;132:587–600
  111. Sasaki T, Giltay R, Talts U, et al. Expression and distribution of laminin alpha1 and alpha2 chains in embryonic and adult mouse tissues: an immunochemical approach. Exp Cell Res. 2002;275:185–199
  112. Ootani A, Li X, Sangiorgi E, et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med. 2009;15:701–706
  113. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature. 2009;457:608–611

 Conflicts of interest The authors disclose no conflicts.

PII: S0016-5085(10)00336-7

doi:10.1053/j.gastro.2010.03.002

Gastroenterology
Volume 138, Issue 5 , Pages 1681-1696, May 2010

Article Tools

* Image Usage & Resolution