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. 2015 Jun 22;10(6):e0130618.
doi: 10.1371/journal.pone.0130618. eCollection 2015.

Deletion of IFT80 Impairs Epiphyseal and Articular Cartilage Formation Due to Disruption of Chondrocyte Differentiation

Xue Yuan  1 , Shuying Yang  2
Affiliations

Deletion of IFT80 Impairs Epiphyseal and Articular Cartilage Formation Due to Disruption of Chondrocyte Differentiation

Xue Yuan et al. PLoS One. .

Abstract

Intraflagellar transport proteins (IFT) play important roles in cilia formation and organ development. Partial loss of IFT80 function leads Jeune asphyxiating thoracic dystrophy (JATD) or short-rib polydactyly (SRP) syndrome type III, displaying narrow thoracic cavity and multiple cartilage anomalies. However, it is unknown how IFT80 regulates cartilage formation. To define the role and mechanism of IFT80 in chondrocyte function and cartilage formation, we generated a Col2α1; IFT80f/f mouse model by crossing IFT80f/f mice with inducible Col2α1-CreER mice, and deleted IFT80 in chondrocyte lineage by injection of tamoxifen into the mice in embryonic or postnatal stage. Loss of IFT80 in the embryonic stage resulted in short limbs at birth. Histological studies showed that IFT80-deficient mice have shortened cartilage with marked changes in cellular morphology and organization in the resting, proliferative, pre-hypertrophic, and hypertrophic zones. Moreover, deletion of IFT80 in the postnatal stage led to mouse stunted growth with shortened growth plate but thickened articular cartilage. Defects of ciliogenesis were found in the cartilage of IFT80-deficient mice and primary IFT80-deficient chondrocytes. Further study showed that chondrogenic differentiation was significantly inhibited in IFT80-deficient mice due to reduced hedgehog (Hh) signaling and increased Wnt signaling activities. These findings demonstrate that loss of IFT80 blocks chondrocyte differentiation by disruption of ciliogenesis and alteration of Hh and Wnt signaling transduction, which in turn alters epiphyseal and articular cartilage formation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Deletion of IFT80 in the embryonic stage.
(A) Line drawing showing the timing of tamoxifen administrations for IFT80 deletion in embryonic stage. Arrows above the line indicate the time of tamoxifen administration to the pregnant females at 14.5, 16.5, and 18.5 days postcoitus. The blue arrow below the line indicates the harvest time. (B) Image of IFT80 f/f and Col2α1; IFT80 f/f newborn mice exposed to tamoxifen at E14.5, E16.5, and E18.5. Arrows indicate the shortened limbs. (C) Alizarin red and Alcian blue staining of hindlimbs of IFT80 f/f and Col2α1; IFT80 f/f newborn mice. (D) Western blot analysis of IFT80 expression in the cartilage of IFT80 f/f and Col2α1; IFT80 f/f mice. IFT80 protein level was normalized to GAPDH (n = 3, *P<0.001, significantly different from the IFT80 f/f group). (E) qPCR of IFT80 expression. IFT80 expression level was normalized to GAPDH (n = 3, *P<0.001, significantly different from the IFT80 f/f group).
Fig 2
Fig 2. Histological examination of the tibial growth plates of newborn IFT80 f/f mice and Col2α1; IFT80 f/f mice.
(A and A’) Histological examination of tibial growth plates of newborn IFT80 f/f mice (A) and Col2α1; IFT80 f/f mice (A’) by H&E staining. Mice were exposed to tamoxifen at E14.5, E16.5, and E18.5. The growth plate can be divided transversely into resting zone (RZ), proliferation zone (PZ), prehypertrophic zone, (PHZ) and hypertrophic zone (HZ). (B and B’) Safranin O staining of tibial growth plates of newborn IFT80 f/f mice (B) and Col2α1; IFT80 f/f mice (B’). (C-F and C’-F’) Histological examination of chondrocytes morphology in higher magnification view. Chondrocytes in the resting zone of Col2α1; IFT80 f/f mice (C’) displayed hypercellularity compared to those in IFT80 f/f mice (C). Cells were less organized in the proliferation zone of Col2α1; IFT80 f/f mice (D’) compared to those in IFT80 f/f mice (D). In the prehypertrophic and hypertrophic zones, chondrocytes were larger, with less cell density, in Col2α1; IFT80 f/f mice (E’ and F’) than those in IFT80 f/f mice (E and F). (G) Quantitative analysis of cartilage length (n = 3). (H) Quantitative analysis of each zone’s lengths. Data was reported as a ratio of the zone length to the total growth plate length (n = 3).
Fig 3
Fig 3. Endochondral bone ossification was examined of the tibial growth plates of newborn IFT80 f/f mice and Col2α1; IFT80 f/f mice using Alizarin Red/Von Kossa staining.
Mice exposed to tamoxifen at E14.5, E16.5, and E18.5. (A) Alizarin Red staining of the tibial section. Bone volume (BV) and tissue volume (TV) were quantified using Image J (n = 3). (B) Von Kossa staining of the tibial section. Fast green was used as a counter stain. BV/TV were quantified using Image J (n = 3).
Fig 4
Fig 4. Deletion of IFT80 in the postnatal stage.
(A) Line drawing showing the timing of tamoxifen administration for IFT80 deletion in postnatal stage. Arrows above the line indicate the time of tamoxifen injection at P4-7 and P14-17. The blue arrow below the line indicates the harvest time P30. (B) Image of IFT80 f/f and Col2α1; IFT80 f/f P30 mice, which were administered tamoxifen at P4-7 and P14-17. (C) Western blot analysis of IFT80 expression in the cartilage of IFT80 f/f and Col2α1; IFT80 f/f mice at P30. IFT80 protein level was normalized to GAPDH (n = 3, *P<0.001, significantly different from the IFT80 f/f group). (D) qPCR of IFT80 expression. IFT80 expression level was normalized to GAPDH (n = 3, *P<0.001, significantly different from the IFT80 f/f group). (E) The average body weight of tamoxifen-injected IFT80 f/f and Col2α1; IFT80 f/f mice (n = 6, #p = 0.0022, *p<0.0001, significantly different from the IFT80 f/f group). (F) The average body length of tamoxifen-injected IFT80 f/f and Col2α1; IFT80 f/f mice (n = 6, *p<0.0001, significantly different from IFT80 f/f group).
Fig 5
Fig 5. Histological examination of tibial growth plates of P30 mice.
Mice were administered tamoxifen at P4-7 and P14-17. (A-B and A’-B’) H&E staining of tibial growth plates of P30 IFT80 f/f mice (A—B) and Col2α1; IFT80 f/f mice (A’—B’). (C) Quantitative analysis of the length of the proliferation zone and the hypertrophic zone. The length of the proliferation zone in Col2α1; IFT80 f/f mice was significantly reduced compared to IFT80 f/f mice (n = 3). (D and D’) Safranin O stained tibial growth plates of P30 IFT80 f/f mice (D) and Col2α1; IFT80 f/f mice (D’). (E and E’) H&E staining of articular cartilage of P30 IFT80 f/f mice (E) and Col2α1; IFT80 f/f mice (E’). The green arrows indicate the ossified bone, and the blue double-ended arrows measure the distance between the tidemark and the surface of the articular cartilage. (F and F’) Safranin O stained articular cartilage of P30 IFT80 f/f mice (F) and Col2α1; IFT80 f/f mice (F’). (G) Quantification of the distance between the articular surface and the tidemark in the tibias of IFT80 f/f and Col2α1; IFT80 f/f mice (n = 3). (H) Quantification of the number of chondrocytes in the articular cartilage between the articular surface and tidemark (40× magnification fields) (n = 3).
Fig 6
Fig 6. Significant cilia formation defects in the growth plate of Col2α1; IFT80 f/f mice.
(A) Immunofluorescence analysis of primary cilia in the tibial growth plate of newborn mice exposed to tamoxifen at E14.5, E16.5, and E18.5 (n = 3). Immunostaining of primary cilia was performed with acetylated α-tubulin (axoneme, cyan) antibody. DAPI (nuclear marker) staining was used as counterstain. Scale bars represent 100 μm. (B) Quantification of the ciliated cell population in resting zone, proliferation zone and hypertrophic zone (n = 3, *p<0.0001, significantly different from the IFT80 f/f group). (C) Immunofluorescence analysis of primary cilia in the cartilage of P30 mice exposed to tamoxifen from P4-P7 and P14-P17 (n = 3). (D) Quantification of the ciliated cell population in epiphyseal plate and articular cartilage (n = 3, *p<0.0001, significantly different from the IFT80 f/f group).
Fig 7
Fig 7. Deletion of IFT80 in chondrocytes caused cilia loss and defects in chondrogenic differentiation.
(A) Immunofluorescence analysis of primary cilia in chondrocytes derived from P10 IFT80 f/f and Col2α1; IFT80 f/f mice, which were administered tamoxifen at P4-7. (n = 4). Primary cilia were stained with acetylated α-tubulin (axoneme, cyan) antibody. DAPI (nuclear marker) staining was used as counterstain. Scale bars represent 50 μm. (B) Quantification of the ciliated cell population in chondrocytes derived from IFT80 f/f and Col2α1; IFT80 f/f mice (n = 4). (C) Proteoglycan production was assessed with Alcian blue staining at day 21 after chondrogenic induction. Quantitative assessment of Alcian blue staining was performed by measuring the optical density of the dyes extracted by 6 M guanidine-HCl (n = 3). (D) Chondrocyte marker genes expression profiles by qPCR. Col2α1; IFT80 f/f chondrocytes in chondrogenic medium showed significantly lower expression of Sox9, Aggrecan, and type X collagen (n = 3).
Fig 8
Fig 8. Deletion of IFT80 in chondrocytes caused impaired Hh signaling, and enhanced Wnt signaling activity.
Primary chondrocytes were derived from P10 IFT80 f/f and Col2α1; IFT80 f/f mice, which were administered tamoxifen at P4-7. (A) Reporter assay showing Gli responsive luciferase (8×Gli-Luc) activity. Shh (1 μg/mL) treatment resulted in significant increases in luciferase activity in IFT80 f/f chondrocytes, whereas less stimulation with Shh was observed in the Col2α1; IFT80 f/f chondrocytes (n = 3). (B) Wnt luciferase reporter assay with or without Wnt3a stimulation. 100 ng/mL Wnt3a significantly promoted higher luciferase activity in Col2α1; IFT80 f/f chondrocytes compared to IFT80 f/f chondrocytes (n = 3).
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