Title: Leucine-Rich Repeat-Containing G-Protein-Coupled Receptors as Markers of Adult Stem Cells
Abstract: 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. 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. 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). Although quiescent stem cells have been shown to exist in locations such as hair follicles1Tumbar T. Guasch G. Greco V. et al.Defining the epithelial stem cell niche in skin.Science. 2004; 303: 359-363Crossref PubMed Scopus (1658) Google Scholar and bone marrow,2Kiel M.J. He S. Ashkenazi R. et al.Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU.Nature. 2007; 449: 238-242Crossref PubMed Scopus (330) Google Scholar 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.3Alison M.R. Islam S. Attributes of adult stem cells.J Pathol. 2009; 217: 144-160Crossref PubMed Scopus (111) Google Scholar 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? The Wnt signaling pathway regulates the proliferative activity of intestinal crypt cells.4Pinto D. Gregorieff A. Begthel H. et al.Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Crossref PubMed Scopus (813) Google Scholar, 5Korinek 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: 379-383Crossref PubMed Scopus (1333) Google Scholar, 6Hoffman J. Kuhnert F. Davis C.R. et al.Wnts as essential growth factors for the adult small intestine and colon.Cell Cycle. 2004; 3: 554-557Crossref PubMed Google Scholar 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.7Korinek V. Barker N. Morin P.J. et al.Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2953) Google Scholar, 8Morin P.J. Sparks A.B. Korinek V. et al.Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3524) Google Scholar 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 tumors7Korinek V. Barker N. Morin P.J. et al.Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2953) Google Scholar and its deletion in mice leads to the immediate loss of crypt stem cell compartments.5Korinek 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: 379-383Crossref PubMed Scopus (1333) Google Scholar 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.9van 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: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1752) Google Scholar This profile consists of a core of about 80 genes that are regulated by Wnt signaling.10Van der Flier L.G. Sabates-Bellver J. Oving I. et al.The intestinal Wnt/TCF signature.Gastroenterology. 2007; 132: 628-632Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar 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,11van Es J.H. Jay P. Gregorieff A. et al.Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (514) Google Scholar 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.12Barker N. van Es J.H. Kuipers J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (4076) Google Scholar These so-called crypt base columnar cells were identified originally by Leblond et al13Cheng H. Leblond C.P. 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: 537-561Crossref PubMed Google Scholar by electron microscopy more than 35 years ago, but have gone largely unstudied since.14Giannakis M. Stappenbeck T.S. Mills J.C. et al.Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches.J Biol Chem. 2006; 281: 11292-11300Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar In studies of genetic mouse models, Lgr5+ve cells were found to represent the long-lived stem cells of the small intestine and colon.12Barker N. van Es J.H. Kuipers J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (4076) Google Scholar, 15Barker N. Clevers H. Tracking down the stem cells of the intestine: strategies to identify adult stem cells.Gastroenterology. 2007; 133: 1755-1760Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar In 1998, Hsu et al16Hsu S.Y. Liang S.G. Hsueh A.J. 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: 1830-1845Crossref PubMed Google Scholar 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).16Hsu S.Y. Liang S.G. Hsueh A.J. 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: 1830-1845Crossref PubMed Google Scholar In 2000, the same investigators identified a third member of this subfamily, Lgr6.17Hsu S.Y. 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: 1257-1271Crossref PubMed Scopus (316) Google Scholar 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,18Nothacker H.P. Grimmelikhuijzen C.J. 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: 1062-1069Crossref PubMed Scopus (0) Google Scholar mollusk,19Herpin 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: 137-144Crossref PubMed Scopus (0) Google Scholar, 20Tensen C.P. Van Kesteren E.R. Planta R.J. 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: 4816-4820Crossref PubMed Scopus (0) Google Scholar the nematode Caenorhabditis elegans,21Kudo 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: 272-284Crossref PubMed Scopus (0) Google Scholar and Drosophila melanogaster.22Hauser F. Nothacker H.P. Grimmelikhuijzen C.J. 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: 1002-1010Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 23Nishi S. Hsu S.Y. 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: 4081-4090Crossref PubMed Google Scholar 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.24Vassart G. Pardo L. Costagliola S. A molecular dissection of the glycoprotein hormone receptors.Trends Biochem Sci. 2004; 29: 119-126Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar 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.24Vassart G. Pardo L. Costagliola S. A molecular dissection of the glycoprotein hormone receptors.Trends Biochem Sci. 2004; 29: 119-126Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 25Kajava A.V. Structural diversity of leucine-rich repeat proteins.J Mol Biol. 1998; 277: 519-527Crossref PubMed Scopus (450) Google Scholar 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). 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.26Vitt U.A. Hsu S.Y. Hsueh A.J. Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules.Mol Endocrinol. 2001; 15: 681-694Crossref PubMed Scopus (0) Google Scholar 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 3.27Hsu S.Y. Nakabayashi K. Nishi S. et al.Activation of orphan receptors by the hormone relaxin.Science. 2002; 295: 671-674Crossref PubMed Scopus (688) Google Scholar, 28Kumagai J. Hsu S.Y. Matsumi H. et al.INSL3/Leydig insulin-like peptide activates the Lgr8 receptor important in testis descent.J Biol Chem. 2002; 277: 31283-31286Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar 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.28Kumagai J. Hsu S.Y. Matsumi H. et al.INSL3/Leydig insulin-like peptide activates the Lgr8 receptor important in testis descent.J Biol Chem. 2002; 277: 31283-31286Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 29Baker J.D. Truman J.W. Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program.J Exp Biol. 2002; 205: 2555-2565Crossref PubMed Google Scholar Cloning of rickets revealed that it encodes the glycoprotein hormone receptor Drosophila Lgr2,29Baker J.D. Truman J.W. Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program.J Exp Biol. 2002; 205: 2555-2565Crossref PubMed Google Scholar 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.30Honegger H.W. Market D. Pierce L.A. et al.Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle-sclerotizing neurohormone.J Comp Neurol. 2002; 452: 163-177Crossref PubMed Scopus (35) Google Scholar Three follow-up studies31Dewey E.M. McNabb S.L. Ewer J. et al.Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading.Curr Biol. 2004; 14: 1208-1213Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 32Luo C.W. Dewey E.M. 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: 2820-2825Crossref PubMed Scopus (204) Google Scholar, 33Mendive F.M. 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: 2171-2176Crossref PubMed Scopus (130) Google Scholar 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).32Luo C.W. Dewey E.M. 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: 2820-2825Crossref PubMed Scopus (204) Google Scholar 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.34Avsian-Kretchmer O. Hsueh A.J. Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists.Mol Endocrinol. 2004; 18: 1-12Crossref PubMed Scopus (193) Google Scholar 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. 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 characterized35Mazerbourg S. Bouley D.M. 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: 2241-2254Crossref PubMed Scopus (126) Google Scholar, 36Van 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: 35-50Crossref PubMed Scopus (69) Google Scholar (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).37Leighton P.A. Mitchell K.J. Goodrich L.V. et al.Defining brain wiring patterns and mechanisms through gene trapping in mice.Nature. 2001; 410: 174-179Crossref PubMed Scopus (341) Google Scholar Van Schoore et al36Van 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: 35-50Crossref PubMed Scopus (69) Google Scholar 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. Mazerbourg et al35Mazerbourg S. Bouley D.M. 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: 2241-2254Crossref PubMed Scopus (126) Google Scholar 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.35Mazerbourg S. Bouley D.M. 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: 2241-2254Crossref PubMed Scopus (126) Google Scholar In a follow-up study, Mendive et al38Mendive F. Laurent P. Van Schoore G. et al.Defective postnatal development of the male reproductive tract in Lgr4 knockout mice.Dev Biol. 2006; 290: 421-434Crossref PubMed Scopus (112) Google Scholar 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.38Mendive F. Laurent P. Van Schoore G. et al.Defective postnatal development of the male reproductive tract in Lgr4 knockout mice.Dev Biol. 2006; 290: 421-434Crossref PubMed Scopus (112) Google Scholar The investigators concluded that these reproductive tract defects resulted from a loss of Lgr4 regulation of c-AMP–dependent estrogen receptor-α activity.39Li X.Y. Lu Y. Sun H.Y. et al.G protein-coupled receptor 48 upregulates estrogen receptor alpha expression via cAMP/PKA signaling in the male reproductive tract.Development. 2010; 137: 151-157Crossref PubMed Scopus (44) Google Scholar Another mouse line, with the gene trap allele Lgr4Gt(pU-21)1Kymm, was generated by Hoshii et al40Hoshii T. Takeo T. Nakagata N. et al.Lgr4 regulates the postnatal development and integrity of male reproductive tracts in mice.Biol Reprod. 2007; 76: 303-313Crossref PubMed Scopus (80) Google Scholar (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.41Yamashita R. Takegawa Y. Sakumoto M. et al.Defective development of the gall bladder and cystic duct in Lgr4- hypomorphic mice.Dev Dyn. 2009; 238: 993-1000Crossref PubMed Scopus (59) Google Scholar Mice with a similar gene trap Lgr4 allele (Lgr4Gt(LST020)Byg) also were generated by Song et al.42Song 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: 36687-36697Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar 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.43Weng 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: 6081-6086Crossref PubMed Scopus (104) Google Scholar Lgr4 mutant embryos of this strain displayed transient anemia during midgestation and abnormal definitive erythropoiesis.42Song 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: 36687-36697Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Deletion of Lgr4 delayed osteoblast differentiation and mineralization, as well as postnatal bone remodeling, but not in chondrocyte proliferation and maturation.44Luo J. Zhou W. Zhou X. et al.Regulation of bone formation and remodeling by G-protein-coupled receptor 48.Development. 2009; 136: 2747-2756Crossref PubMed Scopus (144) Google Scholar Nishimori et al45Kato 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-e75Crossref PubMed Scopus (66) Google Scholar generated mice with a conditional knockout allele (Lgr4tm1.2knis) that facilitated inducible deletion of the exon encoding the 7TM domain of
Publication Year: 2010
Publication Date: 2010-05-01
Language: en
Type: review
Indexed In: ['crossref', 'pubmed']
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