Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), a Wnt pathway member, has been previously recognised as a stem cell marker in numerous epithelial tissues. In this study, we used Lgr5-EGFP-CreERT2 mice to analyse the distribution of LGR5-positive cells during craniofacial development. LGR5 expressing cells were primarily located in the mesenchyme adjacent to the craniofacial epithelial structures undergoing folding, such as the nasopharyngeal duct, lingual groove, and vomeronasal organ. To follow the fate of LGR5-positive cells, we performed lineage tracing using an inducible Cre knock-in allele in combination with Rosa26-tdTomato reporter mice. The slight expansion of LGR5-positive cells was found around the vomeronasal organ, in the nasal cavity, and around the epithelium in the lingual groove. However, most LGR5 expressing cells remained in their original location, possibly supporting their signalling function for adjacent epithelium rather than exerting their role as progenitor cells for the craniofacial structures. Moreover, Lgr5 knockout mice displayed distinct defects in LGR5-positive areas, especially in the reduction of the nasopharyngeal duct, the alteration of the palatal shelves shape, abnormal epithelial folding in the lingual groove area, and the disruption of salivary gland development. The latter defect manifested as an atypical number and localisation of the glandular ducts. The gene expression of several Wnt pathway members (Rspo1-3, Axin2) was altered in Lgr5-deficient animals. However, the difference was not found in sorted EGFP-positive cells obtained from Lgr5 +/+ and Lgr5 -/- animals. Expression profiling of LGR5-positive cells revealed the expression of several markers of mesenchymal cells, antagonists, as well as agonists, of Wnt signalling, and molecules associated with the basal membrane. Therefore, LGR5-positive cells in the craniofacial area represent a very specific population of mesenchymal cells adjacent to the epithelium undergoing folding or groove formation. Our results indicate a possible novel role of LGR5 in the regulation of morphogenetic processes during the formation of complex epithelial structures in the craniofacial areas, a role which is not related to the stem cell properties of LGR5-positive cells as was previously defined for various epithelial tissues.

Cellular Imaging Core Facility Central European Institute of Technology Masaryk University Brno Czechia

Department of Experimental Biology Faculty of Science Masaryk University Brno Czechia

Laboratory of Cell and Developmental Biology Institute of Molecular Genetics Czech Academy of Sciences Prague Czechia

Laboratory of Genomics and Bioinformatics Institute of Molecular Genetics Czech Academy of Sciences Prague Czechia

Laboratory of Molecular Morphogenesis Institute of Animal Physiology and Genetics Czech Academy of Sciences Brno Czechia

Zobrazit více v PubMed

Acevedo A. C., Da Fonseca J. A. C., Grinham J., Doudney K., Gomes R. R., De Paula L. M., et al. (2010). Autosomal-dominant ankyloglossia and tooth number anomalies. J. Dent. Res. 89 (2), 128–132. 10.1177/0022034509356401 PubMed DOI

Bahar Halpern K., Massalha H., Zwick R. K., Moor A. E., Castillo-Azofeifa D., Rozenberg M., et al. (2020). Lgr5+ telocytes are a signaling source at the intestinal villus tip. Nat. Commun. 11 (1), 1936. 10.1038/s41467-020-15714-x PubMed DOI PMC

Barker N., Clevers H. (2010). Leucine-rich repeat-containing G-protein-coupled receptors as markers of adult stem cells. Gastroenterology 138 (5), 1681–1696. 10.1053/j.gastro.2010.03.002 PubMed DOI

Barker N., Van Es J. H., Kuipers J., Kujala P., Van Den Born M., Cozijnsen M., et al. (2007). Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449 (7165), 1003–1007. 10.1038/nature06196 PubMed DOI

Barker N., Huch M., Kujala P., van de Wetering M., Snippert H. J., van Es J. H., et al. (2010). Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro . Cell Stem Cell 6 (1), 25–36. 10.1016/j.stem.2009.11.013 PubMed DOI

Barker N., Tan S., Clevers H. (2013). Lgr proteins in epithelial stem cell biology. Dev. (Cambridge) 140 (12), 2484–2494. 10.1242/dev.083113 PubMed DOI

Boddupally K., Wang G., Chen Y., Kobielak A. (2016). Lgr5 marks neural crest derived multipotent oral stromal stem cells. Stem Cells 34 (3), 720–731. 10.1002/stem.2314 PubMed DOI

Bolte S., Cordelieres F. P. (2006). A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224 (3), 213–232. 10.1111/j.1365-2818.2006.01706.x PubMed DOI

Bramhall N. F., Shi F., Arnold K., Hochedlinger K., Edge A. S. (2014). Lgr5-positive supporting cells generate new hair cells in the postnatal cochlea. Stem Cell Rep. 2 (3), 311–322. 10.1016/j.stemcr.2014.01.008 PubMed DOI PMC

Carmon K. S., Gong X., Lin Q., Thomas A., Liu Q. (2011). R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc. Natl. Acad. Sci. U. S. A. 108 (28), 11452–11457. 10.1073/pnas.1106083108 PubMed DOI PMC

Carmon K. S., Gong X., Yi J., Wu L., Thomas A., Moore C. M., et al. (2017). LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1-Rac1 pathway. J. Biol. Chem. 292 (36), 14989–15001. 10.1074/jbc.M117.786798 PubMed DOI PMC

Darken R. S., Scola A. M., Rakeman A. S., Das G., Mlodzik M., Wilson P. A. (2002). The planar polarity gene strabismus regulates convergent extension movements in Xenopus. EMBO J. 21 (5), 976–985. 10.1093/emboj/21.5.976 PubMed DOI PMC

de Lau W., Barker N., Low T. Y., Koo B. K., Li V. S. W., Teunissen H., et al. (2011). Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476 (7360), 293–297. 10.1038/nature10337 PubMed DOI

De Visser K. E., Ciampricotti M., Michalak E. M., Tan D. W. M., Speksnijder E. N., Hau C. S., et al. (2012). Developmental stage-specific contribution of LGR5+ cells to basal and luminal epithelial lineages in the postnatal mammary gland. J. Pathol. 228 (3), 300–309. 10.1002/path.4096 PubMed DOI

Emmerson E., Knox S. M. (2018). Salivary gland stem cells: a review of development, regeneration and cancer. Genesis 56 (5), e23211. 10.1002/dvg.23211 PubMed DOI PMC

Ewels P. A., Peltzer A., Fillinger S., Patel H., Alneberg J., Wilm A., et al. (2020). The nf-core framework for community-curated bioinformatics pipelines. Nat. Biotechnol. 38, 276–278. 10.1038/s41587-020-0439-x PubMed DOI

Gaillard D., Shechtman L. A., Millar S. E., Barlow L. A. (2019). Fractionated head and neck irradiation impacts taste progenitors, differentiated taste cells, and Wnt/β-catenin signaling in adult mice. Sci. Rep. 9 (1), 17934. 10.1038/s41598-019-54216-9 PubMed DOI PMC

Glinka A., Dolde C., Kirsch N., Huang Y. L., Kazanskaya O., Ingelfinger D., et al. (2011). LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling. EMBO Rep. 12 (10), 1055–1061. 10.1038/embor.2011.175 PubMed DOI PMC

Haegebarth A., Clevers H. (2009). Wnt signaling, Lgr5, and stem cells in the intestine and skin. Am. J. Pathol. 174 (3), 715–721. 10.2353/ajpath.2009.080758 PubMed DOI PMC

Hao H. X., Xie Y., Zhang Y., Charlat O., Oster E., Avello M., et al. (2012). ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485 (7397), 195–200. 10.1038/nature11019 PubMed DOI

Hermey G., Methner A., Schaller H. C., Hermans-Borgmeyer I. (1999). Identification of a novel seven-transmembrane receptor with homology to glycoprotein receptors and its expression in the adult and developing mouse. Biochem. Biophys. Res. Commun. 254 (1), 273–279. 10.1006/bbrc.1998.9882 PubMed DOI

Howe K. L., Achuthan P., Allen J., Allen J., Alvarez-Jarreta J., Ridwan Amode M., et al. (2021). Nucleic Acids Res 49 (1), 884–891. 10.1093/nar/gkaa942 PubMed DOI

Jho E. H., Zhang T., Domon C., Joo C. K., Freund J. N., Costantini F. (2002). Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol. Cell. Biol. 22, 1172–1183. 10.1128/MCB.22.4.1172-1183.2002 PubMed DOI PMC

Kawasaki M., Porntaveetus T., Kawasaki K., Oommen S., Otsuka-Tanaka Y., Hishinuma M., et al. (2014). R-spondins/Lgrs expression in tooth development. Dev. Dyn. 243 (6), 844–851. 10.1002/dvdy.24124 PubMed DOI

Kikuchi A. (1999). Roles of Axin in the Wnt signalling pathway. Cell. Signal. 11 (11), 777–788. 10.1016/s0898-6568(99)00054-6 PubMed DOI

Kim K., Lu Z., Hay E. D. (2002). Direct evidence for a role of beta-catenin/LEF-1 signaling pathway in induction of EMT. Cell Biol. Int. 26 (5), 463–476. 10.1006/cbir.2002.0901 PubMed DOI

Koo B. K., Clevers H. (2014). Stem cells marked by the R-spondin receptor LGR5. Gastroenterology 147 (2), 289–302. 10.1053/j.gastro.2014.05.007 PubMed DOI

Koo B. K., Spit M., Jordens I., Low T. Y., Stange D. E., van de Wetering M., et al. (2012). Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488 (7413), 665–669. 10.1038/nature11308 PubMed DOI

Kriz V., Korinek V. (2018). Wnt, RSPO and hippo signalling in the intestine and intestinal stem cells. Genes (Basel) 9 (1), 20. 10.3390/genes9010020 PubMed DOI PMC

Kumar K. K., Burgess A. W., Gulbis J. M. (2014). Structure and function of LGR5: an enigmatic G-protein coupled receptor marking stem cells. Protein Sci. 23 (5), 551–565. 10.1002/pro.2446 PubMed DOI PMC

Lee J. H., Tammela T., Hofree M., Choi J., Marjanovic N. D., Han S., et al. (2017). Anatomically and functionally distinct lung mesenchymal populations marked by Lgr5 and Lgr6. Cell 170 (6), 1149–1163. 10.1016/j.cell.2017.07.028 PubMed DOI PMC

Lenormand A., Khonsari R., Corre P., Perrin J. P., Boscher C., Nizon M., et al. (2018). Familial autosomal dominant severe ankyloglossia with tooth abnormalities. Am. J. Med. Genet. A 176 (7), 1614–1617. 10.1002/ajmg.a.38690 PubMed DOI

Leung C., Murad K. B. A., Tan A. L. T., Yada S., Sagiraju S., Bode P. K., et al. (2020). Lgr5 marks adult progenitor cells contributing to skeletal muscle regeneration and sarcoma formation. Cell Rep. 33 (12), 108535. 10.1016/j.celrep.2020.108535 PubMed DOI

Lewis J. E., Wahl J. K., Sass K. M., Jensen P. J., Johnson K. R., Wheelock M. J. (1997). Cross-talk between adherens junctions and desmosomes depends on plakoglobin. J. Cell Biol. 136 (4), 919–934. 10.1083/jcb.136.4.919 PubMed DOI PMC

Liebner S., Cattelino A., Gallini R., Rudini N., Iurlaro M., Piccolo S., et al. (2004). Beta-catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse. J. Cell Biol. 166 (3), 359–367. 10.1083/jcb.200403050 PubMed DOI PMC

Love M. I., Huber W., Anders S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550. 10.1186/s13059-014-0550-8 PubMed DOI PMC

Lu L., Li Y., Du M. J., Zhang C., Zhang X. Y., Tong H. Z., et al. (2015). Characterization of a self-renewing and multi-potent cell population isolated from human minor salivary glands. Sci. Rep. 5, 10106. 10.1038/srep10106 PubMed DOI PMC

Luo J., Yang Z., Ma Y., Yue Z., Lin H., Qu G., et al. (2016). LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption. Nat. Med. 22 (5), 539–546. 10.1038/nm.4076 PubMed DOI

Madisen L., Zwingman T. A., Sunkin S. M., Oh S. W., Zariwala H. A., Gu H., et al. (2010). A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13 (1), 133–140. 10.1038/nn.2467 PubMed DOI PMC

Mohamed O. A., Clarke H. J., Dufort D. (2004). Beta-catenin signaling marks the prospective site of primitive streak formation in the mouse embryo. Dev. Dyn. 231 (2), 416–424. 10.1002/dvdy.20135 PubMed DOI

Morita H., Mazerbourg S., Bouley D. M., Luo C-W., Kawamura K., Kuwabara Y., et al. (2004). Neonatal lethality of LGR5 null mice is associated with ankyloglossia and gastrointestinal distension. Mol. Cell. Biol. 24 (22), 9736–9743. 10.1128/MCB.24.22.9736-9743.2004 PubMed DOI PMC

Nagano K. (2019). R-spondin signaling as a pivotal regulator of tissue development and homeostasis. Jpn. Dent. Sci. Rev. 55 (1), 80–87. 10.1016/j.jdsr.2019.03.001 PubMed DOI PMC

Nawshad A., Hay E. D. (2003). TGFbeta3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development. J. Cell Biol. 163 (6), 1291–1301. 10.1083/jcb.200306024 PubMed DOI PMC

Nawshad A., LaGamba D., Hay E. D. (2004). Transforming growth factor beta (TGFbeta) signalling in palatal growth, apoptosis and epithelial mesenchymal transformation (EMT). Arch. Oral Biol. 49 (9), 675–689. 10.1016/j.archoralbio.2004.05.007 PubMed DOI

Nihara J., Kawasaki M., Kawasaki K., Yamada A., Meguro F., Kudo T., et al. (2021). Expression of R-spondins/Lgrs in development of movable craniofacial organs. Gene Expr. Patterns. 41, 119195. 10.1016/j.gep.2021.119195 PubMed DOI

Nusse R. (2022). Wnt target genes. Available at: https://web.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes (Accessed 7 15, 2022).

Patro R., Duggal G., Love M. I., Irizarry R. A., Kingsford G. (2017). Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419. 10.1038/nmeth.4197 PubMed DOI PMC

Schneider C. A., Rasband W. S., Eliceiri K. W. (2012). NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. 10.1038/nmeth.2089 PubMed DOI PMC

Stirling D. R., Swain-Bowden M. J., Lucas A. M., Carpenter A. E., Cimini B. A., Goodman A. (2021). CellProfiler 4: improvements in speed, utility and usability. BMC Bioinforma. 22 (1), 433. 10.1186/s12859-021-04344-9 PubMed DOI PMC

Szenker-Ravi E., Altunoglu U., Leushacke M., Bosso-Lefèvre C., Khatoo M., Thi Tran H., et al. (2018). RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6. Nature 557 (7706), 564–569. 10.1038/s41586-018-0118-y PubMed DOI

Tan C., Costello P., Sanghera J., Dominguez D., Baulida J., de Herreros A. G., et al. (2001). Inhibition of integrin linked kinase (ILK) suppresses beta-catenin-Lef/Tcf-dependent transcription and expression of the E-cadherin repressor, snail, in APC-/- human colon carcinoma cells. Oncogene 20 (1), 133–140. 10.1038/sj.onc.1204052 PubMed DOI

Wang D., Cai C., Dong X., Yu Q. C., Zhang X. O., Yang L., et al. (2015). Identification of multipotent mammary stem cells by protein C receptor expression. Nature 517 (7532), 81–84. 10.1038/nature13851 PubMed DOI

Wansleeben C., Meijlink F. (2011). The planar cell polarity pathway in vertebrate development. Dev. Dyn. 240 (3), 616–626. 10.1002/dvdy.22564 PubMed DOI

Wodarz A., Nusse R. (1998). Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14, 59–88. 10.1146/annurev.cellbio.14.1.59 PubMed DOI

Yee K. K., Li Y., Redding K. M., Iwatsuki K., Margolskee R. F., Jiang P. (2013). Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue. Stem Cells 31, 992–1000. 10.1002/stem.1338 PubMed DOI PMC