© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 385-393 doi:10.1242/dev.108530
RESEARCH ARTICLE
Zbtb20 regulates the terminal differentiation of hypertrophic
chondrocytes via repression of Sox9
ABSTRACT
The terminal differentiation of hypertrophic chondrocytes is a tightly
regulated process that plays a pivotal role in endochondral ossification.
As a negative regulator, Sox9 is essentially downregulated in terminally
differentiated hypertrophic chondrocytes. However, the underlying
mechanism of Sox9 silencing is undefined. Here we show that the zinc
finger protein Zbtb20 regulates the terminal differentiation of
hypertrophic chondrocytes by repressing Sox9. In the developing
skeleton of the mouse, Zbtb20 protein is highly expressed by
hypertrophic chondrocytes from late embryonic stages. To
determine its physiological role in endochondral ossification, we
have generated chondrocyte-specific Zbtb20 knockout mice and
demonstrate that disruption of Zbtb20 in chondrocytes results in
delayed endochondral ossification and postnatal growth retardation.
Zbtb20 deficiency caused a delay in cartilage vascularization and
an expansion of the hypertrophic zone owing to reduced expression
of Vegfa in the hypertrophic zone. Interestingly, Sox9, a direct
suppressor of Vegfa expression, was ectopically upregulated at both
mRNA and protein levels in the late Zbtb20-deficient hypertrophic
zone. Furthermore, knockdown of Sox9 greatly increased Vegfa
expression in Zbtb20-deficient hypertrophic chondrocytes. Our
findings point to Zbtb20 as a crucial regulator governing the
terminal differentiation of hypertrophic chondrocytes at least
partially through repression of Sox9.
KEY WORDS: Bone development, Hypertrophic chondrocyte, Sox9,
Terminal differentiation, Transcription factor
INTRODUCTION
The terminal differentiation of hypertrophic chondrocytes is essential for
endochondral ossification. During endochondral bone development,
chondrocytes proliferate and then differentiate into hypertrophic
chondrocytes. Hypertrophic chondrocytes express specific extracellular
matrix molecules, such as collagen type X (Col10a1) (Elima et al., 1993;
Linsenmayer et al., 1991), and normally undergo a further maturation
process. Terminal hypertrophic chondrocytes at the chondro-osseous
junction actively express additional molecules, such as matrix
metalloproteinase 13 (Mmp13) (Stickens et al., 2004), osteopontin
(Opn; also known as Spp1) (Chen et al., 1993) and vascular endothelial
growth factor (Vegfa) (Gerber et al., 1999), which are crucial for the
1
Department of Pathophysiology, Second Military Medical University, Shanghai
200433, China. 2Department of Cell Biology, Second Military Medical University,
Shanghai 200433, China. 3Genetic Laboratory of Development and Diseases,
Institute of Biotechnology, Beijing 100071, China. 4Department of Orthopedics,
Changhai Hospital, Shanghai 200433, China. 5Department of Pathology, Changhai
Hospital, Shanghai 200433, China.
*These authors contributed equally to this work
‡
Authors for correspondence ([email protected]; [email protected])
Received 29 January 2014; Accepted 25 November 2014
invasion of blood vessels, osteoclasts and osteoblast precursors from the
perichondrium. Defects in the terminal differentiation of hypertrophic
chondrocytes result in severely delayed formation of the primary
ossification center (POC) and secondary ossification center (SOC)
(Hattori et al., 2010).
Hypertrophic conversion of proliferating chondrocytes during
development of the POC is regulated by Indian hedgehog
(Ihh) and parathyroid hormone related peptide (PTHrP; also
known as Pthlh) (Vortkamp et al., 1996) and several transcription
factors including Runx2 and Runx3 (Takeda et al., 2001; Yoshida
et al., 2004; Zheng et al., 2003) and Mef2c (Arnold et al., 2007).
However, the molecular mechanisms regulating the late and terminal
differentiation of hypertrophic chondrocytes are still poorly
understood.
Sox9 is a multifunctional factor that regulates multiple processes
in endochondral ossification. It plays an essential role in early
chondrogenesis (Akiyama et al., 2002), promotes the hypertrophy of
prehypertrophic chondrocytes (Dy et al., 2012) and inhibits the
subsequent terminal differentiation and Vegfa expression of
hypertrophic chondrocytes (Hattori et al., 2010; Ikegami et al.,
2011; Kim et al., 2011). Sox9 mRNA is highly expressed in
chondrocytes of the proliferating and prehypertrophic zones but
declines abruptly in the hypertrophic zone. Downregulation of Sox9
in the hypertrophic zone of the normal growth plate is essential to
allow vascular invasion, bone marrow formation and endochondral
ossification. However, to our knowledge, the mechanism underlying
its downregulation in hypertrophic chondrocytes remains largely
unknown.
Zinc finger and BTB domain-containing protein 20 (Zbtb20, also
known as DPZF, Hof and Zfp288) is a member of a subfamily of
zinc finger proteins containing C2H2 Krüppel-type zinc fingers and
BTB/POZ domains (Mitchelmore et al., 2002; Zhang et al., 2001).
We and others have reported that Zbtb20 functions primarily as
a transcriptional repressor and plays an essential role in the
specification of pyramidal neurons in the developing hippocampus
(Nielsen et al., 2007; Xie et al., 2010, 2008; Zhang et al., 2012).
Zbtb20 null mice exhibit severe postnatal growth retardation,
metabolic dysfunction and lethality, suggesting that Zbtb20
plays nonredundant roles in multiple organ systems (Sutherland
et al., 2009). Furthermore, conditional gene targeting demonstrates
that Zbtb20 regulates insulin secretion in pancreatic β cells (Zhang
et al., 2012), promotes Toll-like receptor-mediated innate
immune response in macrophages (Liu et al., 2013), and
modulates pain sensation in nociceptive sensory neurons (Ren
et al., 2014).
The growth retardation of Zbtb20 null mice prompted us to
investigate its potential role in skeletal development. Here, we
demonstrate previously undescribed expression of Zbtb20 in
developing cartilage. Conditional deletion of Zbtb20 in developing
cartilage resulted in delayed endochondral ossification. Molecular
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Guangdi Zhou1,*, Xuchao Jiang1,*, Hai Zhang1, *, Yinzhong Lu1, Anjun Liu1,2, Xianhua Ma1, Guan Yang3,
Rui Yang1, Hongxing Shen4, Jianming Zheng5, Yiping Hu2, Xiao Yang3, Weiping J. Zhang1,‡ and Zhifang Xie1,2,‡
analysis showed that Sox9 mRNA and protein are ectopically
upregulated in the Zbtb20-deleted late hypertrophic zone, which
impeded the terminal differentiation of hypertrophic chondrocytes,
subsequent growth plate vascularization and endochondral
ossification. Thus, our findings suggest that Zbtb20 regulates the
terminal differentiation of hypertrophic chondrocytes at least
partially through repressing Sox9.
RESULTS
Expression of Zbtb20 in the developing mouse skeleton
To investigate the potential role of Zbtb20 in skeletal development,
we first characterized its expression pattern in the developing
cartilage by immunostaining. Before embryonic day (E) 13.5,
Zbtb20 protein was undetectable in the cartilage anlage of the long
bones (supplementary material Fig. S1A). It was initially detected
as early as E14.5, when the chondrocytes in the center of the
cartilage anlage start to undergo hypertrophic differentiation
(Fig. 1A). Zbtb20 protein was specifically expressed in the nuclei
of hypertrophic chondrocytes (characterized by an increase in cell
size and by expression of Col10a1), but was undetectable in the
reserve and proliferating columnar chondrocytes as well as in
Co1la1-positive osteoblasts in the perichondrium at embryonic
stages. After birth, the reserve zone began to show expression of
Zbtb20 protein (supplementary material Fig. S1B). The staining
intensity was very weak at birth [ postnatal day (P) 0.5] but rapidly
increased thereafter. At P4, Zbtb20 protein was expressed at high
levels in both the reserve and hypertrophic zone, and to a much
lesser extent in the proliferating zone (Fig. 1B). This expression
pattern of Zbtb20 in the growth plate was maintained at 3 weeks and
3 months of age (Fig. 1C; supplementary material Fig. S1C). These
data suggest that Zbtb20 might play an important role in bone
formation.
Chondrocyte-specific deletion of Zbtb20 results in delayed
endochondral ossification and in postnatal growth
retardation
To explore the role of Zbtb20 in skeletal development in vivo, we
deleted the gene specifically in the cartilaginous template of
endochondral bones by mating mice with a floxed Zbtb20 allele
(Xie et al., 2008) with Col2-Cre transgenic mice, a line that
Development (2015) 142, 385-393 doi:10.1242/dev.108530
was reported to mediate loxP recombination specifically in
cartilaginous structures at E13.5 (Yang et al., 2008; Zhang et al.,
2005), when Zbtb20 protein expression is not detectable in
chondrocytes. Immunohistochemical analysis showed that
Zbtb20 protein is undetectable in chondrocytes of Zbtb20
chondrocyte-specific knockout embryos and postnatal mice
(Zbtb20flox/flox;Col2-Cre, hereafter referred to as Col-ZB20KO)
(Fig. 1D,E), confirming the efficient disruption of the Zbtb20 gene
in chondrocytes.
Born at the expected Mendelian ratios, Col-ZB20KO mice did not
differ in size or weight from their littermate controls at birth
(supplementary material Fig. S2A,B), with normal patterning of
hindlimbs and forelimbs by visual inspection. From P14 onwards,
Col-ZB20KO mice displayed significantly shortened tails, although
they still had similar body weights to wild-type littermates (Fig. 2A,B;
supplementary material Fig. S2B). From P21 onwards, the mutants
showed a slight but significant decrease in the length of long bones
(Fig. 2C,D). There was no significant difference between female
and male mice in phenotype. Heterozygous mice were
morphologically indistinguishable from wild-type littermates in
the appearance and length of long bones (supplementary material
Fig. S2C).
We then performed whole-mount Alcian Blue/Alizarin Red
staining to examine skeletal development in detail. The mutant
newborns revealed a remarkable defect in the ossification of
several cartilage-based structures. In the axial skeleton, the
defects in endochondral ossification were evident in the mutant
vertebral column and sternum (Fig. 2E; supplementary material
Fig. S2D). In the appendicular skeleton at the level of the
hindlimbs, Col-ZB20KO newborns showed an impairment or
absence of endochondral ossification centers in the calcaneus,
the talus and middle phalanges (Fig. 2E). The ossification of
calvaria, clavicle and mandible, however, was indistinguishable
between mutant and wild-type control mice, suggesting that
intramembranous ossification is unaffected in Col-ZB20KO
mice (supplementary material Fig. S2E-G). At P14 and P21,
the impairment of endochondral ossification was still apparent in
the mutants and additionally manifested by a severe delay in
the formation of the SOC (Fig. 2F; supplementary material
Fig. S2H).
Fig. 1. Generation of chondrocyte-specific
Zbtb20 knockout mice. (A) Zbtb20 protein
expression in hypertrophic chondrocytes in the
normal femur at E14.5. Arrows indicate Zbtb20+ cells
(red). Col10a1 and Col1a1 mRNA expression, as
shown by in situ hybridization, indicates the
differentiation of hypertrophic chondrocytes and
osteoblasts at E14.5, respectively. (B,C) Zbtb20
protein was differentially expressed by growth plate
chondrocytes in normal tibia at P4 (B) and P21 (C).
Arrows indicate Zbtb20+ cells. Zbtb20 was highly
expressed in the reserve and hypertrophic zones,
and to a much lesser extent in the proliferating zone.
(D,E) Specific disruption of Zbtb20 in chondrocytes
from Col-ZB20KO mice at E15.5 (D) and P6 (E).
Absence of Zbtb20 staining is indicated by
arrowheads in the hypertrophic zone (D,E) and
reserve zone (E) in the mutant tibia. HZ, hypertrophic
zone; PZ, proliferating zone; RZ, reserve zone. Scale
bars: 100 µm.
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Delayed vascular invasion and expanded hypertrophic zone
in Col-ZB20KO embryos and postnatal mice
To investigate the mechanism underlying the delayed endochondral
ossification in Col-ZB20KO mice, we first performed a histological
examination of the growth plates at different stages. At E15.5, the
central portion of the femur is composed of hypertrophic
chondrocytes, with proliferative chondrocytes in each epiphyseal
region. H&E staining showed that the hypertrophic domains in the
wild-type and Col-ZB20KO femurs were similar, which was
confirmed by the similar expression domains of Col10a1 and Opn
(a marker for mature hypertrophic chondrocytes and osteoblasts)
(McKee et al., 1992) (supplementary material Fig. S3A,B). This
observation indicates normal timing in the initiation of chondrocyte
hypertrophy during the formation of the POC. At E16, however, the
mutants exhibited delayed vascular invasion in tibia [demonstrated
by immunostaining for the endothelial marker CD31 (Pecam1)]
(Cao et al., 2009), resulting in a delay in the formation of the POC
when compared with wild-type littermates (Fig. 3A). By E17.5, the
POCs in both control and mutant tibia had developed but the ColZB20KO tibia contained an expanded hypertrophic zone with a
reduced domain of vascularization (Fig. 3B). At P2, the mutant
hypertrophic zone was 74% longer than that in control mice,
whereas the reserve and proliferating zones were indistinguishable
between the two groups (Fig. 3C,D). This abnormality persisted, but
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Fig. 2. Delayed endochondral ossification and growth retardation caused by conditional deletion of Zbtb20 in chondrocytes. (A) Representative skeletal
preparations from control and mutant mice at P21. Mutant mice had shortened tails (arrowhead). (B) Reduced tail lengths of mutant mice at the indicated
ages (n=6 pairs). (C) Representative images of femur and tibia from control and mutant mice at P21. Both femur and tibia were shorter in mutants.
(D) Reduced femur lengths in mutant mice at the indicated ages (n=6 pairs). (E) Ossification defects in mutant mice demonstrated by whole-mount Alcian Blue
(cartilage) and Alizarin Red (bone) staining. At P0.5, the ossification areas were absent or reduced in the mutant cervical vertebra, tail and hindlimbs, as indicated
by arrowheads. C1 and C2 indicate cervical vertebrae. (F) At P14 and P21, the formation of the tibial secondary ossification center (SOC) was delayed in the
mutant (arrowheads). **P<0.01; *P<0.05. Scale bars: 10 mm in A; 5 mm in C; 1 mm in E,F.
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Development (2015) 142, 385-393 doi:10.1242/dev.108530
Fig. 3. Zbtb20 deletion leads to delayed
vascular invasion and an expanded
hypertrophic zone. (A) Immunostaining of
the endothelial marker CD31 (red) showing
impaired vascular invasion in the mutant
tibia at E16. Nuclei are counterstained with
DAPI (blue). Star indicates the lack of CD31
staining in the center of mutant tibia.
(B) Mutant tibias contained an expanded
hypertrophic zone (arrowheads) with a
reduced domain of vascularization at
E17.5. (C) Representative images of
H&E-stained tibia sections from control and
mutant mice at P2. Mutant tibias had a
lengthened hypertrophic zone
(arrowhead). (D) The increased
hypertrophic zone length in P2 mutant
mice. n=5 pairs; **P<0.01. Scale bars:
100 µm in A; 500 µm in B; 200 µm in C.
to a lesser extent, during the growth of the bones (supplementary
material Fig. S3C).
These findings suggest that the hypertrophic conversion of
columnar chondrocytes remains largely normal in Zbtb20 mutants
during the development of the POC, but subsequent vascular
invasion and ossification are significantly delayed.
Decreased columnar chondrocyte proliferation in ColZB20KO mice
To determine whether the expansion of the hypertrophic zone was
associated with increased chondrocyte proliferation in the ColZB20KO growth plate, we evaluated the proliferation rate of
chondrocytes in vivo by a 2-h BrdU labeling approach. At P2, BrdU
incorporation was comparable between Col-ZB20KO and control
mice in both the reserve and proliferating zones (Fig. 4A,B). At P22,
however, the mutant mice showed a marked decrease in BrdU
incorporation in chondrocytes in the proliferating zone compared
with control mice (Fig. 4A,B). The decreased proliferation rate
of the mutant columnar chondrocytes was consistent with the
observation of decreased long bone length in the mutant mice at
P21.
To exclude the possibility that the expanded hypertrophic zone
resulted from decreased cell death, we examined apoptosis in
hypertrophic chondrocytes in the growth plate using the TUNEL
assay. Very few apoptotic cells were observed at P22, and no
difference was apparent between Col-ZB20KO and control mice
(supplementary material Fig. S4). Together, these data suggest that
the expanded hypertrophic zone in the mutant growth plate is not
due to increased proliferation or decreased apoptosis of columnar
chondrocytes. Therefore, we reason that the hypertrophic zone
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Fig. 4. Decreased proliferation rates in
Col-ZB20KO columnar chondrocytes at P22.
(A) Representative images of BrdU-stained tibia
at the indicated ages. BrdU+ cells in the boxed
areas were counted. For P22, boxed areas are
also magnified in insets. Scale bars: 100 µm.
(B) Percentage of BrdU+ cells within boxed areas
in A. Mutant mice showed a marked decrease in
BrdU+ chondrocytes in the proliferating zone at
P22 but not at P2. **P<0.01; n=5 pairs.
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phenotype may be caused by the differentiation and/or ossification
defect.
Impaired terminal differentiation of hypertrophic
chondrocytes in Col-ZB20KO mice
To determine whether the impaired vascularization in the mutant
hypertrophic zone is associated with the alteration of chondrocyte
biology, we analyzed the differentiation state of hypertrophic
chondrocytes. As mentioned above, hypertrophic chondrocytes
normally undergo a further maturation process. The late differentiated
hypertrophic chondrocytes in the lower hypertrophic zone are
capable of producing a mineralized matrix (Kirsch et al., 1997). von
Kossa staining revealed an expanded mineralized hypertrophic zone
in the mutant (Fig. 5A).
We next examined the expression of a set of differentiation
markers in the Col-ZB20KO mice. At P4, the expression domain of
Col10a1 mRNA was expanded in accordance with a lengthened
hypertrophic zone in the tibia (Fig. 5B). The prehypertrophic zone,
as characterized by high expression of Ihh mRNA (Vortkamp et al.,
1996), appeared normal in the mutant (Fig. 5B). The expression
domains and intensities of Mmp13 and Opn, which are markers for
Development (2015) 142, 385-393 doi:10.1242/dev.108530
terminally differentiated hypertrophic chondrocytes at the chondroosseous border (the lowest region of the growth plate) (Chen et al.,
1993; Mark et al., 1988; Stickens et al., 2004), were comparable
between mutant and control (Fig. 5C; supplementary material
Fig. S5A). We then analyzed angiogenesis-related factors.
Interestingly, in situ hybridization analysis revealed a marked
decrease in Vegfa mRNA expression in the mutant hypertrophic
zone at E16 and P4, which was confirmed by quantitative RT-PCR
analysis in mutant primary hypertrophic chondrocytes at P5
(Fig. 5D-F). However, expression levels of Lect1, which encodes
an angiogenesis-inhibiting factor (Hiraki et al., 1997), were
comparable between control and mutant mice (supplementary
material Fig. S5A). The downregulation of Vegfa was compatible
with delayed cartilage vascularization in the mutant long bone at
E16. Thus, Zbtb20 deletion impairs the terminal differentiation of
hypertrophic chondrocytes.
An expanded hypertrophic zone could also result from a defect in
osteoblast and/or osteoclast function. Therefore, we examined the
expression of osteoblast markers, including Opn for immature
osteoblasts and osteocalcin (also known as Bglap) for terminally
differentiated osteoblasts, in the primary spongiosum (Mark et al.,
Fig. 5. Impaired terminal
differentiation of chondrocytes in
Col-ZB20KO mice. (A) von Kossa
staining showing the expanded
mineralization of the hypertrophic zone
(mHZ) in the growth plate of mutant tibia
at P2. (B-E) In situ hybridization showing
mRNA expression of Col10a1 (B), Ihh
(B), Mmp13 (C) and Vegfa (D,E) in tibia
sections from control and mutant
animals at the indicated ages. Note that
in the mutant the expression domain of
Col10a1 and Ihh was expanded and
Vegfa expression was decreased in the
HZ (arrowheads), whereas Mmp13
expression was comparable between
the two groups. Scale bars: 200 µm.
(F) Real-time RT-PCR analysis of Vegfa
mRNA expression in P5 hypertrophic
chondrocytes. The control Vegfa
expression level was set at 1. **P<0.01;
n=3 pairs. PreH, prehypertrophic zone.
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1988). The mutant showed normal expression domains and
intensities for these genes, as indicative of normal osteoblast
development (supplementary material Fig. S5A). Similarly, the
expression domain of Mmp9, a marker for chondroclast/osteoclast
(Vu et al., 1998), and the number of TRAP+ multinuclear osteoclasts
at the chondro-osseous junction (10.7±4.2 versus 9.4±1.5 TRAP+
cells/section; P=0.6, n=5 pairs) were comparable between control
and mutant (supplementary material Fig. S5A-C).
Together, these data demonstrated that the absence of Zbtb20
causes an impairment in the final steps of chondrocyte terminal
maturation but has little or no impact on the development of
osteoblasts or osteoclasts.
Ectopic dysregulated expression of Sox9 in the late
hypertrophic zone in Col-ZB20KO mice
To elucidate the mechanism by which Zbtb20 regulates the terminal
differentiation and Vegfa expression of hypertrophic chondrocytes,
we examined the expression of some key regulators of chondrocyte
maturation. The expression domains of Runx2 and Mef2c mRNA,
which are positive regulators of chondrocyte maturation that are
normally expressed in the prehypertrophic and hypertrophic zones
(Arnold et al., 2007; Zheng et al., 2003), were broadened in the
mutant, similar to that of Col10a1, whereas the staining intensities
of these genes were indistinguishable between the mutant and
control growth plates (supplementary material Fig. S6).
Sox9 is required for hypertrophy but negatively regulates terminal
differentiation (Dy et al., 2012; Hattori et al., 2010). Consistent with a
previous report (Hattori et al., 2010), in the control growth plate
Sox9 mRNA was highly expressed in chondrocytes of the
proliferating and prehypertrophic zones but downregulated abruptly
390
in the hypertrophic zone. In the mutant, however, the expression
domain extended to the whole hypertrophic zone at P4 (Fig. 6A). This
observation was confirmed by immunohistochemistry showing that
the expression of Sox9 protein expanded to the chondro-osseous
border in the mutant, whereas it was excluded from the late
hypertrophic zone in control mice (Fig. 6B). Ectopic Sox9 expression
in the late hypertrophic zone was also observed in the mutant embryos
at E15.5 (Fig. 6C). The expression domain of Sox5 protein, which is
reported to coexpress and cooperate with Sox9 in chondrogenesis
(Han and Lefebvre, 2008), remained unchanged in the mutant
(Fig. 6D). Thus, Zbtb20 is required specifically for Sox9
downregulation in the hypertrophic chondrocytes. As Sox9 is a
direct suppressor of Vegfa expression (Hattori et al., 2010), the
abnormal downregulation of Vegfa and upregulation of Sox9 in
Zbtb20-deleted hypertrophic zones supports the hypothesis that
Zbtb20 inhibits Sox9 expression, thereby positively regulating Vefga
expression and chondrocyte terminal maturation.
To further examine the functional involvement of Sox9 in the
regulation of chondrocyte terminal maturation by Zbtb20, we next
investigated the effects of siRNA-mediated knockdown of Sox9
in wild-type and Zbtb20-deleted hypertrophic chondrocytes.
Primary hypertrophic chondrocytes isolated from both wild-type
and Col-ZB20KO mice expressed high levels of Col10a1 and
alkaline phosphatase (Alpl) mRNA (Miao and Scutt, 2002), two
widely used markers for hypertrophic chondrocytes, and their
hypertrophic characteristics were maintained at 48 h post-seeding
(supplementary material Fig. S7). The wild-type hypertrophic
chondrocytes transfected with Sox9 siRNA failed to show a
significant increase in Vegfa mRNA levels, probably owing to
their relatively low basal level of Sox9 expression (Fig. 7). By
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Fig. 6. Ectopic upregulation of Sox9 expression in the late
hypertrophic zone in Col-ZB20KO mice. (A) In situ
hybridization showing the expression domain of Sox9 mRNA in
control and mutant tibia at P4. In contrast to the control, Sox9
mRNA expression was expanded to the late hypertrophic zone in
the mutant (arrowhead). (B-D) Immunostaining of Sox9 and
Sox5 (red) in the hypertrophic zone of the tibia at P4 and E15.5.
Dotted lines demarcate the prehypertrophic and hypertrophic
zones. In the mutant, Sox9 protein expression was upregulated in
the late hypertrophic zone (B,C), whereas Sox5 protein was
unaffected (D). Nuclei were counterstained with DAPI (blue).
Scale bars: 200 µm.
Fig. 7. Knockdown of Sox9 restores Vegfa expression in Col-ZB20KO
hypertrophic chondrocytes. Primary wild-type (WT) and mutant
hypertrophic chondrocytes were transfected with control siRNA or Sox9 siRNA
and Sox9 and Vegfa expression levels were analyzed after 48 h. (A) Western
blot analysis showed that Sox9 protein levels are markedly reduced by Sox9
siRNA in wild-type or mutant hypertrophic chondrocytes. The blot is
representative of three independent experiments, with semi-quantification
shown to the right. (B) mRNA expression levels of Sox9 and Vegfa were
determined by quantitative RT-PCR. Data are presented as fold increase
relative to control siRNA-treated cells (set at 1) for each group. The mean value
of three independent experiments is shown. **P<0.01, *P<0.05.
contrast, the mutant hypertrophic chondrocytes treated with Sox9
siRNA exhibited a dramatic increase in Vegfa mRNA levels
(Fig. 7). Taken together, these findings indicate that Zbtb20
positively regulates chondrocyte terminal maturation at least
partly via downregulation of Sox9 expression.
Indirect regulation of Sox9 expression by Zbtb20
To explore the molecular mechanisms underlying the regulation of
Sox9 expression by Zbtb20, we first performed chromatin
immunoprecipitation (ChIP) analysis to determine whether Zbtb20
directly binds the Sox9 promoter in vivo. Using ChIP grade antiZbtb20 antibodies (Xie et al., 2008), we did not detect any
significant occupancy by Zbtb20 in the 1 kb region upstream of the
Sox9 transcription start site in isolated mouse primary chondrocytes
(supplementary material Fig. S8A-C), whereas the positive control
with anti-acetyl-histone H3 antibody showed robust enrichment
of acetyl-H3 in the Sox9 promoter. A reporter assay using a
luciferase reporter construct (Sox9-Luc) harboring the mouse Sox9
promoter region (−2196 to +27) showed that overexpression of
Zbtb20 in HEK293T cells had no significant effects on the
transcriptional activity of the Sox9 promoter (supplementary
material Fig. S8D). Taken together, these data suggest that
Zbtb20 regulates Sox9 expression via an indirect, as yet
unidentified, mechanism.
DISCUSSION
In this study, we examined the role of Zbtb20 in chondrocytes. Our
findings provide compelling evidence that Zbtb20 is a novel
essential regulator of cartilage development. First, Zbtb20 is
expressed in developing chondrocytes, with expression most
abundant in hypertrophic chondrocytes. Second, and most
Development (2015) 142, 385-393 doi:10.1242/dev.108530
importantly, conditional deletion of Zbtb20 in developing cartilage
results in delayed endochondral ossification and postnatal growth
retardation, with a delay in cartilage vascularization and an
expansion of the hypertrophic zone associated with reduced
expression of Vegfa in the hypertrophic zone. Lastly, Sox9, a
direct suppressor of Vegfa expression, is ectopically upregulated in
the late hypertrophic zone by the loss of Zbtb20, which at least
partially impairs the terminal differentiation of hypertrophic
chondrocytes and subsequent growth plate vascularization.
The initiation of chondrocyte hypertrophy during the
development of the POC is regulated by genes including Ihh,
Runx2/3 and Mef2c. However, the most terminally differentiated
hypertrophic chondrocytes actively express genes that allow the
invasion of blood vessels, osteoclasts and osteoblast precursors from
the perichondrium, suggesting additional regulatory mechanisms at
this late stage. Sox9 has previously been reported to promote
hypertrophy but to inhibit terminal differentiation, indicating that its
downregulation is a necessary event to allow this process to occur
(Dy et al., 2012; Hattori et al., 2010). Interestingly, loss of Zbtb20
resulted in the ectopic expression of Sox9 in the late hypertrophic
zone, suggesting that Zbtb20 positively regulates terminal
maturation, at least partially, by inhibiting Sox9 expression.
Although this fits the notion that Zbtb20 functions primarily as a
transcriptional repressor (Liu et al., 2013; Xie et al., 2008; Zhang
et al., 2012), ChIP and luciferase reporter assays did not support
direct binding of Zbtb20 to the Sox9 promoter to regulate
transcriptional activity. One possibility is that Zbtb20 regulates
Sox9 transcription via a cis-acting element that is located outside of
the sequence that we analyzed, such as a distal enhancer.
Alternatively, Zbtb20 might regulate Sox9 expression via an
indirect mechanism. These possibilities are currently under further
investigation.
However, Col-ZB20KO mice showed less severe retardation in
vascular invasion, when compared with transgenic mice
misexpressing Sox9 in hypertrophic chondrocytes under the
control of a BAC Col10a1 promoter (Hattori et al., 2010). Indeed,
the expression of Vegfa was completely inhibited in the Sox9overexpressing hypertrophic zones but only downregulated in
corresponding Zbtb20-deleted areas. Similarly, expression domains
of Mmp13 and Opn were decreased in the Sox9 transgenic
hypertrophic zones but appeared unchanged in corresponding
Col-ZB20KO zones. These discrepancies could result from
differences in Sox9 expression levels and domain between the two
animal models. In Zbtb20-deleted hypertrophic zones, Sox9 protein
levels were increased but were similar to those in the proliferating
zones, as shown by comparable immunostaining intensity in
these two regions in the same section (Fig. 6B). In the Sox9
overexpression model, however, the Sox9 immunostaining intensity
was stronger than that in the proliferating zone in the same section,
suggesting a much higher dosage of Sox9 in the hypertrophic zones.
Furthermore, in Col-ZB20KO mice upregulation of Sox9 was
restricted to the late hypertrophic zones, whereas in the transgenic
mice Sox9 was highly overexpressed by all chondrocytes expressing
Col10a1, which spans from the prehypertrophic to the terminal
hypertrophic zones; indeed, the prehypertrophic zone, as
characterized by strong expression of Ihh and Runx2, was
extended and hypertrophic chondrocytes were retained in a
prehypertrophic/early hypertrophic state in the Sox9 transgenic
cartilage (Hattori et al., 2010). In addition, the other reported
hypertrophic chondrocyte-specific Sox9 overexpression mouse
model, which was constructed by mating CAG-mRFP1floxed-Sox9EGFP transgenic mice and Col10a1-Cre mice, exhibited delayed
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chondrocyte terminal differentiation but did not show Mmp13
downregulation (Kim et al., 2011). These discrepancies between the
different animal models indicate that some of the functions of Sox9
might be dosage dependent.
A microdeletion in human chromosome 3q13.31, which
encompasses five RefSeq genes including ZBTB20, has recently
been reported (Molin et al., 2012). Patients with this microdeletion
share several major characteristics, including significant developmental
delay. We thus speculate that ZBTB20 might play crucial roles in human
skeletal development, similar to that in mice.
MATERIALS AND METHODS
Generation of Zbtb20 conditional knockout mice
Mouse strains carrying floxed Zbtb20 were described previously (Xie et al.,
2008). To achieve chondrocyte-specific deletion of floxed Zbtb20, mice were
crossed with transgenic mice expressing Cre recombinase under the control
of regulatory regions of the mouse Col2a1 gene (Zhang et al., 2005). All
animal experimental procedures were preformed according to institutional
guidelines of the Second Military Medical University, Shanghai, China.
Sex-matched littermates carrying floxed Zbtb20 were used as controls.
Immunohistochemistry
Tissue was fixed with 4% paraformaldehyde ( pH 7.4) and decalcified in
0.5 M EDTA from P5 onwards, then embedded in paraffin or OCT (Thermo
Scientific, 22-110-617). Immunostaining for paraffin sections or
cryosections (CD31) was performed using the protocol described
previously (Xie et al., 2010). The following antibodies were used: Zbtb20
monoclonal antibody 9A10 (made in our lab, 1:1000) (Xie et al., 2008),
CD31 (Abcam, ab28364, 1:2000), Sox5 and Sox9 (Santa Cruz, sc-20091,
sc-20095; 1:2000), All secondary antibodies were from Vector Laboratories
(PI-1000, PI-2000; 1:500).
In situ hybridization
Riboprobes labeled with digoxygenin-UTP were transcribed from cDNA
clones (supplementary material Table S1). Tissue samples were fixed
overnight in 4% paraformaldehyde ( pH 9.0) and decalcified in 0.5 M EDTA
from P5 onwards. In situ hybridization of cryosections was performed as
previously described (Xie et al., 2010).
Skeletal analyses
Fetal and postnatal skeletons were freed from adherent tissue, fixed in 95%
ethanol and stained for cartilage with Alcian Blue and counterstained for
bone with Alizarin Red as described previously (Ovchinnikov, 2009).
Development (2015) 142, 385-393 doi:10.1242/dev.108530
paraffin knee joint sections was performed using a similar protocol to
that described previously (Xie et al., 2010), except that antigen retrieval
used 95% (v/v) formamide. BrdU+ cells were counted in designated
areas on five semi-serial sections through the center of proximal tibia or
distal femur from each animal. Apoptotic chondrocytes were detected
using the In Situ Cell Death Detection Kit (Roche Diagnostics).
Real-time RT-PCR
Hypertrophic cartilage was microdissected from P5 long bones and ribs, and
digested with collagenase as described (Belluoccio et al., 2010). Total RNA
was extracted using TRIzol (Life Technologies) and digested with DNase I
to eliminate any contaminating genomic DNA. cDNA was synthesized
by reverse transcription and amplified in triplicate using the SYBR Green
PCR assay (QuantiFast SYBR Green PCR Kit, Qiagen, 204057). RNA
expression levels were normalized to that of internal control 36B4 (Rplp0).
The primers used are listed in supplementary material Table S2.
RNA interference analysis
Pooled primary resting and hypertrophic chondrocytes from P2 wild-type or
P7 Col-ZB20KO mice were isolated as described above and subjected to
further digestion with 1.0 mg/ml of pronase (Roche) for 3 h at 37°C with slow
agitation. The resting chondrocytes were harvested, while the hypertrophic
chondrocytes were washed twice in PBS and electroporated with 500 nM Sox9
small interfering RNA (siRNA; sc-36534, Santa Cruz) or control siRNA (sc36869, Santa Cruz) diluted in BTXpress High Performance Electroporation
Solution (Harvard Apparatus). Electroporation was performed in a 2 mm
cuvette with settings of one pulse of 170 Volts for 15 ms using a BTX
ECM830 electroporator. Chondrocytes were then plated and cultured in
DMEM supplemented with 10% FBS (Hyclone), 25 μg/ml L-ascorbic acid
2-phosphate, 10 mM β-glycerophosphate and 1× penicillin/streptomycin/
amphotericin B solution (Sigma). The cells were collected 48 h after
electroporation and mRNA or total proteins were extracted. Sox9, Vegfa,
Col10a1 and Alpl mRNA levels were analyzed by real-time RT-PCR,
standardized to 36B4. Sox9 protein levels were assessed by western blotting.
The intensity of the signals was evaluated by densitometry and semi-quantified
using the ratio between the protein of interest and β-actin for each experiment.
Reporter gene assay
The Sox9 luciferase reporter was constructed by cloning the mouse Sox9
promoter region (−2196 to +27) into the XhoI and BglII sites of the pGL4.10
vector (Promega). HEK293T cells were co-transfected with 1 µg reporter
construct, 100 ng β-galactosidase expression vector as internal control and
1 µg either Zbtb20 expression vector or a mock vector. Luciferase and βgalactosidase were measured 48 h post-transfection as described previously
(Xie et al., 2008).
Histological techniques
ChIP assay
Fragmented chromatin from mouse P5 primary chondrocytes was incubated
overnight with specific antibodies: anti-acetylated histone H3 (Millipore,
06-942), anti-Zbtb20 (Xie et al., 2010), or normal mouse IgG (Upstate,
12-371), followed by incubation with Dynabeads Protein G (Invitrogen).
Purified chromatin DNA was subjected to conventional and quantitative
PCR with primers for core promoter regions of Sox9 (supplementary
material Table S2). Two independent ChIP reactions were performed.
Statistical analysis
Results are expressed as mean±s.d. Statistical comparison between
genotypes was performed with a two-tailed Student’s t-test. Proliferative
index was analyzed by one-way analysis of variance (ANOVA). Data from
the RNA interference experiment were analyzed using a two-way ANOVA.
P<0.05 was considered significant.
Acknowledgements
Proliferation and apoptosis assays
We thank Y. Zhang, Q Hao and L Gao for technical assistance.
Cell proliferation was analyzed using BrdU (5-bromo-2′-deoxyuridine)
incorporation. BrdU was administered by intraperitoneal injection
(100 mg/kg) 2 h prior to sacrifice. BrdU immunoperoxidase staining of
Competing interests
392
The authors declare no competing financial interests.
DEVELOPMENT
Tissue was fixed and decalcified as for immunohistochemistry. Paraffin
sections (4 µm) were stained with Hematoxylin and Eosin (H&E), Alcian
Blue or Safranin O as described (Schmitz et al., 2010). To measure growth
plate length, five semi-serial sections (20 µm between each level) through
the center of proximal tibia from each mouse were cut, stained with H&E,
and photographed for measurement using Image-Pro Plus software (Media
Cybernetics). For von Kossa staining, nondecalcified tissue was used.
Sections were deparaffinized and immersed in 1% silver nitrate solution
under ultraviolet light for 45 min. Slides were rinsed in distilled water and
immersed in 3% sodium thiosulfate for 5 min. Slides were rinsed again in
distilled water and counterstained with Nuclear Fast Red for 5 min.
Osteoclast activity was detected by staining for tartrate-resistant acid
phosphatase (TRAP) using a leukocyte acid phosphatase kit (SigmaAldrich). At least five semi-serial sections through the center of proximal
tibia from each mouse were used to reveal mean TRAP+ cell counts at the
chondro-osseous border.
Author contributions
Z.X. and W.J.Z. conceived and designed the experiments. Z.X., G.Z., X.J.,
H.Z., Y.L., X.M., A.L., G.Y., R.Y. and H.S. performed the experiments. J.Z., Y.H.
and X.Y. analyzed the data. Z.X. and W.J.Z. wrote the paper.
Funding
This work was supported by grants from the China National Natural Science
Foundation [31171395, 31025013, 81130084, 31470759] and the National Key
Basic Science Research and Development Program [2012CB524904,
2013CB530603].
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.108530/-/DC1
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