Figure 1.
Generation of the Fam20c floxed alleles and Fam20c null alleles.
(A) Targeting construct. Dark boxes: Exons; dark triangles: loxP sites. A mcl-neo cassette flanked by Frt sites (white triangles) was inserted between exons 9 and 10. A PGK-DTA cassette was downstream to the 3′ homologous arm. The mcl-neo cassette was removed from the targeted allele after correct targeting. (B) PCR screening for targeted ES clones. The correct targeting was confirmed by PCR using 5′ screen primers (5′S-F and 5′S-R) and 3′ screen primers (3′S-F and 3′S-R). The correct targeting produced a 5.2 kb fragment for the 5′ screening, and a 4.5 kb fragment for the 3′ screen. WT and random insertion had no PCR products. Two correctly targeted ES clones (Clone 286 and Clone 297) were identified and both went through germline transmission. (C) Genotyping strategy. The alleles were genotyped by PCR using a mixture of three primers: “a”, “b” and “c” (see Figure 1A). The primers “a” and “b” produced a 400 bp fragment for the floxed allele. The primers “a” and “c” produced a 500 bp fragment for WT allele. The null alleles did not produce any PCR products due to the loss of the binding sequences for primer “a”. The Cre-loxP recombination was confirmed by PCR using primers Rec-F and Rec-R. A 260 bp fragment was produced for the null allele, but no PCR products for the WT allele. Lane 1 indicated the Fam20cflox/flox genotype. Lane 2 referred to the Fam20cflox/+. Lane 3 indicated the Fam20cΔ/Δ. Lane 4 demonstrated the WT. Lane 5 showed the Cre-LoxP recombination on Fam20c floxed allele.
Figure 2.
Validation of FAM20C inactivation.
(A) RT-PCR was performed with the cDNAs reversely transcripted from the total RNAs extracted from the femurs of 3-week-old Sox2-Cre-Fam20cΔ/Δ (cKO) mice and WT littermates, using two sets of primers: Set 1 primers (the forward primer was in exon 4, and the reverse in exon 7) produced a 388 bp fragment for WT mice, and gave rise to no product from the cKO mice (due to exon 7 ablation); Set 2 primers (the forward in exon 4, and the reverse in exon 10) produced a 840 bp fragment for the WT mice, and a 405 bp fragment for the cKO mice. (B) ISH on femurs. The osteoblasts in the trabecular bone area of 3-week-old cKO mice had negative staining for Fam20c mRNA (left), in contrast with the strong staining in the WT mice (right). (C) IHC on femurs. The osteoblasts and osteocytes of cKO mice (left) showed negative staining for FAM20C protein, while positive staining was observed in the WT littermates (right). Scale bars: 50 µm.
Figure 3.
Gross defects in the Sox2-Cre-Fam20c-cKO mice.
(A) The 4-week-old Sox2-Cre-Fam20cΔ/Δ (cKO) mouse on the left was smaller compared with the WT littermate on the right. (B) The 4-week-old cKO mouse (left) had flat face (undeveloped nose, a typical manifestation of rickets) compared with the WT littermate (right). (C) Body weight monitoring from newborn (Nb) to postnatal 10 weeks revealed significant growth retardation in the cKO mice. (D) Plain X-ray examination of a 5-month-old cKO mouse (left) revealed smaller skeleton, hypomineralization and distorted spine (arrowheads) compared with its WT littermate (right).
Figure 4.
Alizarin red/alcian blue staining of the skeleton in the 1-week-old Sox2-Cre-Fam20c-cKO mice.
(A) The Sox2-Cre-Fam20cΔ/Δ (cKO) mice (upper) showed smaller size and remarkably delayed ossification (more blue staining) in all bones (arrowheads) when compared with WT littermates (lower). (B) The rib cartilages of cKO mice (upper) stained blue, while those of their WT littermates (lower) showed broad areas with the red staining indicating ossification (arrowheads), suggesting an aberrant endochondral ossification in the cKO mice. (C) The femur of cKO mice (left) had shorter length and delayed secondary ossification center (arrow, blue stained), in contrast with the red secondary ossification center of their WT littermates (right). (D) The ossification centers in the carpus of the cKO mice (left) had more blue-stained areas than that of the WT littermates (right). (E) The skull of the cKO mice (left) had smaller size and delayed suture closure (arrows), when compared with the WT littermates (right), indicating that the intramembranous ossification was disturbed in the cKO mice. Scale bars: 1 cm in A, 2 mm in E, 1 mm in B–D.
Figure 5.
Bone defects in the 6-week-old Fam20c-cKO mice revealed by X-ray and histology.
(A) Plain X-ray of the hinder legs. The tibia of the Sox2-Cre-Fam20cΔ/Δ (global cKO) mice (left) showed shorter length, hypomineralization, thinner cortical bone (arrow) and underdeveloped secondary ossification center (arrowhead) compared with their WT littermates (right). The long bones of the Col1a1-Cre-Fam20cΔ/Δ (mineralized tissue-specific cKO) mice (middle image) showed defects very similar to those of the global-cKO mice. (B) Plain X-ray of the tail of Sox2-Cre-Fam20cΔ/Δ mice (left) showed hypomineralization, shorter length, and underdeveloped secondary ossification centers (arrowheads), compared with the WT littermates (right). (C,D) Micro-CT analyses. The tibia of the Sox2-Cre-Fam20cΔ/Δ mice (left) showed shorter length, thinner cortical bone (arrow), more porous areas on both the outer and inner surfaces (indicating more hypominerlized areas) and smaller secondary ossification centers (arrowheads), compared with their WT littermates (right). (E) H&E staining of the sagittal sections of tibias. Tibias of the Sox2-Cre-Fam20cΔ/Δ mice (left image) and Col1a1-Cre-Fam20cΔ/Δ mice (middle image) were smaller and underdeveloped compared with the WT littermates (right image). (F) Higher magnification of the metaphysis areas in E showed that the growth plates of the global cKO mice (left image) and mineralized tissue-specific cKO mice (middle image) had a thinner zone of proliferative chondrocytes (dashed lines) and a wider zone of hypertrophic chondrocytes (solid lines) than the WT littermates (right image). (G) Higher magnification of the cortical bone areas in E showed that the cortical bone of the global cKO mice (left image) and mineralized tissue-specific cKO mice (middle image) had more osteoids (grey areas indicated by arrowheads) compared with the WT littermates (right image). Scale bars: 1 mm in A–D, 200 µm in E, 50 µm in F and G.
Table 1.
Quantitative μ-CT analyses of the cortical bone (the midshaft region) of the tibias from 3-week-old and 6-week-old WT and Fam20c-cKO mice.
Figure 6.
Reduced mineralization level in the Sox2-Cre-Fam20c-cKO mice.
(A) Goldner-Masson trichrome staining of sagittal sections from the undecalcified tibias of the cKO mice (left) and their WT littermates (right). The cKO mice showed more red staining (unmineralized osteoid) and less green staining (mineralized bone) compared with the WT. (B) Higher magnification of the cortical bone areas in A showed that the cortical bone of the cKO mice (upper) had more osteoid (red stained areas indicated by arrowheads), compared to the WT (lower). (C) IHC staining against biglycan on the sagittal sections of tibias from 3-week-old cKO mice (left) and their WT littermates (right). Note that the cKO mice had more biglycan. (D) Higher magnification of the cortical bone areas in C showed that the cortical bone of cKO mice (upper) had more biglycan (arrowheads), compared with the WT (lower). (E) Double fluorescence labeling of the tibia from 6-week-old cKO mice (left) and WT littermates (right). The first injection (calcein) produced a green label, while the second injection (Alizarin Red) gave rise to a red label. The distance between the green and red labeling indicated the mineral deposition in the period between the two injections (7 days). The tibia cortical bone of the cKO mice (left) showed narrower distance and blurry boundary between the two labels compared with the WT (right). (F) The quantitative measurements of the distance between the two injections revealed a significantly lower mineral deposition rate in the cKO mice compared with their WT littermates. ***P<0.005. Scale bars: 200 µm in A and C, 50 µm in B, D and E.
Figure 7.
Growth plate defects in the 3-week-old Sox2-Cre-Fam20c-cKO mice.
(A) ISH staining of Col II on the sagittal sections of tibia showed less Col II in the growth plate of cKO mice. The pink/red color indicates positive ISH staining. (B) Higher magnification of the boxed area in A revealed less Col II in both the proliferative chondrocytes (dashed lines) and hypertrophic chondrocytes (solid lines) in the growth plate of the cKO mice compared with WT littermates. (C) ISH staining of Col X showed lower expression in the growth plate of the cKO mice. (D) Higher magnification of the boxed area in C revealed less Col X in the hypertrophic chondrocytes (solid lines) of the cKO mice compared with WT. (E) BrdU staining on the sagittal sections of tibia. (F) Higher magnification of the boxed area in E revealed fewer proliferating cells in the zone of proliferative chondrocytes (dashed lines) of the cKO mice compared with WT. (G) TUNEL assay revealed fewer apoptotic cells in the zone of hypertrophic chondrocytes (arrowheads) of the cKO mice (left) compared with WT littermates (right). Counter stained with DAPI. (H) Statistical counting of apoptotic cells in G showed significantly less number in the growth plate of the cKO mice. *** P value<0.001. Scale bars: 200 µm in A, C, E and G. 50 µm in B, D and F.
Figure 8.
FAM20C is essential to the differentiation of bone cells.
(A,B) SEM analyses of tibia from 6-week-old Sox2-Cre-Fam20c-cKO mice (upper) and WT littermates (lower). The osteocytes of the cKO mice showed abnormal shape (green arrows), wider periosteocytic region (red asterisk), and loss of osteocyte processes (red arrowheads), appearing immature and poorly differentiated. (C,D) Backscatter SEM analyses of the alveolar bone showed that the osteocytes in the 6-week-old Sox2-Cre-Fam20c-cKO mice (upper) had periosteocytic lesions (the “halo” defects, i.e., wider unmineralized regions surrounding the ostecytes) appearing as larger lacunae (arrows) compared with their WT littermates (lower). The black areas represent the unmineralized areas (osteocytes, periosteocytic region, osteocyte processes), while the grey/white areas represent the matrix that is well mineralized. (E–J) ISH staining of Col1a1, OCN, and DMP1 on the sagittal sections of tibia from 3-week-old cKO mice (upper panels) and WT littermates (lower panels), revealed significant downregulation of these terminal differentiation markers in the cKO mice. The pink/red color represents the positive ISH staining. (K) IHC staining of FGF23 in the tibia from 3-week-old cKO mice and their WT littermates revealed more FGF23 protein in the long bone of the cKO (left) than in the WT (right) mice. The brown color represents the positive IHC staining. (L) Higher magnification of the box areas in K showed more FGF23 protein in the osteocytes (arrowheads) and the bone matrices (arrows) of the cKO mice (upper) than in the WT (lower). Scale bars: 5 µm in A and B, 10 µm in C and D, 50 µm in L, 200 µm in E–K.
Table 2.
Alterations of selected genes in the calvaria of 3-week-old Fam20c-cKO mice.
Figure 9.
Lentiviral shRNA-mediated “knockdown” of FAM20C leads to similar alterations in the expression of DMP1 and FGF23 in human and mouse osteogenic cell lines.
The cells were divided into three groups: uninfected cells, cells infected with control lentivirus expressing the scrambled shRNA and cells infected with FAM20C-shRNA lentivirus containing a mixture of 3 pieces of shRNA targeting different regions of the FAM20C mRNA. The mRNA levels of DMP1 and FGF23 in each group were determined by real-time PCR. The expression levels of the uninfected cells without osteogenic induction were taken as 1, while that of the cells infected with the control virus or FAM20C-shRNA virus were expressed as folds of change to the uninfected cells. (A) The knockdown of FAM20C led to remarkable downregulation of DMP1 in mouse MC3T3-E1 cells. Without the osteogenic induction, the expressional level of DMP1 had no significant difference among the three groups of cells. During the 3-week osteogenesis-induction process, DMP1 was significantly upregulated in the uninfected cells and cells infected with the control lentivirus, while its expression was remarkably reduced in the cells infected with FAM20C-shRNA lentivirus at 1-, 2- and 3-weeks after the start of osteogenic induction. (B) Inactivation of FAM20C led to remarkable downregulation of DMP1 in hMSC cells. The expression level of DMP1 had no significant difference among three groups in the first 2 weeks of culture in the osteogenic medium. After 3 weeks of osteogenic induction, the DMP1 expression was remarkably reduced in the cells infected with FAM20C-shRNA lentivirus when compared with the uninfected cells and cells infected with the control lentivirus. (C) Inactivation of FAM20C led to remarkable downregulation of DMP1 in Saos-2 cells. DMP1 expression was downregulated in human osteoblasts (Saos-2) infected with FAM20C-shRNA lentivirus before the osteogenic induction started, compared with the uninfected cells and cells infected with the control virus. The downregulation of DMP1 in the FAM20C-knockdown cells became more prominent after 1 week of osteogenic induction. (D) Inactivation of FAM20C led to significant upregulation of FGF23 in mouse MC3T3-E1 cells. The expression level of FGF23 had no significant difference among three groups in the first 2 weeks of osteogenic induction. After inducing the cells for osteogenic differentiation for 3 weeks, FGF23 was significantly upregulated in the cells infected with FAM20C-shRNA lentivirus, compared with the uninfected cells and cells infected with the control lentivirus. (E) Inactivation of FAM20C led to significant upregulation of FGF23 in hMSC cells. The FGF23 elevation also occurred in the hMSC cells after 3-week osteogenic induction. Note that upregulation of FGF23 in the hMSC cells was not as remarkable as in the MC3T3-E1 cells at the same time point. (F) Inactivation of FAM20C led to significant upregulation of FGF23 in Saos-2 cells. Without osteogenic induction, FGF23 level had no significant difference among the 3 groups. After 1 week of osteogenic induction, FGF23 was significantly upregulated in the cells infected with FAM20C-shRNA lentivirus, compared with the uninfected cells and cells infected with the control lentivirus. *: P<0.05; **: P<0.01; NI: without osteogenic induction; Control: uninfected control cells; Control virus: cells infected with control lentivirus; FAM20C-shRNA virus: cells infected with FAM20C-shRNA lentivirus.
Figure 10.
Recombinant FAM20C promotes the differentiation of MC3T3-E1 cells.
(A) Alizarin red was used to stain the mineralized nodules formed by MC3T3-E1 cells treated with different concentration of recombinant FAM20C. The lower panel showed the representative culture wells from each of the experimental groups as well as the control group. The upper graph displayed the quantitative measurement of Alizarin red dye released from the mineralized nodules formed by MC3T3-E1 cells. Note that the administration of FAM20C improved the mineralized nodule formation in a dose-dependent manner. (B) Real-time PCR using RNA extracted from MC3T3-E1 cells treated with recombinant FAM20C (800 ng/ml) revealed upregulation of DMP1, OCN and BSP in the experimental groups. *: P value<0.05; **: P value<0.01; ***: P value<0.001.
Table 3.
Serum biochemistry results in the 18-day-old and 42-day-old WT and Fam20c-cKO mice.
Table 4.
Renal mRNA levels of Klotho, NaPi-2a, 1α-hydroxylase, and 24-hydroxylase in the 18-day-old and 42-day-old WT and Fam20c-cKO mice.
Figure 11.
Expression of the Fam20c transgene rescued the defects of Sox2-Cre-Fam20c-cKO mice.
(A) The expression level of the Fam20c transgene was evaluated by real-time PCR using mRNA extracted from the long bones of 6-week-old mice. One line of the transgenic mice (cTg) with the highest expression level of the Fam20c transgene (∼8 folds over that of their WT littermates) was used to rescue the defects in the cKO mice. (B) Plain X-ray analyses of tibias from 6-week-old mice. The bone of mice expressing the Fam20c transgene in the wild type background (cTg) had no difference from the WT. Note the tibia of the cKO mice expressing the Fam20c transgene (cTg+cKO) had no difference from the WT, indicating that the expression of the transgene fully rescued the bone defects of Sox2-Cre-Fam20c-cKO mice (cKO). (C) H&E staining on the sagittal sections of tibias from 6-week-old WT, cTg, cKO and cTg+cKO mice. The tibia cortical bone of the cKO mice had more osteoids (grey areas indicated by arrowheads) and thinner cortical bone, while the cortical bones of the WT, cTg and cTg+cKO mice showed normal structures. These findings further demonstrate that expressing the Fam20c transgene completely rescued the bone defects of the Fam20c cKO mice. (D) Anti-FGF23 IHC staining on the sagittal sections of tibias in the 3-week-old WT, cTg, cKO and cTg+cKO mice. The cortical bone of tibias in the WT, cTg and cTg+cKO mice showed very similar level of FGF23 expression while that of the cKO mice demonstrated a significantly elevated FGF23 expression. These results indicate that expressing the Fam20c transgene rescued the altered FGF23 expression in the cKO mouse bone. Scale bars: 100 µm in C and D.
Figure 12.
FAM20C may mediate phosphate homeostasis via FGF23.
FAM20C, DMP1 and PHEX may share similar mechanisms in their involvement in the bone-kidney axis when regulating phosphate homeostasis via FGF23. FAM20C secreted by osteoblasts/osteocytes may regulate the phosphaturic hormone FGF23 expression in these cells. FGF23 targets the Klotho/FGF receptor (FGFR) complexes in the kidney, reducing the expression of the renal sodium-phosphate cotransporters NaPi-2a/2c in the proximal tubules, thereby accelerates phosphate excretion into the urine and helps maintaining the serum phosphate levels in the normal range. While inactivation of FAM20C led to remarkable reduction of DMP1 in the mice and osteogenic cell lines, and recombinant FAM20C significantly increased the expression of DMP1 in MC3T3-E1 cells, it is unclear if FAM20C regulates FGF23 and phosphate homeostasis via DMP1 (dotted arrow) or FAM20C directly regulates FGF23 (dashed line).