Visualizing Cathepsin K-Cre Expression at the Single-Cell Level with GFP Reporters

Abstract

The Cre/lox system is a fundamental tool for functional genomic studies, and a number of Cre lines have been generated to target genes of interest spatially and temporally in defined cells or tissues; this approach has greatly expanded our knowledge of gene functions. However, the limitations of this system have recently been recognized, and we must address the challenge of so-called nonspecific/off-target effects when a Cre line is utilized to investigate a gene of interest. For example, cathepsin K (Ctsk) has been used as a specific osteoclast marker, and Cre driven by its promoter is widely utilized for osteoclast investigations. However, Ctsk-Cre expression has recently been identified in other cell types, such as osteocytes, periosteal stem cells, and tenocytes. To better understand Ctsk-Cre expression and ensure appropriate use of this Cre line, we performed a comprehensive analysis of Ctsk-Cre expression at the single-cell level in major organs and tissues using two green fluorescent protein (GFP) reporters (ROSA nT-nG and ROSA tdT) and a tissue clearing technique in young and aging mice. The expression profile was further verified by immunofluorescence staining and droplet digital RT-PCR. The results demonstrate that Ctsk-Cre is expressed not only in osteoclasts but also at various levels in osteoblast lineage cells and other major organs/tissues, particularly in the brain, kidney, pancreas, and blood vessels. Furthermore, Ctsk-Cre expression increases markedly in the bone marrow, skeletal muscle, and intervertebral discs in aging mice. These data will be valuable for accurately interpreting data obtained from in vivo studies using Ctsk-Cre mice to avoid potentially misleading conclusions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: AGING; CATHEPSIN K; CRE; GFPS; OSTEOBLASTS; OSTEOCLASTS.

Fig. 1

Visualization of Ctsk⁺ cells in Ctsk‐Cre⁺:tdT mice. Whole‐femur imaging was performed on frozen sections from mice aged 1 month (A) and 10 months (B), with representative areas shown at higher magnifications as indicated (×400 and ×1000). The representative areas include the growth plates (a), trabecular bones (b), cortical bones (c), and bone marrow (d), with enlarged images of osteocytes with dendrites. Arrows: white: tdT⁺ cells with more than two nuclei on the resorption bays of bone surfaces are osteoclasts; green: cuboid‐shaped tdT‐positive cells with a single nucleus on the bone surfaces are osteoblasts; empty white: osteocytes embedded within bone matrix; empty green: flat cells lining bone surface are bone lining cells. CA, cartilage; C, cortical bones; P, periosteum; BM, bone marrow. Scale bars, 500 μm for whole‐femur images with a magnification of ×40; 20 μm for representative areas at magnifications of ×400 and ×1000; and 10 μm for images of osteocytes.

Fig. 2

Capturing Ctsk expression in spine. Images were taken of spine sections from Ctsk‐Cre⁺:tdT mice aged 1 month and 10 months. The representative areas from each section are shown in the growth plate (A), IVD (B), and trabecular bone (C). Arrows: white: tdT‐positive cells with more than two nuclei on resorption bays of bone surfaces, osteoclasts; green: cuboid‐shaped tdT‐positive cells with a single nucleus on bone surfaces, osteoblasts; empty white: osteocytes embedded within bone matrix; empty green: flat cells lining bone surface, bone lining cells; and empty arrowheads: IVD. M, muscle; GP, grown plate; AF, annulus fibrosus; NP, nucleus pulposus. Scale bars, 500 μm for whole images of spines and 20 μm for magnified images of representative areas. Magnifications: ×40 for whole‐spine images; ×400 for representative images.

Fig. 3

Confocal imaging of Ctsk⁺ cells in Ctsk‐Cre⁺:nT‐nG mice. (A) Documentation of Ctsk⁺ cells (nG⁺) in femoral sections. Imaging of Ctsk⁺ cells was performed for young (A, aged 1 month) and aging mice (B, aged 8 months). The representative areas are shown as follows: a: articular cartilage; b: growth plate; c: trabecular bones; and d: cortical bone close to growth plate. White arrows indicate multinucleated nGFP‐expressing cells (mnGFPs). AC, articular cartilage; GP, grown plate; C, cortical bones; P, periosteum. Scale bars, 20 μm. Magnification: ×400. (B) Imagining Ctsk⁺ cells in whole tails. Whole tails were imaged for mice aged 1 month (a) and 8 months (b). High‐magnification images of fibrous layer from each are shown in (c, d), respectively. White arrows indicate bright nG expression under growth plates. Scale bars, 200 μm for (a, b); 20 μm for (c, d). Magnification: ×200 for (a, b) and ×400 for (c, d). (C) Alteration of Ctsk expression in intervertebral discs (IVDs) with aging. The IVDs were imaged in 1‐month‐old mice (a) and 8‐month‐old mice (b). Dotted ellipses indicate NP area. Empty white arrows indicate area of AF. AF, annulus fibrosus; NP, nucleus pulposus. Scale bars: 200 μm. Magnification: ×200.

Fig. 4

Representative images of Ctsk⁺ cells in sutures. Representative image of cranial suture of Ctsk‐Cre⁺:nT‐nG mice aged 1 month (A). Arrows: yellow: bone lining cells; blue: cuboidal nG+ cells on bone surface; white: nG⁺ cells embedded within bone matrix. Scale bars, 20 μm. Magnification: ×400. Representative images of sutures from Ctsk‐Cre⁺:tdT mice (B). tdT expression (a) and immunofluorescence staining with Sp7 antibody (b). Empty white arrows indicate Sp7‐positive cells. p, periosteum; s, suture. Scale bars, 20 μm. Magnification: ×1000.

Fig. 5

Identification of tdT⁺ cells by immunofluorescence staining. In femoral/tibial sections from Ctsk‐Cre⁺:tdT mice, a set of antibodies was applied to identify Ctsk⁺ cells, as described below. (A) Osteocalcin, showing that a cluster of tdT⁺ cells that stained positively for osteocalcin was osteoblasts in the epiphysis. (B) Osteocalcin, revealing that a group of cuboidal tdT⁺ cells that stained positively for osteocalcin was osteoblasts on the surface of trabecular bone within the bone marrow. (C) TRACP, showing multinucleated tdT⁺ cells stained positively for TRACP were osteoclasts. (D) Calcitonin R, illustrating that tdT⁺ cells stained positively for calcitonin R. (E) EMCN, demonstrating EMCN expression on bone surface, particularly areas covering surface of each individual cell. (F) CD31, indicating CD31 expression in type H vessels and osteoblasts located on bone surface of epiphysis. Arrows: green: cuboid‐shaped tdT⁺ cells are osteoblasts positive for osteocalcin; small green: cells positive for osteocalcin without tdT expression; white: multinucleated tdT⁺ cells in resorption bays that were positive for TRACP or calcitonin R were osteoclasts; empty white: osteocytes embedded within bone matrix; empty green: flat cells lining bone surface. V, vessel; BM, bone marrow. Scale bars, 20 μm. Magnification: ×1000.

Fig. 6

Ctsk⁺ cells in osteoclast or osteoblast cultures. Ctsk expression was examined in cultures of osteoclasts and osteoblasts. The mononuclear cells or mBMSCs from the BM were induced to differentiate into either osteoclasts (A) or osteoblasts (B). For osteoclast culture, bone marrow cells were harvested from Ctsk‐Cre⁺:nT‐nG mice aged 2 months and induced to differentiate into osteoclasts with M‐CSF and RANKL. In the early stage of osteoclast culture, the nGFP⁺ nuclei accumulated in the center of osteoclasts (a), and then these nuclei were found close to the cellular membrane after 2 days of further culture (b). In addition, nGFP⁺ was observed in small multinuclear osteoclasts (empty white arrows) and mononuclear cells (pink arrowheads). The yellow arrow indicates the nuclear membrane. At the end of osteoclast culture, TRACP staining was performed for 4% PFA‐fixed cells (c), showing Tracp⁺ staining of mononuclear cells (pink arrowhead) and small (white arrowhead) and large multinuclear osteoclasts. In osteoblast cultures, mBMSCs were derived from Ctsk‐Cre⁺:tdT mice aged 2 and 11 months (B). The tdT⁺ cells were detected on days 4, 8, and 16 during osteoblast differentiation culture. Scale bars, 50 μm. Magnification: ×200 for images in (A) and (B).

Fig. 7‐1

Ctsk⁺ cells in BAT. Ctsk⁺ cells were detected in either Ctsk‐Cre⁺:tdT (A) or Ctsk‐Cre⁺:nT‐nG (B) mice. The frozen sections were further stained with the Ucp1 antibody (C). In Ctsk‐Cre⁺:tdT mice aged 1 month (A), a representative area is amplified at higher magnification in the left panel. Arrowheads: white: arteriole; yellow: brown cells; empty white arrowhead: white adipose cells. In Ctsk‐Cre⁺:nT‐nG mice (B), the nT‐ and nG‐positive cells from the 1‐month (a) and 8‐month (b) groups and their comparison (c, n = 3). tdT⁺ cells were identified as brown cells by immunofluorescence staining with an anti‐Ucp1 antibody (C‐a), and macrophages were recognized by IF with an anti‐F4/80 antibody (C‐b). Arrows: wide white: tdT⁺ cells stained positively for Ucp1; white arrowheads: dendritic structure similar to peripheral nerve fibers; white: F4/80 positive staining cells without tdT expression. Scale bars, 50 μm for (A) and (B); 20 μm for (C). Magnifications: ×200 for (A) and (B); ×1000 for (C).

Fig. 7‐2

Imaging of Ctsk‐expressing cells in WAT.tdT⁺ cells in Ctsk‐Cre⁺:tdT mice (A) and Ctsk‐Cre⁺:nT‐nG mice (B). A piece of the WAT from Ctsk‐Cre⁺:tdT mice is shown at a magnification of ×40, with two areas in higher‐magnification images (×200): a: area containing more white adipocytes; b: area containing more beige cells. In Ctsk‐Cre⁺:nT‐nG mice, the Ctsk⁺ cells are recognized as nG+ cells derived from nT (B), with (a) 1‐month mice; (b) 8‐month mice, and (c) their comparison (n = 3), showing a remarkable increase in the number of nT+ cells during aging (n = 3). tdT⁺ beige cells were confirmed by IF with antibodies to identify beige cells, macrophages, and endothelial cells, respectively (C): (a) Ucp1, (b) F4/80, and (c) CD31. Arrows: white, staining of Ucp1 in tdT⁺ cells; white arrowhead, positive staining for only Ucp1 (a); white arrow: positive cells for F4/80; white arrowhead: F4/80‐stained fiber structure; yellow arrow: tdT⁺ cells, yellow arrowhead: dendrite‐shaped tdT⁺ cells (b); yellow arrowhead: arteriole; blue arrow: cells stained positive for CD31 (c). Scale bars, 500 μm for left panel of (A), 50 μm for A‐(a, b) and B‐(a, b); 20 μm for (C) magnifications: ×40 for left panel of (A); ×200 for A‐(a, b) and B‐(a, b); and ×1000 for (C).

Fig. 7‐3

Representative images of Ctsk expression in lung. tdT+ cells were examined in lung sections of Ctsk‐Cre+:tdT mice (A). tdT was highly expressed in certain cells in alveoli and bronchioles (a), and a representative area is shown at a higher magnification (b). Arrows: yellow: type II alveolar cells or macrophages in alveoli. A dotted circle indicates a bronchiole. In Ctsk‐Cre+:nT‐nG mice (B), quantitative analysis of nT and nG expression was performed in young (a) and aged (b) mice (c, n = 3). White arrowhead: bronchioles. Antibodies against either CD44 or F4/80 were used to identify macrophages or type II pneumocytes by IF (C), showing CD44 in alveoli (a) and bronchioles (b); F4/80 in alveoli (c); F4/80 in cells adjacent to bronchioles (d). Arrows: in (C‐a‐c) empty white: cells that only stained positively for CD44 but not tdT expression were macrophages; white: CD44‐ or F4/80‐stained cells with tdT expression were type II pneumocytes or macrophages; in (d) white: Clara secretory cells/Goblet cells, mucus‐producing cells; white arrowhead: ciliated cells; empty yellow: basal cells; empty green: neuroendocrine cells/putative early progenitors; empty white arrowheads: macrophages. AD, alveolar duct; AS, alveolar sac; A, alveoli. Scale bars, 500 μm for (C‐a), 50 μm for (A, B); and 20 μm for (C‐c,d). Magnifications: 200 for (A) and (B); 200 for (A, B, C‐a); 400 for (C‐b); 1000 for (C‐c,d).

Fig. 7‐4

Imaging of Ctsk expression in skeletal muscle sections. tdT expression was detected in Ctsk‐Cre⁺:tdT mice (A). An image of a lobe of muscle fibers at low magnification is presented (a). A representative area is shown at a high magnification (b), showing strong tdT expression in the connective tissue, whereas lower tdT expression was observed in the sarcoplasm of muscle fibers. White arrowhead: perimysium. In Ctsk‐Cre⁺:nT‐nG mice (B), Ctsk expression is recognized in nG⁺ cells switched from nT⁺ cells in mice aged either 1 month (a) or 8 months (b). The expression of nT‐ and nG‐positive cells was quantitatively analyzed (c) (n = 3), showing a significant reduction in nG expression in the 8‐month‐old mice. Immunofluorescence was performed with antibodies against α‐SMA and CD31 to identify satellite cells and smooth muscle of the arteriole (C). (a) Cells positive for both tdT and α‐SMA in skeletal muscle; (b) cells positive for both α‐SMA and tdT in artery; and (c) cells positive for CD31 in muscle adjacent to artery. Arrows: empty white: satellite cells in (a) and (c); empty white: cells positive for both tdT and α‐SMA of artery in (b); white: smooth muscle, red: internal elastic lamina/tunica intima in (c). Scale bars, 500 μm for left panel of (A), 50 μm for (B, C); and 20 μm for right panel of (A). Magnifications: ×40 for left panel of (A); ×200 for (B); and ×400 for right panel of (A) and (C).

Fig. 7‐5

Representative images of Ctsk expression in kidney. Ctsk expression in Ctsk‐Cre+:tdT mice at low magnification (40) (A). Whole kidney image is shown in left panel with two representative areas: Ctsk expression in adrenal gland (a) and renal cortex with fibrous capsule (b). Immunofluorescence was carried out with antibodies against type I collagen and F4/80 in renal parenchyma. The results are shown in (B): a: type I collagen staining; b: type I collagen staining in arteriole; and c: F4/80 staining. P, proximal convoluted tubule; D, distal convoluted tubules; A, arteriole; V, vein. Arrows in (B‐a): empty white arrows: renal corpuscles; in (b): yellow: endothelial cells; red: outer elastic layer (Tunica externa); white: smooth muscle layer (Tunica media); blue: inner elastic layer (Tunica intima); in (c): white empty: positive staining for F4/80. Scale bars, 500 μm for whole kidney image and 20 μm for (A‐a, b); 50 μm for images for (B) and (C‐a); 20 μm for (C‐b–d). Magnifications: 40 for whole kidney and 400 for representative images in (A‐a, b), and (B‐c). 1000 for images in (a), (b) and (d) in (C).

Fig. 7‐6

Detection of Ctsk expression in pancreas. In Ctsk‐Cre⁺:tdT mice (A), tdT was highly expressed in blood vessels (white arrowheads) and islets (white empty arrowheads), but it was weakly expressed in acinar cells. The left panel of (A) is an image of a pancreas section at a low magnification (×40). Representative images: (a) blood vessel (white arrowhead); (b) two islets (empty white arrowheads). In Ctsk‐Cre⁺:nT‐nG mice, Ctsk expression is indicated by nG (B). Its expression was examined in the mice aged 1 month (a) and 8 months (b) and their comparison (c), in which strong expression was detected in the islets of Langerhans in the young mice, but its expression was reduced in the aging mice. Immunofluorescence with antibodies against NGN3 or F4/80 was carried out to verify tdT⁺ cells or macrophages (C). tdT⁺ cells were identified as endocrine cells in the islets in the pancreatic islets of Langerhans, as they were positively stained for NGN3 (a: lower magnification and b: higher magnification; empty white arrowheads: islets). The F4/80‐stained cells were macrophages (c, an islet; d, an acinar area near the islet; empty white arrows‐macrophages). Scale bars as indicated in the images, 500 μm for the left panel of (A) and 20 μm for (A‐a, b); 50 μm for (B‐b); and (C‐a); 20 for (C‐b–d); and 10 for right panels of (B). Magnifications: ×40 for left panel in (A), ×200 for (A‐a, b), and (B‐a, b); ×400 for representative images of islets; ×1000 for (C‐b–d).

Fig. 7‐7

Fluorescence images of liver. tdT expression in Ctsk‐Cre⁺:tdT mice (A). Although tdT was expressed at low levels in hepatocytes (a), it was highly expressed in interlobular connective tissue (b). Ctsk expression in Ctsk‐Cre⁺:nT‐nG mice (B), aged at 1 month (a) and 8 months (b), with their comparison (c) (n = 3). nG was coexpressed with nT in hepatocytes and was significantly increased in aging mice. Immunofluorescence was performed to identify macrophages and endothelial cells with antibodies against F4/80 (a), Col1a1 (b‐artery), or CD31 (c: arterioles and d: hepatocytes located at edge of liver lobe), respectively. Arrowheads in (C‐a): empty white indicates cells stained positively for F4/80 but no tdT expression; white indicates cells expressing tdT but no F4/80 staining. Scale bars, 20 μm for (A); 50 μm for (B) and (C). Magnifications, ×400 for (A) and (C); ×200 for (B).

Fig. 7‐8

Ctsk expression detected in heart and testis. Ctsk expression was imaged in myocardium of Ctsk‐Cre⁺:tdT mice (a). Its expression was detected in cardiac muscle fibers. It was also co‐expressed with α‐SMA by immunofluorescence with an antibody against α‐SMA (indicated by empty white arrows). In addition, tdT expression was found in the coronary arteries, with α‐SMA expression (indicated by white arrows). RV, right ventricle; LV, left ventricle; M, myocardium. In addition, Ctsk expression was detected in testis sections (seminiferous tubules) from Ctsk‐Cre⁺:tdT mice (b). Its expression was localized in the interstitial tissue (IC of Leydig cells) and the seminiferous tubules (ST). It was also found in developing spermatozoa (SPZ). Arrows: yellow, spermatogonia (SG); white, myeloid cells (M); red, Sertoli cells (SC); and green, late spermatids (LS). Scale bar: 500 μm for and 50 μm for (b). Magnification: (a) ×40 and (b) ×400.

Fig. 7‐9 (A and B)

Ctsk expression in horizontal sections of brain. Ctsk⁺ cells imaged in whole‐brain horizontal section of Ctsk‐Cre⁺:tdT mice (left panel, low magnification) with three representative areas at higher magnification (A): (a) meninge; (b) blood vessel; and (c) falx cerebri in longitudinal fissure. Ctsk expression in Ctsk‐Cre⁺:nT‐nG mice (B), aged 1 month (a) and 8 months (b) and their comparison (c) (n = 3). Scale bars: 500 μm for whole‐brain image in left panel of (A); 50 μm for images in (A‐a–c, B). Magnifications: ×40 for left panel of (A); ×200 for (A‐a–c, B).

Fig. 7‐9 (C)

(C) Identification of tdT⁺ cells by immunofluorescence with antibodies against IBA1 (a marker of microglia) (a, b), NeuN (neuronal nuclei, a marker of CNS neuronal cells) (c, d, e), Neurogenin 3 (neurogenic astrocytes and oligodendrocytes) (f), and GFAP (a marker for astrocytic cells) (g, h) in the following areas: (a) in the olfactory bulb, immunofluorescence with an IBA1 antibody. Arrows: white: tdT⁺ cells in sixth layer (anterior commissure); empty arrowhead: OB with strong tdT expression and colocalized IBA1 staining. Layers: 1: glomerular layer; 2: external plexiform layer; 3: mitral cell layer; 4: internal plexiform layer; 5: granule cell layer; 6: anterior commissure. Scale bar: 100 μm. Magnification: ×200. (b) In hippocampus (Cornu ammonis 3, CA3), immunostaining with IBA1 antibody. Arrows: white arrows: pyramidal cells positive for both tdT (nuclei) and IBA1 (cell body); empty white arrows: positive for IBA1; and empty arrowheads: blood vessels or neural dendrites. SR, strata radiatum; SL, strata lucidum; SO, stratum oriens; SP, stratum pyramidale layers. Scale bar: 50 μm; Magnification: ×400. (c) In several areas, staining with NeuN antibody at lower magnification. Areas: 1: hippocampus; 2: midbrain; 3: striatum; 4: pallidum; 5: hypothalamus; 6: thalamus; 7: anterior commissure tempered limb; 8: anterior commissure olfactory limb. IPN, interpeduncular nucleus; SNR, substantia nigra reticular part; NST, nigrostriatal tract. Scale bar: 500 μm. Magnification: ×40. (d) In medulla, staining with NeuN antibody. Arrows: white: motor neurons; empty arrowhead: blood vessels. Scale bar: 100 μm. Magnification: ×200. (e) In pons, staining with NeuN antibody. Arrows: white: neural cells positive for NeuN; empty arrowheads: blood vessels. Scale bar: 50 μm. Magnification: ×400. (f) Staining with Neurogenin 3 antibody was performed in olfactory bulb. Arrows: empty white: neural cells stained positive for Neurogenin 3 (periglomerular cells); white empty arrowheads: blood vessels. Layers: 1: olfactory nerve layer; 2: glomerular layer; 3: external plexiform layer; 4: mitral cell layer; 5: internal plexiform layer; 6: granule cell layer. Scale bar: 100 μm. Magnification: ×200. (g) In olfactory bulb, staining with GFAP antibody. Arrows: empty white arrows: cells positive for GFAP (astrocytes); empty white arrowheads: blood vessels. Layers: 1: olfactory nerve layer; 2: glomerular layer; 3: external plexiform layer; 4: mitral cell layer; 5: internal plexiform layer. Scale bar: 50 μm. Magnification: ×400. (h) In cerebellum, staining with GFAP antibody. Arrows: white: cells positive for GFAP (astrocytes); empty white arrows: Purkinje neurons (P) positive for both tdT and GFAP; empty white arrowheads: vascellum. White square indicating a P cell with an amplified image shown on left. Layers: ML, molecular layer; GL, granular layer. Scale bar: 100 μm. Magnification: ×200.

Fig. 8

Validation of Ctsk expression in tissues and organs of B6 mice by dd‐RT–PCR. The expression profiles of Ctsk described above were validated by dd‐RT–PCR. dd‐RT–PCR was performed as described in Methods using mRNA from 2‐month‐old B6 mice (male). The expression data were categorized into three groups: high, middle, and low.