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

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.

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.

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.

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.

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.

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.