Figures and data
Generation of CRISPR/Cas9-mediated Fshr-ZsGreen knockin reporter mice.
A. CRISPR/Cas9-mediated targeting strategy to generate Fshr-P2A-ZsGreen knockin mice. B. Detection of integration by PCR in F0 and F1 mice: (a) Schematic of PCR primer design specific to Fshr-P2A-ZsGreen and the wild-type allele. The results of genomic DNA PCR genotypes using the primer pair EGE-YHN-013-A-L-GT-F/EGE-YHN-013-A-Mut-R (b) and another primer pair EGE-YHN-013-A-R-GT-F/EGE-YHN-013-A-R-GT-R (c). C. Southern blot confirmation of the correct integration of the P2A-ZsGreen allele in F0 and F1 mice. The Southern blot results demonstrated the successful generation of the targeted P2A-Fshr-ZsGreen allele: (a) Restriction sites in the wild-type sequence and targeted vector; (b) Southern blot analysis probes, expected restriction fragment lengths as indicated and blotted images. D. The 2nd PCR genotyping strategy using primers: (a) schematic for the design of PCR primer sets for the Fshr-ZsGreen allele; (b and c) genotyping readout of heterozygous mice; (d) genotyping readout of homozygous mice. Abbrev.: PC-positive control, WT-wild type.
Imaging of Fshr-ZsGreen expression in the reproductive system.
Frozen sections of the testis and ovary were immunostained with an anti-Stra 8 antibody, followed by imaging of Fshr-ZsGreen expression and its colocalization with Stra8 staining. A. Fshr-ZsGreen expression and its colocalization with Stra8 in the testis. The whole image of the sectioned testis is shown in A-a. The representative area of seminiferous tubes with interstitial cells (Leydig cells) is presented at a higher magnification (400X) (A-b), and an imaged seminiferous tube is presented at a magnification of 1000X (A-c). White empty arrowheads indicate Leydig cells. In addition, Fshr-ZsGreen expression was further confirmed by FISH, as shown in B (sense probe as a negative control) and C (anti-sense probes). The entire image of the sectioned testis is taken at a lower magnification of 40X, and two representative areas of seminiferous tubes with Leydig cells are demonstrated at a higher magnification of 1000X. White arrows indicate spermatozoa. Abbrev.: LCs-Leydig cells, and ST-seminiferous tubule. The images for Fshr-ZsGreen expression and its colocalization with Stra8 are shown in C. In C., the entire picture of the sectioned ovary is shown in C-a. The representative images of the section are taken at a higher magnification of 400X as described in the following: b. an area containing a corpus luteum, tertiary and ruptured follicles and a partial oviduct; c. an area with more corpora lutea and d. oviducts. Furthermore, representative images of the ovary at a higher magnification of 1000X are also taken as described below: e. primordial and secondary follicles, f. a mature follicle, g. follicle cells in a corpus luteum and h. ciliated epithelial cells in the oviduct. Magnifications: 100X for a, 200X for a, c and d, and 1000X for e to h. Scale bars: 100 μm for a, b, c and d, 20 μm for e to h.
Fshr-ZsGreen expression in skeletal tissues.
A. Detection of Fshr-ZsGreen expression in frozen sectioned skeletal tissues by fluorescence confocal microscopy. The upper panels show Fshr-ZsGreen-positive cells in the representative areas of skeletal tissues at low magnification (400X), while the lower panels show their corresponding cells at high magnification (1000X). These representative areas demonstrate the Fshr-ZsGreen-positive cells in chondrocytes of the growth plate (a and e), on the surfaces of sponge bone under the growth plate (b and f), in trabecular bone in bone marrow (c and g), and in the cortex (d and h). Abbreviations: OBs-osteoblasts; OCs-osteoclasts; BM-bone marrow; CB-cortical bone; and P-periosteum. Arrows: white empty arrowhead indicates GFP-positive osteocytes. Magnifications: 400X for a to d and 1000X for e to h; scale bars: 50 μm for a to d and 20 μm for e to h.
B. Confirmation of GFP-positive cell identities with antibodies against either osteocalcin or Trap as markers for osteoblasts or osteoclasts. Colocations of GFP expression with osteocalcin in mature chondrocytes (a), osteoblasts, bone lining cells and osteocytes on the surface of or within sponge bone (b), trabecular bone (c), and cortical bone (d). Colocations of Trap with multinucleated GFP-positive cells are shown in e and f, which represent osteoclasts on the resorptive areas and in bone marrow adjacent to bone surfaces and bone lining cells over trabecular bone. Arrows: white empty arrows indicate early chondrocytes with GFP expression but undetectable osteocalcin expression, while white arrows point to mature chondrocytes with both strong GFP and osteocalcin expression. Magnifications: 1000X for a to f. Scale bars: 20 μm for a to f.
C. Identification of stem/progenitor cells. a. Colocalization of Fshr-ZsGreen with CD34-positive staining in bone marrow, indicated by empty white arrows; an empty white arrowhead indicates multinucleated Fshr-ZsGreen cells with CD34-positive staining. b. CD34-positive stained cells in growth plates. Empty white arrows indicate spindle-shaped ZsGreen-positive chondrocytes stained positively for CD34, where empty white arrowheads point to round cells with CD34-positive staining located in the bottom of the growth plate. c. Colocalization of Fshr-ZsGreen with CD133 staining in the cells of bone marrow and on the bone surface. Empty white arrows indicate bone marrow cells positive for both Fshr-ZsGreen and CD133 staining, and empty white arrowheads indicate Fshr-ZsGreen-positive cells on the trabecular bone surface with positive staining for CD133. d. Positive staining for CD133 on Fshr-ZsGreen-positive chondrocytes on the surface of articular cartilage. e. CD133 staining in the weak Fshr-ZsGreen-positive fibroblast-like cells in the periosteum.
Fshr-ZsGreen expression in adipose tissues.
A. Confocal imaging of Fshr-ZsGreen expression in frozen sections of inguinal WAT. The whole image of a piece of the inguinal WAT was imaged under confocal microscopy with a low magnification (40X), and three representative areas were also presented with a higher magnification (400X) showing low and high cellular densities (low-b and high-a and c). These images demonstrate Fshr-ZsGreen expression in the cellular membranes of adipocytes.
B. IF staining of Fshr-ZsGreen-positive adipocytes with an antibody against mouse Ucp1. To identify the beige cells in WAT, we performed IF staining with an antibody against mouse Ucp1. Positive staining for Ucp1 was colocalized within Fshr-ZsGreen-positive cells in the areas with a higher cell density (a and b) imaged at low magnification, as shown in the left panels, while higher magnification images are shown in the right panels (a and d). In addition, strong Fshr-ZsGreen expression was observed in the arterioles of WAT (c and d). Arrows: white empty arrows indicate beige cells; white arrows point to white adipocytes, and empty arrowheads indicate GFP-positive arterioles. Magnifications: 400X for a and c; 1000X for b and d. Scale bars: 50 μm for a and c and 20 μm for b and d.
C. Fluorescence images of Fshr-ZsGreen expression in frozen sectioned BAT. A whole image of BAT was imaged under confocal fluorescence microscopy at low magnification (a, 40X). Three representative areas are also presented at a higher magnification (b, c and d, 400X), which clearly show the Fshr-ZsGreen expressed in the cells of BAT and skeletal muscles in the right parts of the images (b and c). Abbreviation: M-muscle. Arrows: white arrows indicate strong Fshr-ZsGreen expression in BAT cells. Magnifications: 40X for a and 40°C for b, c and d. Scale bars: 100 μm for a and 50 μm for b, c and d.
D. Identification of brown adipocytes in BAT by IF staining. Brown fat cells were identified using an antibody against mouse Ucp1. Ucp1 staining is shown at lower magnification (40X) in the left panel covering a whole piece of BAT. Three representative areas are imaged at higher magnifications (a, b and c, 400X and 1000X), showing Fshr-ZsGreen colocalization with Ucp1-positive staining. Magnifications and scale bars are indicated in the figure.
E. Examination of peripheral neural fibers within BAT. To determine whether Fshr-ZsGreen is expressed in the peripheral nerves in BAT, we performed IF staining with an antibody against tyrosine hydroxylase (TH), a marker for peripheral sympathetic neurons. A whole piece of BAT was imaged at a lower magnification (40X) after being stained for TH, as shown in the left panel. Representative areas are presented in the right panels with higher magnifications (400X –a, b and c and 1000X-d, e and f, respectively). Arrows: red outlined arrowheads indicate Fshr-ZsGreen– and TH-positive large peripheral nerves; green outlined arrowheads point to Th-stained nerve fibrils accompanying Fshr-ZsGreen-positive nerve fibrils around a Fshr-ZsGreen-positive arteriole; red arrows indicate Fshr-ZsGreen– and TH-positive nerve fibrils; and white arrows indicate brown adipocytes with both Fshr-ZsGreen and TH expression. Magnifications: 40X for the whole image of BAT in the left panel; 400X for a, b and c; and 1000X for a, e, and f. Scale bars: indicated in the images.
F. Further confirmation of Fshr-ZsGreen expression in the peripheral nerves. To confirm Fshr-ZsGreen expression in peripheral neurons, we also employed another antibody against Peripherin (Peri), a 57-kD type III intermediate filament that is a specific marker for peripheral neurons, to further identify peripheral neurons in BAT. The left panel shows an entire image of the BAT stained for Peri at a lower magnification (40X). The three representatives are shown in the right panel: a. the first area located in the edge of the BAT with more white adipocytes; b. the second area with more brown adipocytes and a large peripheral nerve; c. the last area enriched with brown adipocytes. Their corresponding higher magnifications (1000X) are shown in a, e and f, respectively. Abbrev.: LNF-large never fibril. Arrows: Empty white arrows-small peripheral fibrils; white arrow-Fshr-ZsGreen-positive fibrils and empty white arrows-both Fshr-ZsGreen– and Peri-positive brown adipocytes. Magnifications: 40X for the t panel; 400X for a, b, and c and 1000X for d, e and f. Scale bars: 1000 μm for the whole images in the left panel; 50 μm for a, b and c, and 20 μm for d, e and f.
Imaging of Fshr-ZsGreen expression in the heart and aorta.
Fshr-ZsGreen expression was imaged in frozen sections of the heart (A): cardiomyocytes (a) and smooth muscles (b). Then, IF staining with antibodies against α-SMA (B) and EMCN (C) was carried out to identify cardiomyocytes and smooth muscles. The whole image of the heart with Fshr-ZsGreen expression and staining for α-SMA is shown in the left upper panel of B. Its representative areas are presented at two magnifications of 400X (B-a to h) and 1000X (B-i to n), respectively, in the following: (1) 400X magnification: a-cross-oriented cardiomyocytes; b-longitudinally oriented cardiomyocytes; c-cross-oriented smooth muscle of the ascending aorta with brown adipose tissue; d-smooth muscle of the left pulmonary artery with brown adipose tissue; e-brown adipose tissue attached to a large artery; e-layer of endothall cells of the superior vena cava (SVC); g. connective tissues between arteries containing brown adipose, different sized nerves and arteries; h. another part of the SVC with layers of smooth muscles and connective tissue. (2) 1000X magnification: i, cardiomyocytes with an arteriole; j. brown adipose tissue attached to the circulation system above the heart; k. the layers of endothelial cells and smooth muscles of a bronchial artery; m. transverse section of nerve fibers; n. the layers of ECs and SM of the SVC. In addition, imaging of the IF staining for EMCN is shown in C. The whole image of Fshr-ZsGreen and EMCN staining in the heart at a magnification of 40X in the left upper panel, from which a representative of pulmonary artery at a magnification of 400 is shown in a at a magnification of 400X (a); a representative of aorta is presented in b. Representative images at higher magnification of 1000X: c-cardiomyocytes in transverse orientation with a venule and d-a layer of endothelial cells of pulmonary artery. Abbrev.: CM-cardiomyocytes; SM-smooth muscle; ECs-endothelial cells; N-never; B-brown adipose. Magnifications and scale bars are indicated as in the figure.
Two orientations of the ascending aorta were examined for Fshr-ZsGreen after staining for α-SMA or EMCN: the longitudinal section (D-a to c) and the cross section (D-d to f)). D-a shows the entire image of the ascending aorta stained for α-SMA at a magnification of 100X, while representative images at a higher magnification of 1000X are presented in D-b for α-SMA staining and D-c for EMCN staining. The entire image of the transversely sectioned aorta (D-d) and representative images at a higher magnification of 1000X for α-SMA (D-e) and EMCN (D-f). Abbrev.: TI-tunica intima, TM-tunica media, AV-aortic valve, SM-smooth muscles, Sub. CT-subendothelial connective tissue. Arrows: empty white arrows indicate arterioles positively stained for α-SMA in b; empty arrowheads point to endothelial cells positively stained for either α-SMA or EMCN. Magnifications: 100X for a and d; 1000X for b, c, e and f. Scale bars: 100 μm for a and d and 20 μm for b, c, e, and f.
Fshr-ZsGreen expression in the lung and kidney.
Detection of Fshr-ZsGreen expression and IF staining with PD-L1 was performed in lung sections at different magnifications. The whole image of the frozen sectioned lung is shown in the left panel at 40X magnification (A-left panel). Representative areas are shown in the right panels at magnifications of 400X (Figure 6A-a to d) and 1000X (A-e to h). They are the ciliated columnar cells of primary bronchi (a and c), the bronchioles with alveoli (b and f), respiratory bronchiole with bronchial gland (c and g) and alveoli (d and h). Arrows: empty white arrowheads-type I pneumocytes, white arrowheads-type II pneumocytes and white arrows-macrophages. Abbrev.: C-ciliated epithelium; PA-pulmonary arteriole; L-lumen; RB-respiratory bronchiole; BG-bronchial gland; A-alveoli; AD-alveolar duct. Magnifications: 40X for the left mage of the whole section, 400X for a to d, and 1000X for e to h. Scale bars: 1000 mm for the left panel, 50 μm for a to d and 20 μm for e to h.
Fshr-ZsGreen expression and staining with Col1a1 were examined in the sectioned kidney under confocal fluorescence microscopy at three magnifications (B). The images in the top panel are images of the whole section at 40X magnification (a-d). The images in the middle panel show the colocalization of Fshr expression with positive staining for Col1a1 in the glomerulus and renal tubes (proximal and distal convoluted tubes) at 400X magnification (e and f), while the bottom panel shows images of the glomerulus (g) and arteriole (h) at 1000X magnification. Magnification: 1000X. Scale bars: 100 mm for a-d, 50 mm for e and f and 20 μm for g and h. Abbrev. A-arteriole; PCT-proximal convoluted tubule; DCT-distal convoluted tubule; g-glomerulus.
Identification of Fshr-ZsGreen expression in the liver.
Frozen sectioned liver tissue was stained for Col1a1 (A), CD31 (B) or KCNMA1 (C), followed by florescent imaging for Fshr-ZsGreen and each of these stained molecules simultaneously. In the section stained for Col1a1 (A), two representative areas are shown at magnifications of 400X and 1000X as indicated: (1) hepatic cells with a central vein (CV) (a and c) and (2) hepatic artery (b and d). In the section stained for CD31 (B), a representative area of hepatic cells with a CV is shown at lower (a) and higher (b) magnifications. In the section stained for KCNMA1, three representative areas are presented at two magnifications as follows: (1) hepatic cells with a CV (a and b); (2) hepatic cells with peripheral nerve fibers (c and d); and (3) small nerve fibers located around a vein (e and f).
Visualization of Fshr-ZsGreen expression in the pancreas.
Visualized Fshr-ZsGreen expression was obtained in frozen sections of the pancreas under fluorescence microscopy after immunostaining with antibodies against NG3, insulin or glucagon at two magnifications (400X and 1000X). Images of Fshr-ZsGreen expression with NG3, insulin and glucagon staining are shown in A, B and C, respectively.
Fshr-ZsGreen expression in the thyroid.
Fshr-ZsGreen expression was detected by its colocalization with immunostaining of TSH in frozen thyroid sections. The left panel is an entire image of the section, and two representative areas are in the right panels at higher magnifications, demonstrating Fshr-ZsGreen expression in follicular cells and parafollicular cells (C cells) at the edge (a and b) and the center of the thyroid (c and d). Arrows: white arrowheads indicate follicular cells, and white arrows indicate parafollicular cells. Magnification: 40X for the whole image.
Detection of Fshr-ZsGreen expression in the skin.
Fluorescence images of Fshr-ZsGreen were taken in two types of skin (A): thick skin (A-a) and thin skin (A-b). Then, IF staining was performed using an antibody against CD34 to identify stem cells with Fshr-ZsGreen expression in the hair follicles (B) at lower (B-a) and higher (B-b) magnifications (400X and 1000X, respectively). Abbrev.: HF-hair follicle; SG-sweat gland; FT-fat tissue; DP-dermal papillae. Magnifications: 400X for A and B-a, 1000X for B-b. Scale bars: 50 μm for A and B-a and 20 μm for B-b.
Fshr-ZsGreen expression in skeletal muscle.
The whole image of frozen sectioned skeletal muscle (gastrocnemius) was taken at a lower magnification (40X) after the section was stained with anti-α-SMA antibody, as shown in the left panel (a). Then, longitudinal sections (b and c) and cross-sections (d and e) were imaged at higher magnifications (400X and 1000X), respectively. In addition, frozen sections stained with antibodies against PAX7 (f and g) and TH (h and i) were imaged at two magnifications (400X and 1000X) as indicated. Arrows: (1) In b-e, empty white arrowheads indicate both Fshr-ZsGreen– and α-SMA-positive satellite cells; empty white arrows point to only Fshr-ZsGreen-positive satellite cells without α-SMA staining; white arrowheads indicate muscle fibrils with strong Fshr-ZsGreen expression but no α-SMA staining. (2) In f and g, white arrowheads indicate muscle fibrils with strong Fshr-ZsGreen expression, blue arrows point to muscle fibrils with both Fshr-ZsGreen and TH expression, and red arrowheads indicate peripheral nerves with both Fshr-ZsGreen and TH expression. Magnifications: 40X for a, 400X for b, d, f and h, and 1000X for c, e, g and i. Scale bars: 1000 μm for a, 50 μm for b, d, f and h, and 20 μm for c, e, g and i.
Examination of Fshr-ZsGreen expression in the spleen and bone marrow
To identify Fshr-ZsGreen expression in immune cells of the spleen, IF staining was performed in frozen sections of the spleen. Two antibodies against either CD11B or CD3 were used to identify myeloid-lineage cells or T cells. Fshr-ZsGreen expression in myeloid-lineage cells is shown in A, in which the left panel is the whole image of the spleen at a low magnification and its three representative areas are presented at higher magnifications (400X and 1000X): (1) an area located at the edge showing strong Fshr-ZsGreen and CD11B expression (a and b); (2) an area of red pulp c and d); (3) an area of white pulp (e and f). Abbrev.: RP-red pulp, WP-white pulp, BM-bone marrow, F-fibroelastic capsule and MZ-marginal zone. Scale bars are indicated in each image.
Fshr-ZsGreen expression in the representative areas of the brain.
Fshr expression in three representative areas of the brain-the olfactory bulbs (a, d, and g), pallidum (b, e and h) and hippocampus (c, f and j). Each section was immunofluorescently stained with antibodies against GFAP, Iba1 or NeuN that recognized markers for astrocytes (a-c), microglia (d-f) or neuron (g-j), respectively. Their colocalizations are indicated by white arrowheads. Abbrev.: OLB-olfactory bulb. Magnifications: 400X for a, c and f, and 1000X for b, d, e, f g and h. Scale bars are indicated in each image.
Confirmation of Fshr-ZsGreen expression by IF staining with a specific antibody against mouse Fshr and ddRT-PCR.
The Fshr-ZsGreen expression described above was further confirmed by IF staining using an antibody against mouse Fshr and ddRT‒PCR. The frozen tissue sections used for this confirmation include bone, BAT, thyroid, cardiac muscles, kidney, liver, lung, aorta, ovary and testis (A), demonstrating Fshr-ZsGreen colocalization with Fshr-positive staining in these tissues/organs.
Fshr expression at the mRNA level in different tissues/organs was examined by ddRT‒PCR (B). The results indicate that Fshr is expressed in all examined tissues/organs. Based on their expression levels, they can be categorized into three groups: (1) high (H), including lung and kidney, ranging from 1005 to 1843 copies/μL; (2) middle (M), including thoracic (T) vertebra, skull, femur, jejunum, liver, tooth, tibia and muscle, ranging from 173 to 475 copies/μL; and (3) low (L), including duodenum, pancreas, brain, WAT, thyroid, BAT, tongue, bladder, heart, stomach, spleen, skin, cartilage and tail, ranging from 5.4 to 81 copies/μL.
The first set of Integration detection PCR primer design and sequences:
The 2nd set of junction PCR primer design and sequences for genotyping F0 and F1:
PCR primer design and sequences for genotyping heterozygotes and homozygotes:
The primary antibodies used for IFs.
The sequences of sense and antisense probes for FISH in testis:
-No non-specific Fshr-ZsGreen expression in the examined tissues/organs from B6 mice
