RESEARCH ARTICLE 3991
Development 136, 3991-4000 (2009) doi:10.1242/dev.042150
Two populations of endochondral osteoblasts with
differential sensitivity to Hedgehog signalling
Christina Lindsey Hammond* and Stefan Schulte-Merker
Hedgehog (Hh) signalling has been implicated in the development of osteoblasts and osteoclasts whose balanced activities are
critical for proper bone formation. As many mouse mutants in the Hh pathway are embryonic lethal, questions on the exact effects
of Hh signalling on osteogenesis remain. Using zebrafish, we show that there are two populations of endochondral osteoblasts
with differential sensitivity to Hh signalling. One, formed outside the cartilage structure, requires low levels of Hh signalling and
fails to differentiate in Indian hedgehog mutants. The other derives from chondrocytes and requires higher levels of Hh signalling
to form. This latter population develops significantly earlier in mutants with increased Hh signalling, leading to premature
endochondral ossification, and also fails to differentiate in Indian hedgehog mutants, resulting in severely delayed endochondral
ossification. Additionally, we demonstrate that the timing of first osteoclast activity positively correlates to Hh levels in both
endochondral and dermal bone.
INTRODUCTION
Bone formation in vertebrates is regulated by a balance between the
activities of cells that secrete bone matrix, the osteoblasts, and those
that reabsorb it, the osteoclasts. The tight regulation of the activities
of these two cell types is of crucial importance.
Osteoclasts are of haematopoietic origin from the monocyte/
macrophage lineage, and their differentiation is controlled by
RANKL (Tnfsf11 – Mouse Genome Informatics) and Csf1 (Kong
et al., 1999; Yasuda et al., 1998). Osteoblasts, by contrast, are
derived from mesenchymal cells; Runx2 is the master regulator of
bone and cartilage cell fate, whereas osterix (Sp7 – Mouse Genome
Informatics) is the master regulator of osteoblastogenesis
(Nakashima et al., 2002). Additionally there are two types of bone:
endochondral bone, in which osteoblasts lead to mineralisation of
an existing cartilage matrix; and dermal bone, in which bone is
formed de novo.
Until recently the majority of the work on bone development has
been undertaken either in the mouse, or using mammalian in vitro
cell-culture systems; however, as in many other cases in
developmental biology, the zebrafish is an excellent model in which
to study bone formation, particularly as transgenic lines allow us to
follow the behaviour of cells in vivo. Despite some differences, e.g.
the appendicular skeleton, zebrafish and the related teleost medaka
show remarkable similarities in bone formation to higher
vertebrates, particularly in their craniofacial development (Renn and
Winkler, 2009; Wagner et al., 2003; Yelick et al., 1996; Yelick and
Schilling, 2002).
The Hedgehog (Hh) signalling pathway, principally responding
to Indian hedgehog, has long been linked to endochondral bone
formation. Ihh is expressed by chondrocytes; signals to both
chondrocytes and the adjacent perichondral cells and exerts control
over the timing of chondrocyte differentiation (St-Jacques et al.,
1999; Vortkamp et al., 1996). Analysis of the knockout mouse
Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences & University
Medical Centre, 3584CT Utrecht, The Netherlands.
*Author for correspondence ([email protected])
Accepted 22 September 2009
showed that Indian hedgehog regulates chondrocyte proliferation
and is required for osteoblast differentiation (St-Jacques et al., 1999).
More recently, the Hedgehog receptor protein patched 1 (patched
homolog 1, Ptch1 – Mouse Genome Informatics) has been
implicated in the regulation of both osteoblast and osteoclast fate in
the mouse (Mak et al., 2008a; Ohba et al., 2008). Two papers
analysing different Ptch1 mouse mutants reached somewhat
different conclusions from their analyses. In mouse, Ptch1 null
animals die before bone formation; thus only heterozygous carriers
of the Ptch1 gene or conditional knockouts are available for analysis.
The Chung group, studying Ptch1 heterozygous animals saw
increased bone mass in these animals, which they attributed to
increased osteoblast differentiation, through a mechanism whereby
reduction of the Gli3 repressor led to increased sensitivity to Runx2
expression (Ohba et al., 2008). By contrast, the Yang group,
analysing a conditional knockout, in which Ptch1 was deleted in
cells expressing osteocalcin (Bglap1 – Mouse Genome Informatics),
i.e. mature osteoblasts, saw reduced bone mass in the adult animals,
which they attributed to increased differentiation of osteoclasts
under the control of RANKL (Mak et al., 2008a).
As many of the murine Hedgehog signalling pathway mutants are
embryonic lethal, we chose to undertake studies in the zebrafish,
which have a number of advantages that make them ideal for studies
of this nature. Importantly, zebrafish carrying null mutations for a
number of members of the Hedgehog signalling pathway survive
long enough to undertake an analysis of early bone differentiation.
These include three mutants thought to lead to activation of the
pathway: patched 1, patched 2 and dre (suppressor of fused) (sufu –
ZFIN) (Koudijs et al., 2008; Koudijs et al., 2005) and an ihha mutant
carrying an early stop, which we believe to be a null mutant. As an
independent approach we also made use of drugs that act directly on
smoothened to titrate the effects on the pathway. Cyclopamine is a
small molecule antagonist that directly binds to smoothened and
inhibits its function (Chen et al., 2002). Purmorphamine, by contrast,
is a smoothened agonist (Sinha and Chen, 2006), which has been
previously observed to induce osteogenic differentiation in cell
culture in some studies (Wu et al., 2004), while inhibiting osteoblast
differentiation in others (Plaisant et al., 2009). Thus different culture
systems can give conflicting results on the effect of activation of
DEVELOPMENT
KEY WORDS: Hedgehog, Chondrocyte, Osteoblast, Zebrafish
3992 RESEARCH ARTICLE
MATERIALS AND METHODS
In situ hybridisation
In situ labelling was performed as previously described (Schulte-Merker,
2002). The markers used were osterix, osteocalcin, RANKL and
collagen1alpha2 (for primers, see Table S1 in the supplementary material).
In all cases in situs were carried out with mutant and siblings, analysed blind
and subsequently genotyped by DNA isolation and sequencing, using the
primers shown in Table S1 in the supplementary material.
Mutant lines
ptc1, ptc2 and dre mutant lines are ptc1hu1602, lep(ptc2)tj222 and dretm146d,
respectively (Koudijs et al., 2008; Koudijs et al., 2005). Ihhahu2131 stocks
were obtained from the Sanger Centre (Cambridge, UK). Lines were crossed
to the transgenic lines Tg(osterix:nuGFP) line (Spoorendonk et al., 2008) or
Tg(kdr-l:gfp)s843, originally referred to as Tg(flk1:EGFP)s843 (Jin et al.,
2005).
Drug treatment
Cyclopamine (Sigma-Aldrich) was used at a concentration of 75 M,
added directly to the E3 medium in which the larvae were grown.
Purmorphamine (Calbiochem) was used at a concentration of 20 M. For
both drugs, treatment began at 2 days post-fertilisation (dpf) to prevent
gross effects on patterning or heart formation, and solutions were replaced
approximately every 12 hours. Controls were incubated in E3, to which
appropriate amounts of ethanol (cyclopamine) or DMSO
(purmorphamine) were added.
BrdU labelling
BrdU labelling was performed as previously described (Kimmel et al.,
1998). In short, BrdU was diluted to a working concentration of 3 mM in E3
medium. Embryos were incubated in this solution overnight and fixed
subsequently.
Immunohistochemistry
Embryos were briefly fixed in 4% PFA and stored in MeOH. Embryos were
rehydrated, blocked in PBS with 5% lamb serum and incubated with 1/100
anti-BrdU primary antibody (DAKO), anti-GFP (1/500 Torrey Pines
Biolabs) or anti-Collagen II (1/500 DSHB) overnight at 4°C. Embryos were
washed extensively then incubated in Alexa-Fluor secondary antibodies
(Molecular probes) diluted 1/500 in blocking solution for 3 hours at room
temperature. Embryos were washed extensively in the dark, with DAPI
(Sigma-Aldrich) added at 1/1000 to one wash, then mounted for analysis.
For DAB staining the secondary antibody was anti-mouse IgG-biotin,
followed by incubation in ABC reagent (DAKO) and development of the
DAB stain.
BAC transgenesis
mCherry was recombined directly after the ATG site of the Collagen 2a1 on
a bacterial artificial chromosome (BAC) clone, using similar principles to
those previously (Kimura et al., 2006). The BAC was CH73-184B14,
containing around 39.5 kb upstream of Col2 and around 11 kb downstream.
Primer sequences for cloning available on request.
Alcian Blue and Alizarin Red staining
Bone and cartilage labelling was performed as described previously
(Spoorendonk et al., 2008; Walker and Kimmel, 2007).
TRAP staining
Tartrate-resistant acidic phosphatase (TRAP) staining was performed as
described (Albertson and Yelick, 2005), with the following modifications:
zebrafish were fixed in cold methanol (–20°C) overnight, rehydrated with
PBS, and incubated in freshly made TRAP medium, for 1.5 hours at 37°C.
Fish were subsequently bleached in 10% H2O2 for 4 hours and post-fixed in
4% PFA.
Microscopy
In situ hybridisations were analysed and photographed with a Leica 480C
camera on a Zeiss Axioplan microscope. For cell number analyses in
transgenic lines, images were captured on a Leica TCS-SPE confocal
system, and the stacks were analysed and cells counted using Velocity
software. In the cell-counting experiments, a minimum of four different
individuals for each genotype were counted. Results are presented as
mean±1 standard deviation (s.d.), significance was ascertained by
performing two-tailed paired Student’s t-tests of each data set to the wildtype situation.
RESULTS
Increased Hh signalling leads to premature
chondral mineralisation
Recently two papers have implicated the Hedgehog membrane
receptor patched 1 (Ptch1) in mouse bone development, but came to
somewhat contradictory conclusions. The first, studying
heterozygous deficiency for Ptch1, concluded that decreased Ptch1
leads to increased bone deposition (Ohba et al., 2008), whereas the
other found that conditional knockout of Ptch1 in osteocalcinexpressing cells leads to decreased bone density and increased
osteoclast activity (Mak et al., 2008a). Owing to embryonic lethality
before the onset of bone mineralisation, it is impossible to resolve
these issues in homozygous Ptch1 mutant mice. In zebrafish,
however, ptc1 mutants can typically survive to around 12 dpf, and
ptc2 mutants are even sub-viable, with small numbers reaching 3
months of age; as ptc1, ptc2, dre and ihha are expressed in positions
where cartilage later forms (see Fig. S1A in the supplementary
material) (Avaron et al., 2006; Thisse and Thisse, 2005), and as bone
development in zebrafish is first apparent from 3 dpf, this gives us a
window of opportunity in which to study bone development in a
variety of Hh mutants.
In wild-type zebrafish, the first evidence of mineralisation
detected by Alizarin Red staining is at 3 dpf in the cleithrum (data
not shown). By 4 dpf the dermal bones, cleithrum, operculum and
notochord tip are mineralised, and at this stage ptc1, ptc2 and dre
mutants were indistinguishable from their wild-type siblings (Fig.
1A). However, by 7 dpf all three mutants could be distinguished by
premature mineralisation of the ceratohyal and, in the case of ptc2
and dre, the hyosimplectic – both bones of chondral origin (Fig. 1A,
black arrowheads).
Osterix expression is increased in endochondral
bones
In order to better understand why an increase in Hh signalling leads
to increased bone deposition in chondral bones, we performed in situ
hybridisations for osterix. Osterix is a marker of early osteoblast
development and is thought to be the master regulator of osteoblast
differentiation (Nakashima et al., 2002). In ptc1, ptc2 and dre
mutants at 3.5 dpf, expression of osterix mRNA expression was
DEVELOPMENT
smoothened on osteoblast precursors. We therefore sought to
investigate the effect of manipulating smoothened activity in vivo
in the zebrafish.
Together the study of these mutants and titration of smoothened
activity allows us to build up a comprehensive picture of the effects
both of an increase or depletion of Hh signalling on early bone
formation in vivo. We show that, at least in the early stages of
development, alteration of Hh signalling has no effect on dermal
bone formation. However, in chondral bone elements Hh signalling
critically controls both the differentiation of osteoblasts and the
onset of osteoclast activity. In addition we demonstrate that many
cells that are located within the cartilage element retain a level of
plasticity that allows them, on receipt of high levels of Hh signalling,
to differentiate as osteoblasts.
Development 136 (23)
Hedgehog signalling and osteogenesis
RESEARCH ARTICLE 3993
Fig. 1. Levels of Hh signalling are
critical for endochondral ossification
and proliferation of chondrocytes.
(A)Alizarin Red staining of representative
Hh mutants reveals no differences at 4
dpf. At 7 dpf, however, premature
mineralisation of endochondral bone
elements can be seen (black arrowheads
mark the ceratohyal, purple arrowheads
mark the hyosymplectic), while the extent
of ossification in the cleithrum is
unchanged (red arrowheads). (B)In situ
analysis of mutants at 4 dpf reveals
increased expression of osterix in the
endochondral bone, where premature
mineralisation is later seen (black
arrowheads point to ceratohyal, purple to
hyosymplectic), while osterix expression is
unchanged or slightly reduced in dermal
bone elements such as the cleithrum (red
arrowheads). Insets show ventral views of
osterix expression in the ceratohyal.
(C)Alcian Blue/Alazarin Red double
staining of 17 dpf ihha and wild-type
sibling fish. The second panel is an
enlargement of the boxed area for each
genotype. (D)Whole-mount antibody
staining for BrdU incorporation between
4.5 and 5 dpf in wild type and ptc2
mutants. Excessive proliferation can be
seen in the ptc2 mutant in the tectum
(black arrowhead) and jaw cartilages
(blue arrowhead). (E)Representative
single confocal images of the Meckel’s
cartilage in 5 dpf zebrafish. Proliferating
chondrocytes (blue arrowheads) are
labelled with anti-BrdU (green), the
dashed red line shows the shape of a
single chondrocyte. (F)Quantitation of
BrdU-positive cells in the Meckel’s
cartilage at 5 dpf. Data are mean±s.d.
taken of at least five fish per genotype.
*P<0.01 versus wild type. wt, wild type.
Loss of Indian hedgehog leads to a severe
retardation of endochondral mineralisation
In mouse loss of Indian hedgehog (Ihh) leads to a reduction in
chondrocyte proliferation and a failure of osteoblast differentiation
in endochondral bones (St-Jacques et al., 1999), and further studies
have shown Ihh to promote chondrocyte hypertrophy before bone
formation (Mak et al., 2008b). The zebrafish ihhahu213allele contains
a premature stop codon at amino acid 44, and is thus likely to be a
functional null. We crossed ihhahu2131 carriers and examined bone
development at a variety of stages (Fig. 1C and data not shown). At
stages before the onset of endochondral mineralisation, no
difference between siblings and ihhahu2131 mutants was seen.
However, from 8 dpf the mutants were distinguished by a nearcomplete lack of endochondral ossification. This became more
pronounced over time, until 17 dpf, by which point all branchial
arches were mineralised in siblings, whereas in the mutant only very
small areas of endochondral mineralisation could be detected (Fig.
1C). Surprisingly, the mutant fish could survive and from 21 dpf
began to mineralise all bone elements, perhaps owing to partial
redundancy between the ihha and ihhb genes, such that by 30 dpf
the fish were almost indistinguishable in the extent of their
mineralisation from siblings, although the fish were frequently much
smaller (data not shown). These results show that, in the zebrafish
ihha is crucial for timely onset of endochondral mineralisation,
analogous to the situation in mammals.
Loss of ptc1 or ptc2 leads to increased, and loss of
ihha leads to decreased, chondrocyte proliferation
Hh signalling has long been implicated in maintaining the balance
between proliferation and differentiation of a number of cell types
(Agathocleous et al., 2007). Indeed, the zebrafish ptc2 and dre
mutants were identified in a screen for changes in proliferation
(Koudijs et al., 2005). We therefore decided to study proliferation in
the jaw cartilages in the various Hh mutants. In addition to the
DEVELOPMENT
upregulated in the endochondral bones (Fig. 1B, black arrowheads)
while remaining at wild-type levels in dermal bone elements, such
as the cleithrum (Fig. 1B, red arrowheads).
3994 RESEARCH ARTICLE
Development 136 (23)
Fig. 2. Mutants with altered Hh signalling
show no differences in the number of
osterix-positive cells in dermal bone
elements. (A)Confocal stacks of the cleithra of
osterix-nuclear GFP fish at 4 dpf. Inset image on
left is a merged image of Tg(osx:nuGFP) (green),
Alazarin Red and brightfield images. (B)Confocal
stacks of the operculum of osterix-nuclear GFP
fish at 4 dpf. Inset image on left is a merged
image of Tg(osx:nuGFP) (green), Alazarin Red
and brightfield images. (C)Quantification of the
number of osterix-positive nuclei in the cleithrum
(below) and operculum (above) at 3 and 4 dpf.
Data is shown as mean±s.d. taken of at least
four fish per genotype. *P<0.05 versus wild type.
wt, wild type.
The number of osterix-expressing osteoblasts is
independent of Hedgehog signalling levels in
dermal bones
To investigate the effects of Hh signalling on osteoblast numbers,
we crossed ptc1, ptc2 and ihha carriers to a transgenic line in which
nuclear GFP is fused to the medaka osterix promoter (Renn and
Winkler, 2009; Spoorendonk et al., 2008), allowing us to count the
number of osterix-expressing cells in different bone elements.
Comparison of the number of osterix-expressing cells in dermal
bone elements such as the cleithrum (Fig. 2A) and the operculum
(Fig. 2B) between the different lines revealed no significant
differences at the stages examined (Fig. 2C), although in ihha
mutants at later stages the size of the operculum was reduced despite
the number of cells remaining the same (see Fig. S2A in the
supplementary material), perhaps suggesting that the osteoblasts are
less active. However, in a number of the ptc1 mutants ectopic tissue
between the eyes was marked by osterix (see Fig. S2B in the
supplementary material) and subsequently mineralised (data not
shown). We conclude that Hh signalling levels are not critical for
numbers of early dermal bone osteoblasts.
Two distinct populations of osterix-expressing
cells in endochondral bone with different
sensitivity to Hedgehog signalling
These data suggested a differential effect of Hh signalling on dermal
versus endochondral bone. In order to better understand the way in
which altered Hh signalling was influencing endochondal osterix
expression, we investigated the ceratohyal and Meckel’s cartilage,
which mineralise prematurely in mutants with increased Hh
signalling. We observed that there are two different populations of
osterix-expressing cells associated with these cartilage elements.
The first appears to be the equivalent of the mammalian bone collar.
We see this population as a host of cells strongly expressing osterix
and surrounding the edge of the cartilage element (red arrowheads
in Fig. 3A-D,F). These cells were first seen at 5 dpf in wild type,
ptc1 and ptc2 mutants but not in ihha–/–, and increased in number
over days 6-7. In the ptc1 and ptc2 mutants a slight increase in the
number of these cells is seen at 5 dpf but by 6 dpf the difference in
number was no longer significant (Fig. 3F). The second population
of osterix-expressing cells retain chondrocyte morphology and are
interspersed with other cells in the cartilage elements (blue arrows
in Fig. 3A,C,D,E). This second population of cells was first seen in
ptc mutants at 3 dpf (Fig. 3C). Significantly in siblings the first time
the equivalent cells were observed was 6 dpf when around half of
the siblings had one or two osterix-positive cells (Fig. 3A,E). At this
stage these cells were present in significantly higher numbers in the
ptc mutants (blue arrows in Fig. 3A,E). By contrast, the first stage
in which multiple osterix-positive cells were reliably seen in siblings
was 9 dpf (Fig. 3D). The presence of osteoblasts within the
endochondral bone around 3 days earlier in mutants than siblings
corresponds with the position and timing of endochondral
ossification (detected by Alizarin Red) in the different fish
genotypes (Fig. 3 and Fig. 1A).
The number of both populations of osterixexpressing osteoblasts is significantly decreased
in ihha mutants in endochondral bones
We also counted the number of both populations of osterix-positive
cells in ihha mutants. We found that both populations of cells were
absent in all stages studied, to 9 dpf (Fig. 3E,F and data not shown);
by which time osteoblasts were populating the maxilla, a dermal
bone that lies closely behind the Meckel’s cartilage (white arrows in
Fig. 3E). Again, the lack of both types of osterix-expressing cells
corresponds with the failure to make endochondral bone in these
mutants. From this we conclude that differentiation of the bone
collar cells also require Hh signalling, as although their numbers
were unaffected in the mutants with increased Hh signalling they
were absent in the ihha mutant. However, as the morphology of the
bone collar cells is similar to other cells in the vicinity, we could not
distinguish between loss of the cells and presence of the cells but
loss of the markers.
The changes to the number of both populations
of cells can be phenocopied by titrating
smoothened activity by using small molecule
inhibitors and activators
To demonstrate the dose dependence for Hedgehog signalling on
the differentiation of the different populations of osteoblasts, we
used drugs to titrate smoothened activity. Cyclopamine is a plantderived alkyloid demonstrated to bind smoothened and block its
DEVELOPMENT
previously reported increased proliferation in the ciliary marginal
zones and tecta of the ptc2 mutants (Fig. 1D, black arrowhead), we
detected a significant increase in proliferation in the jaw cartilages
at all stages studied (Fig. 1D-F and data not shown), whereas in ihha
mutants we saw a reduction in the levels of proliferation in the
cartilage (Fig. 1F and data not shown), mirroring the situation in the
mouse (St-Jacques et al., 1999).
Hedgehog signalling and osteogenesis
RESEARCH ARTICLE 3995
activity (Chen et al., 2002), whereas purmorphamine also binds
smoothened but leads to its activation (Sinha and Chen, 2006).
Addition of cyclopamine (75 m) to ptc1,2 double heterozygous
incrosses led to rescue of the premature mineralisation (data not
shown) and reduction of the number of chondrocytes expressing
osterix to near wild-type levels (Fig. 3H) while not significantly
altering the number of bone collar osteoblasts in mutants or siblings
(Fig. 3H).
By contrast, treatment of wild-type embryos with 20 M
purmorphamine from 2 dpf to 6 dpf led to a significant increase in
the number of osterix-positive cells located within the collagen
matrix (Fig. 3G and Fig. 4C), without a significant concomitant
increase in the number of bone collar osteoblasts, suggesting that
there may be a maximum number of these cells, which is already
reached in the mutants. Treatment of ihha mutants from 2 to 6 dpf
with 20 m purmorphamine led to a near-complete rescue of bone
DEVELOPMENT
Fig. 3. There are two populations of endochondral osteoblasts with differential sensitivity to Hh signalling. (A)Confocal stacks from
wild-type, ptc1–/– and ptc2–/– 6 dpf larvae in an osterix-nuclear GFP background, imaged for GFP expression. The far right image is a single confocal
plane to allow distinstintion of the cell morphology. At this stage in both ptc mutants and wild-type larvae, similar numbers of bone collar
osteoblasts are present (red arrowheads). However, in ptc1–/– and ptc2–/– larvae, there is a significant increase in the number of chondrocytes that
express osterix (blue arrows). A single chondrocyte is outlined in black in the image of the ptc1 mutant. (B)A ventral view of one side of the
Meckel’s cartilage and ceratohyal of a 6 dpf wild-type larva stained with Alcian Blue and Alizarin Red; a single chondrocyte is outlined in black to
demonstrate the morphology of the chondrocyte cells. (C)Confocal stacks of the ceratohyal from ptc1–/– and wild-type embryos at 3 dpf. In ptc1–/–
embryos (but not in wild-type siblings) cells can be found that retain the shape of chondrocytes but that express osterix (blue arrow). (D)Confocal
stack of the Meckel’s cartilage of a 9 dpf wild type. Expression of osterix is visible in cells within the cartilage element (blue arrows) and outside (red
arrowhead). (E)Confocal stacks of the Meckel’s cartilage of wild-type and ihha–/– 9 dpf larvae. By this stage the wild-type embryo has osterixexpressing bone collar cells (red arrowhead) and transdifferentiating chondrocytes (blue arrows), while the ihha–/– larvae has neither. White
arrowheads mark the maxilla, a dermal bone, which forms adjacent to the Meckel’s cartilage. (F)Quantification of number of osteoblasts at 6 dpf
showing number of osteoblasts in the bone collar of the Meckle’s cartilage and the number of chondrocytes differentiating as osteoblasts in the
Meckel’s cartilage. Data are presented as means±s.d. taken from at least six fish per genotype; asterisk represents P<0.05 in paired Student’s t-tests
of mutant versus wild type. (G)Quantification of osteoblast number at 6 dpf in the Meckel’s cartilage of embryos treated with 20m
purmorphamine or controls. Data are presented as means±s.d. taken from at least four fish per genotype; * represents P<0.05 in paired Student’s
t-tests of treated versus untreated. (H)Quantification of osteoblast number at 6 dpf in the Meckel’s cartilage of embryos treated with 75M
cyclopamine or controls. Data are presented as means±s.d. taken from at least four fish per genotype; * represents P<0.05 in paired Student’s ttests of treated versus untreated. wt, wild type.
3996 RESEARCH ARTICLE
Development 136 (23)
collar osteoblast number to wild-type levels (Fig. 3G and Fig. 4C);
however, the effect of this dose of purmorphamine in the mutants led
to only slight increases in the number of osteoblasts within the
cartilage matrix (Fig. 3G and Fig. 4C).
The osterix-positive cells located in the cartilage
are surrounded by collagen II-positive matrix, and
go on to express bone markers
To investigate whether this second population of endochondral
osteoblasts displayed molecular differences to the bone collar
osteoblasts in addition to their morphological differences, we
studied a number of markers of cartilage and bone. Type II collagen
is the main constituent of cartilage matrix (Mackie et al., 2008), we
therefore stained the various mutants for collagen type II and GFP
to show the osterix-positive cells. Again, it was clear that there are
two populations of cells: one entirely enclosed by the cartilage
matrix, and the other on the surface (Fig. 4A). Maximal activation
of the Hedgehog signalling pathway is seen in embryos that are
mutant for both ptc1 and ptc2 (Koudijs, 2008). Ptc1/2 double
mutants survived only to 4 dpf and had gross malformation of the
head, such that individual bone elements could not be distinguished;
however, by 4 dpf there were disorganised collagen II-positive
condensations (Fig. 4A). Staining for osterix-positive cells revealed
that every cell in the collagen II-positive condensation also
expressed osterix (Fig. 4A).
To ascertain whether the cells encircled by cartilage matrix were
themselves secreting it, we made BAC constructs in which the
mCherry fluorescent protein is driven by the promoter of
collagen2a1. Injection of this construct led to mosaic expression of
mCherry in the collagen2-expressing chondrocytes. When injected
into a ptc2 heterozygous incross, cells could be seen in cartilage
elements that contained both GFP and cherry in mutant embryos
(Fig. 4B). This shows that the osterix-positive cells located within
the cartilage matrix themselves express collagen2, making it
unlikely that the matrix surrounding these cells is derived solely
from neighbouring cells.
Because in rare cases hypertrophic chondrocytes can express
Osterix (Shibata et al., 2006; Shibata and Yokohama-Tamaki, 2008),
we also studied later markers of osteoblast maturation that do not
overlap with hypertrophic chondrocytes. One such marker is
osteocalcin, which is exclusively expressed in osteoblasts
(Hoffmann et al., 1996). We assayed osteocalcin expression in
mutants and saw that in ptc1 and ptc2 mutants in addition to strong
osteocalcin expression in the teeth (Fig. 4E, black arrows) and bone
collar (Fig. 4E, red arrowheads), there was expression in cells in the
cartilage elements (Fig. 4E,F, blue arrows). Further supporting this
concept that these chondrocytes are differentiating as osteoblasts we
also saw expression of type 1 collagen, another marker specific to
osteoblasts, both in cells of the bone collar and in this population of
cells in the cartilage (Fig. 4D and data not shown), demonstrating
DEVELOPMENT
Fig. 4. The osterix-expressing cells with chondrocyte morphology
express markers of chondrocyte and osteoblast differentiation.
(A)Confocal stacks taken of the ceratohyal of 5 dpf embryos stained
for collagen II in red, GFP (osterix) in green and DAPI in blue. In wildtype embryos osteoblasts can be seen lying on the surface of the
collagen II-positive matrix (white arrows). In ptc1 mutant embryos,
similar cells can be seen (white arrows), and also osterix-positive cells,
which are completely surrounded by collagen II-positive matrix can be
seen (blue arrows). In ptc1/2 double mutants the cartilage structure is
disorganised, but all cells surrounded by collagen II-positive matrix are
also positive for osterix (blue arrows). In the ihha–/– mutants, neither
population of osterix-positive cells are seen, despite the normal
appearance of the collagen II-positive cartilage matrix. (B)Confocal
stack from a 4 dpf ptc2 mutant osterix-nuGFP larva, injected with a
BAC containing mCherry inserted after the ATG of collagen2a1, leading
to mosaic expression through the cartilage. White arrows mark cells
expressing osterix, which do not contain col2 mCherry. Yellow arrow
marks a cell expressing col2 which is negative for osterix, whereas blue
arrows mark cells expressing both mCherry and GFP. (C)Confocal stacks
from the ceratohyal of collagen 2 (red) and anti-GFP osterix (green)
antibody stained 6 dpf larvae treated from 2-6 dpf with
purmorphamine. White arrows indicate bone collar osteoblasts
overlying the cartilage matrix. Blue arrows mark cells expressing osterix,
which are surrounded by cartilage matrix. (D)Col1a2 in situ
hybridisation in 9 dpf wild-type embryo, showing the palatoquadrate.
Two populations of col1a2 expressing-osteoblasts can be seen. One
population is located outside but adjacent to the cartilage matrix (black
arrows), whereas the other comprises cells located within the cartilage
matrix (red arrows). (E)Ventral views of 5 dpf embryos in situ hybridised
for osteocalcin, anterior to right. In all genotypes osteocalcin staining
can be seen in the teeth (black arrows). Particularly strong staining in
the osteoblasts of the bone collar (red arrowheads) is present in wildtype and ptc2–/–, embryos but is absent for ihha–/–. In ptc2 mutant
embryos weaker osteocalcin expression can be seen in cells within the
cartilage (blue arrows), which are not seen in wild-type embryos at this
stage. (F)High magnification image of Meckel’s cartilage from a ptc2–/–
5 dpf embryo showing osteocalcin expression in cells within the
cartilage element (blue arrows). wt, wild type.
Fig. 5. Osteoclasts are active earlier in ptc mutants and later in
ihha mutants than in the wild-type situation. (A)Whole mount
stained for tartrate resistant acid phosphatases (TRAP). Activity is first
present in the fifth branchial arch and in teeth (black arrows).
Additionally, by 14 dpf activity can be seen in the Meckel’s cartilage
(blue arrow in inset, region enlarged is marked by a dashed box) and
operculum (red arrow) in the ptc2–/– mutant. Dashed box in the top
right image denotes the region shown for 10 and 12 dpf. (B-D)Closeup views of the regions of TRAP activity: (B) dissected teeth and fifth
branchial arch, (C) neural arch, (D) tail fin. wt, wild type.
that these cells are indeed mature osteoblasts. These results taken
together demonstrate that cells that are differentiating as
chondrocytes retain a degree of plasticity that allows them, on
receipt of high levels of Hh, to differentiate as osteoblasts.
Interestingly, in situ hybridisations for gli transcription factors
showed increased expression of both gli1 and gli3 in chondroprogenitors of mutants with increased Hh signalling at 3 dpf, and a
slight reduction in expression of the repressive gli2 (see Fig. S1B in
the supplementary material). This shift in favour of activator forms
such as gli1 over repressive factors such as gli2 bears similarities to
mouse, in which altered processing of Gli3 to its activator form was
seen (Ohba et al., 2008). Taken together, these data suggest that the
two populations of osterix-expressing cells require different levels
of Hh signalling for their differentiation: bone collar cells require
low levels of ihh, whereas the chondro-osteoblasts require higher
levels of the Hh signal.
The increased mineralisation in ptc1 and ptc2
mutants cannot be accounted for by a deficit in
the differentiation of osteoclasts
As bone deposition and modelling is a balance in the activities of
both osteoblasts and osteoclasts, there is an alternative hypothetical
explanation for the increase in mineralisation: namely a decrease or
a delay in osteoclast differentiation. In the mouse conditional Ptch1
knockout in osteocalcin-expressing cells, decreased bone density
RESEARCH ARTICLE 3997
Fig. 6. Model illustrating the different populations of osterixexpressing cells during cartilage ossification in situations of
normal, high or low levels of hedgehog signalling during
zebrafish development.
was observed and attributed to increased osteoclastogenesis (Mak et
al., 2008a). We sought to test whether this was the case in fish. In
zebrafish two different populations of osteoclasts are believed to
exist: a mononuclear population forms earlier than the
multinucleated osteoclasts; both populations have been described as
forming relatively late in development between 12 and 20 dpf
(Witten et al., 2001). To understand the effect Hh signalling has on
osteoclast activity, we performed TRAP staining on mutants at a
variety of ages, from 8 to 14 dpf (Fig. 5 and data not shown). In
accordance with published literature, in wild-type larvae we first
observed osteoclast activity at 12 dpf in the fifth branchial arch, at
the position where the teeth join the bone (Fig. 5A,B). In the ptc2
mutant we could consistently observe TRAP activity from 10 dpf,
indicating that by this stage osteoclasts are already differentiated and
active in the mutant (Fig. 5A). Over time both the levels of activity
and the number of bone elements displaying osteoclast activity
increased, such that at 14 dpf in the ptc2 mutants activity was
observed in the Meckel’s cartilage (blue arrow in Fig. 5A), the
neural arches (Fig. 5C) and the tail fin (Fig. 5D). By contrast, in ihha
mutants TRAP activity was not seen until 14 dpf (Fig. 5A). Thus
increased systemic Hedgehog activity, in addition to promoting
osteoblastogenesis, also promotes increased osteoclast activity.
Interestingly, TRAP activity was also seen in the operculum, a
dermal bone, earlier in the ptc2 mutant than in siblings or wild-type
fish (Fig. 5A, red arrow), despite there being no difference in the
number of dermal bone osteoblasts at early stages between the
genotypes (Fig. 2). This suggests that the effect on Hh signalling on
osteoclastogenesis is direct, and is independent of osteoblast
number. Additionally, we studied adult fish heterozygous for ptc1,
ptc2 or both. In contrast to mouse (Ohba et al., 2008), there were no
obvious morphological or skeletal changes in the heterozygous fish
compared to siblings (data not shown); this could be because fish are
DEVELOPMENT
Hedgehog signalling and osteogenesis
less dose-sensitive to loss of ptc1 and 2, or because zebrafish do not
have trabecular bone, in which the greatest differences were
observed in mice (Ohba et al., 2008). We also generated an in situ
probe to RANKL (TNFSF11), and assayed expression levels in the
various mutants; however, we could not detect expression in or
around bone elements before 8 dpf (data not shown) consistent with
our TRAP staining, which suggests that osteoclast formation begins
around 10-12 dpf in the zebrafish. We could, however, detect
expression in the brain from 6 dpf (data not shown). We therefore
conclude that differences in RANKL expression are not responsible
for the early changes to bone deposition seen in the hedgehog
mutants.
The difference in the number of osteoblasts in
endochondral bone elements cannot be explained
by increased vascularisation of the cartilage
elements
One hypothesis for how osteoblasts come to be in endochondral
bone is that they are brought in by blood vessels that invade the
cartilage. In some cases anti-angiogenic agents can block ectopic
ossification (Mori et al., 1998). To test whether the increased
mineralisation and increased osteoblast number in the endochondral
bones in ptc mutants is preceded by increased or premature
vascularisation of the cartilage, we crossed the ptc mutants into the
kdr-l:eGFP transgenic line that marks blood vessels (Jin et al.,
2005). In neither ptc1 nor ptc2 mutants, ectopic or premature
vascular sprouting was seen when compared to wild-type embryos
(see Fig. S3A in the supplementary material). Moreover, in the ptc1
mutants, vascularisation close to the endochondral bone
mineralisation was actually somewhat reduced, with the gill vessels
failing to loop around the branchial arches (see Fig. S3A in the
supplementary material). Indeed, the first cartilage elements to
mineralise were not closely associated with any blood vessels at
onset of osteoblast appearance (see Fig. S3B in the supplementary
material). This result, taken together with the previous data, strongly
suggests that the osteoblasts that contribute to premature
endochondral ossification in ptc mutants derive from chondrocytes
that then differentiate as osteoblasts in situ; we have summarised the
data in a model (Fig. 6).
DISCUSSION
Our data demonstrate that there are two populations of osteoblasts
in zebrafish that make endochondral bone: those lying outside the
cartilage matrix, which we refer to as the bone collar, and those that
lie within the cartilage, have chondrocyte morphology and on receipt
of Hh signalling express markers of osteoblast differentiation. A key
finding of our study is that the two populations of osteoblasts have
differential sensitivity to Hh signalling. Osteoblasts of the bone
collar require Hh signalling, as they do not form in the ihha mutant
and their numbers are reduced following cyclopamine treatment.
However, they are less sensitive to increased Hh signalling than the
latter population. We presume that for the osteo-chondrocyte
precursors a certain threshold of Hh signalling needs to be reached
before they are able to differentiate as osteoblasts.
As chondrocytes mature, they cease to proliferate and
differentiate into prehypertrophic chondrocytes expressing Ihh
(Kobayashi et al., 2005; Maeda et al., 2007). In zebrafish, ihh
expression is readily detectable at the relevant stages in
chondrocytes (Avaron et al., 2006). We suggest that during wild-type
chondrocyte maturation these cells begin to secrete Ihh, which, upon
reaching a certain threshold, acts both in an autocrine and paracrine
manner to allow a number of these cells and those surrounding them
Development 136 (23)
to differentiate as osteoblasts. Consistent with this idea, in ptc
mutants, which are sensitised to Hh signalling, these cells express
osteoblast markers earlier and in larger numbers than in siblings. An
alternative model postulates hypertrophic chondrocytes to secrete a
signal that induces osteoblast differentiation and bone formation
close to the position of the hypertrophic chondrocytes (Chung,
2004). We would argue that, a number of the chondrocytes
themselves begin to express markers of osteoblast differentiation
such as osterix and osteocalcin. This, indeed, appears also to be true
in other teleosts (Gavaia et al., 2006), and the amphibian Xenopus
tropicalis, in which weak expression of osterix is seen in the lacuna
of chondrocytes at the same time as the surrounding bone collar
forms (Miura et al., 2008). In amniotes a number of chondrocytes
display properties typically seen in osteoblasts (Galotto et al., 1994).
Furthermore, there is also evidence that the hypertrophic
chondrocytes located adjacent to the perichondrum differentiate as
osteoblasts and contribute to the earliest endochondral bone matrix
(Bianco et al., 1998).
Lending further credence to the idea that some osteoblasts
develop from the cartilage itself are a number of results, including
the fact that mice null for Runx2 fail to make bone (Kim et al., 1999;
Otto et al., 1997). In situations in which Runx2 is knocked out in
cells of the chondrocyte lineage endochondral bone formation is
severely delayed (Ueta et al., 2001), whereas overexpression of
Runx2 in cells of the same lineage leads both to rescue of the Runx2
null phenotype and to premature ossification of cartilage elements;
showing that expression of Runx2 in cells of the chondrocyte lineage
alone can re-establish endochondral bone formation in a cellautonomous fashion (Takeda et al., 2001). This argues against a
mechanism by which all osteoblasts are brought in by the blood
vessels, and is suggestive of a mechanism by which some of the
osteoblasts differentiate in situ. We do not debate the close
physiological connection between cartilage and blood vessels.
Vasculogenesis and endochondral bone formation are closely linked,
with hypertrophic chondrocytes able to stimulate vasculogenesis via
Vegf, and Vegf itself also able to stimulate proliferation of
osteoblasts and osteogenesis (Takeda et al., 2001; Wang et al., 2007).
Moreover, Runx2 mutant mice do not express Vegf in the cartilage,
a feature that is fully revertable upon introduction of Runx2,
demonstrating that Runx2 regulates cartilage expression of Vegf
(Zelzer et al., 2004; Zelzer and Olsen, 2005). However, from our
data it is clear that the presence of blood vessels within the cartilage
itself is not a prerequisite for all endochondral osteogenesis,
although proximity to vasculature to maintain normoxic conditions
is likely to be a requirement for normal bone formation.
In the mouse, two recent studies, both interfering with Ptch1
levels and focusing on bone homeostasis, led to somewhat
contradictory results. Ohba et al. (Ohba et al., 2008) reported
increased bone deposition through increased osteogenesis upon
lowering Ptch1 levels by 50%, whereas Mak et al. (Mak et al.,
2008a) showed osteopenia and reduced bone mass through
stimulation of osteoclastogenesis. Our studies in zebrafish help in
clarifying the role of Ptch and Hh signalling, as we can study the
effect of systemic loss of ptc1 and ptc2 and the titration of
smoothened activity. We show that increased Hh signalling
promotes the formation of both osteoblasts and osteoclasts,
confirming the main results from each paper. Our data, though, more
closely reflect the findings of Ohba et al. (Ohba et al., 2008), who
showed an increase in bone mass upon increased Hedgehog
signalling, despite the fact that we did not see any obvious defects
in heterozygotes. We believe that by knocking out Ptch1 only in
mature osteoblasts Mak et al. (Mak et al., 2008a) would not have
DEVELOPMENT
3998 RESEARCH ARTICLE
observed the Hh-sensitive differentiation of a population of
chondrocytes into osteoblasts. In order to see this it would require
Ptch1 to be conditionally knocked out under the control of an earlier
marker expressed in both osteoblasts and chondrocytes such as
Runx2. Thus, we suspect that their model would underestimate the
number (and activity) of mature osteoblasts that would be present in
a mouse with systemic loss of Ptch1. We believe that our findings
help to explain the discrepancies between the two papers.
It appears that the increased activity of osteoclasts upon increased
Hh signalling is not limited to endochondral bone but also occurs in
dermal bones such as the opercula, where we see no difference in
osteoblast number. Thus, we conclude that misregulation of Hh
leads to a situation where the number of osteoblasts and osteoclasts
are no longer proportional, particularly in dermal bone, in which
osteoblast activity is increased with no concomitant increase in
osteoblast number. This is particularly interesting in the context of
the recent finding that Ihh is expressed during dermal bone
development in the mouse (Abzhanov et al., 2007). As a
consequence, the effect on dermal bones is likely to be a net loss of
bone mass over time, as seen with osteopenia observed in
membranous bones as well as endochondral ones in the mouse (Mak
et al., 2008a).
In summary we demonstrate in a number of different ways that
systemically increased levels of Hh signalling lead both to increased
differentiation of osteoblasts from cells of a chondrogenic lineage,
and to increased activity from osteoclasts, confirming and extending
the findings from mice (Mak et al., 2008a; Ohba et al., 2008).
Importantly we show that not only does loss of ptc1 cause this
phenotype but also a number of other manipulations that increase
Hh signalling, demonstrating that the effects seen in mice are not due
to intrinsic effects of Ptch1 but rather to a general increase in Hh
signalling. We unite these results with experiments in ihha mutants
and cyclopamine-treated larvae, which suggest that the effects
of increased craniofacial endochondral osteoblast and
osteoclastogenesis are likely to be mediated via increased Indian
hedgehog signalling.
Acknowledgements
Many thanks to Josi Peterson-Maduro for her help and expertise in cloning the
BAC transgenic, to members of the Schulte-Merker laboratory and C. Winkler
for helpful discussions, and to the Sanger Institute for providing the Ihhahu2131
mutant fish. This work was supported by an EMBO long-term fellowship
(C.L.H.). S.S.-M. gratefully recognizes the support of the Smart Mix Programme
of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry
of Education, Culture and Science.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/23/3991/DC1
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