1 Department of Orthopaedic Surgery, University of California, San Francisco,
California, 94143-0514, USA
2 Genetics Unit, Shriners Hospital, Montreal, Quebec H3G 1A6, Canada
3 Department of Plastic and Reconstructive Surgery, Stanford University,
Stanford, CA 94305, USA
* Author for correspondence (e-mail: jhelms{at}stanford.edu)
Accepted 16 December 2004
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SUMMARY |
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Key words: Renal capsule transplantation, Lineage analysis, X-gal, Null mutant mouse, Pericyte, Vasculature, Bone
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Introduction |
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The vasculature is another tissue essential for the program of
skeletogenesis. The initiation of skeletal tissue formation requires that
blood vessels vacate the area of the future condensation
(Yin and Pacifici, 2001), but
the differentiation of cells into osteoblasts requires vascular invasion at a
later stage (Gerber et al.,
1999
; Vu et al.,
1998
). This co-dependency among tissues that comprise the skeleton
suggests why it is difficult to distinguish the role cartilage plays in the
orchestration of vascular and perichondrial development, relative to its own
developmental progression. We set out to determine the extent to which indian
hedgehog (IHH) functions as a `molecular coordinator' of chondrocyte
differentiation, perichondrial development and vascular remodeling during the
process of fetal skeletogenesis.
Hedgehog proteins are prime candidates for coordinating cell
differentiation and thus orchestrating tissue formation
(Bumcrot and McMahon, 1995). In
the appendicular skeleton, IHH is secreted by pre-hypertrophic and early
hypertrophic chondrocytes (Chung et al.,
2001
; Kobayashi et al.,
2002
; Long et al.,
2004
; Vortkamp et al.,
1996
). IHH is involved in osteoblast differentiation, as
illustrated by the fact that Ihh-/- appendicular skeletal
elements do not ossify (Chung et al.,
2001
; St-Jacques et al.,
1999
). Although some data suggest that the absence of ossification
results from a primary defect in osteoblast differentiation
(Chung et al., 2001
;
Long et al., 2004
), secondary
defects might also contribute to the phenotype. For example, the
Ihh-/- phenotype may be the result of a failure to specify
appendicular perichondrial cells to an osteoblast lineage, rather than an
actual failure of specified osteoblasts to differentiate
(Long et al., 2004
;
Naski et al., 1998
). In either
of these two scenarios, IHH would act directly on the osteoblast.
Alternatively, IHH may only secondarily block ossification because of its
primary role in regulating chondrocyte differentiation
(Long et al., 2001
;
Vortkamp et al., 1996
).
Another possibility is that IHH influences vascular development
(Byrd et al., 2002
;
Dyer et al., 2001
;
Pola et al., 2001
), which, in
turn, affects osteoblast differentiation. To understand the extent to which
IHH controls osteogenesis and angiogenesis in addition to chondrogenesis, we
took a closer look at the Ihh-/- phenotype for new clues
regarding these tissue interactions. A series of embryonic tissue
manipulations shed new light on the function of this morphogen during fetal
skeletogenesis.
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Materials and methods |
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Renal capsule transplantations
Stylopod cartilage anlagen from E14.0 wild-type, PtchlacZ,
Ihh-/-, Ihh-/-;Rosa26, wild type;Rosa26,
or Ihh-/-;PtchlacZ embryos were dissected and
transplanted underneath the renal capsule of adult wild-type or Rosa26 mice,
as previously described (Colnot et al.,
2004). Mice used as hosts and donors for the renal capsule
transplantations were both bred in the C57B6 background to avoid immune
rejection. All procedures followed standard Stanford and UCSF CAR/LARC
protocols. Skeletal elements were collected after 24 hours, 48 hours and 4-14
days and processed for cellular and molecular analyses.
SHH-N treatment
To determine if a hedgehog (HH) signal was provided by the kidney
environment, we grafted E14.0 PtchlacZ and
Ihh-/-;PtchlacZ cartilage elements under the
renal capsule of wild-type hosts in the absence or presence of exogenous SHH-N
protein (Curis, Inc.). As Ptch is a downstream target of HH
signaling, up-regulation of the PtchlacZ transgene is indicative of
activation of the HH signaling pathway in vivo. The SHH-N protein was added as
a positive control to demonstrate that the tissue was responsive to a hedgehog
signal. Affi-Gel Blue beads (BioRad) were soaked in a solution containing 400
µg/ml of the recombinant SHH-N protein in PBS with 0.1% bovine serum
albumin. Two beads were placed on each side of the cartilage elements at the
time of transplantation into the renal capsule.
Tissue processing, histology and histochemistry
Embryonic limbs and skeletal elements collected at various time points
after transplantation in the renal capsule were fixed in 4% paraformaldehyde
at room temperature for 1 hour or at 4°C overnight, decalcified at 4°C
in 19% EDTA, pH 7.4, for 1 to 7 days, dehydrated and embedded in paraffin.
Five micron-thick sections were collected on superfrost-plus slides and
analyzed for histology using Safranin-O/Fast green (SO) and trichrome (TC)
staining. Tartrate-resistant acid phosphatase (TRAP) staining, MMP9 and PECAM
immunohistochemistry were performed as previously described
(Colnot et al., 2003). Smooth
muscle actin (SMA) and COL4 immunohistochemistry was performed according to
established protocols (Marcucio and Noden,
1999
) at dilutions of 1:200 (mouse anti-
SMA, clone 1A4;
Sigma) and 1:400 (rabbit anti-collagen type IV; cat. no. c7510-51, US
Biological). SMA primary antibody was detected using the Vectastain ABC mouse
IgG detection kit (Vector Labs, Inc.), and COL4 was detected using the
Vectastain ABC rabbit IgG detection kit (Vector Labs). Since the mouse
anti-
SMA was used on mouse tissue, we compared this staining to
negative control samples that were exposed to the Vectastain mouse IgG
detection kit alone (absent primary SMA antibody). For detection of
lacZ or PtchlacZ expression, whole mount X-gal staining was
performed as described above. Samples were then embedded, sectioned and
counterstained with Eosin.
In situ hybridization
35S-labelled antisense riboprobes corresponding to cDNAs for
collagen type I (Col1), collagen type IIa1 (Col2),
osteopontin (Op; Spp1 - Mouse Genome Informatics), matrix
metalloproteinase 13 (Mmp13), indian hedgehog (Ihh), patched
1 (Ptch) and vascular endothelial growth factor (Vegf) were
hybridized on tissue sections and the in situ hybridization signal was
visualized as described previously
(Albrecht et al., 1997;
Ferguson et al., 1999
).
Lineage analyses
To distinguish cells derived from the graft and cells derived from the
host, E14.0 wild type;Rosa26 or Ihh-/-;Rosa26 embryos were
transplanted into wild-type hosts (Jackson Laboratory, Maine, USA). They were
processed for X-gal staining as described above and by Colnot et al.
(Colnot et al., 2004).
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Results |
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Our molecular analyses, however, belied this theory. Although cartilage maturation was definitely delayed, most Ihh-/- chondrocytes still progressed to the point of expressing markers of late hypertrophy such as matrix metalloproteinase 13 (Mmp13) and osteopontin (Op; Fig. 1A-C,E-G). In addition, Ihh-/- chondrocytes expressed vascular endothelial growth factor (Vegf), indicating that regardless of the fact that it was disorganized, Ihh-/- hypertrophic cartilage still switched from an anti-angiogenic to an angiogenic state (Fig. 1D,H). We therefore concentrated our efforts on the condition of the vasculature surrounding the mutant appendicular skeleton.
|
|
Using this system we found that the Ihh-/- skeletal anlage were indeed angiogenic: in an ex vivo environment, these elements became vascularized and formed a marrow cavity (Fig. 3A,B,F,G; n=80 wild type and n=80 Ihh-/-). This finding is in keeping with the fact that Vegf expression persists in Ihh-/- hypertrophic cartilage in situ (Fig. 1).
|
On day 10 these same lineage analyses demonstrated that the vasculature had become chimeric. While most of the blood vessels were still derived from the graft, some were derived from the host, at least in the case of wild-type tissues (Fig. 3D, and data not shown). When we examined the Ihh-/- grafts we found that the majority of vessels were derived from the wild-type host and only a small percentage of the total vasculature was composed of Ihh-/- vessels (Fig. 3I, and data not shown). In addition, the Ihh-/- vessels had an abnormal morphology (Fig. 3I). For example, normally wild-type vessels line the bone trabeculae and are positioned perpendicular to the surface of the chondro-vascular junction (Fig. 3D) but Ihh-/- blood vessels had a large lumen and were arranged parallel to the chondro-vascular junction (Fig. 3I).
By day 14 wild-type grafts continued to contain graft-derived endothelial cells in significant numbers (Fig. 3E) but in mutant grafts Ihh-/- endothelial cells had disappeared entirely from the mutant ossification center and had left behind only host-derived vessels that sustained development of the mutant anlage (Fig. 3J). Collectively, these data taken from three time points indicate that Ihh-/- endothelial cells were capable of penetrating the mutant hypertrophic cartilage, but were unable to persist in the Ihh-/- hypertrophic cartilage. These two observations were identical to those we made in the Ihh-/- in vivo setting. In the ex vivo environment, however, wild-type blood vessels were able to replace the mutant blood vessels, and thereby sustained the Ihh-/- anlage in a manner that was not possible in vivo.
We drew three conclusions from this part of the study. First, the ex vivo results confirmed that Ihh-/- appendicular cartilage can undergo an angiogenic switch. Second, terminal differentiation of Ihh-/- hypertrophic chondrocytes can be uncoupled from vascular invasion and expansion, since chondrocyte terminal differentiation continued to progress even though vascular invasion came to an abrupt halt. Third, Ihh-/- blood vessels retained their invasive potential but were unable to persist, despite the presence of wild-type vessels and blood-born growth factors. This last finding suggested that Ihh-/- blood vessels themselves might be defective.
Ihh-/- endothelial cells are capable of participating in mature vessels
In order to further investigate the disappearance of
Ihh-/- vessels both in vivo and ex vivo, we performed an
immunohistochemical comparison of vascular maturity in wild-type and mutant
vessels. At E15.0, endothelial cells at the initial site of vascular invasion
express PECAM and COL4; these same markers are evident at E17.5, at the
chondrovascular junction (Fig.
4A,C, and data not shown). These same vessels had yet to be
covered by SMA-positive pericytes (Fig.
4B, and data not shown). Vessels in the marrow cavity of E17.5
samples, however, were SMA positive (Fig.
4D,E, red arrows). Using these markers of endothelial cell
maturity, we examined Ihh-/- samples from various stages
and found that endothelial cells which formed the vascular islands were
similar in maturation state to those found at the wild-type chondrovascular
junction, in that they were COL4 positive but SMA negative
(Fig. 4H-J). In order to
examine the later stages of ossification in the Ihh-/-
anlage, we turned again to the renal capsule transplant system.
Ihh-/-;Rosa26 elements transplanted at E14.0 into
wild-type hosts and collected after 10 days contained some vessels that
resembled those found in the marrow cavity of E17.0 wild-type elements;
vessels that were both COL4 and SMA positive
(Fig. 4K-M). Because the
transplants were done with Rosa26 grafts, we were able to identify the origin
of these vessels and found that some of the mature, SMA-positive vessels were
derived from the Ihh-/- graft
(Fig. 4L,M, red arrows).
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Osteoblast precursors are present in the Ihh-/- appendicular skeleton and differentiate ex vivo
In addition to revealing the Ihh-/- vascular anomaly,
the ex vivo experiments yielded another unexpected finding: all
Ihh-/- elements ossified in the renal capsule
(Fig. 5A,B,D,E; n=80
wild type and n=80 Ihh-/-). Because these
experiments were performed using Rosa26 donor embryos, we could unequivocally
determine that the osteoblasts were Ihh-/- cells
(Fig. 5C,F). This finding was
unexpected for two reasons. First, these same Ihh-/-
elements never ossify in vivo (St-Jacques
et al., 1999) and second, IHH is thought to be required for
osteoblast differentiation in the limb
(Long et al., 2004
). That
osteoblasts were potentially differentiating in the absence of hedgehog was an
intriguing possibility.
|
|
The rescue of osteoblast differentiation in the renal capsule is hedgehog independent
We set about determining if the renal capsule provided exogenous hedgehog
protein to the transplanted Ihh-/- elements, and
discovered that Ihh RNA was detectable by RT-PCR in the cortex of the
kidney (data not shown). This finding raised the possibility that the protein
might diffuse far enough and penetrate deep enough to act on
Ihh-/- perichondrial cells. We took advantage of the fact
that cells and tissues from PtchlacZ embryos can serve as a
functional readout of hedgehog activation
(Byrd et al., 2002;
Milenkovic et al., 1999
;
Pola et al., 2001
) and devised
a functional assay to test whether exogenous hedgehog protein reached the
Ihh-/- elements in the renal capsule. We first tested the
feasibility of using this reporter assay by transplanting E14.0 wild-type
PtchlacZ skeletal elements to a syngenic wild-type renal capsule and
evaluating the elements after 48 hours. As anticipated, there was abundant
X-gal staining in the cartilage and perichondrium, indicating that
Ptch expression was maintained in the wild-type transplanted element
(Fig. 7A,B; n=6). When
we examined Ihh-/-;PtchlacZ elements we did not
detect any X-gal staining, either in whole mounts or sections
(Fig. 7F,G; n=4). The
most plausible explanation for this finding was that the reporter was not
activated because neither the renal capsule environment nor the skeletal
environment itself (i.e. Ihh-/- chondrocytes) provided
functional hedgehog protein. Alternatively, one might argue that
Ihh-/-;PtchlacZ elements were incapable of
responding to a hedgehog signal, even if it were present in the environment.
To confirm that the Ihh-/-;PtchlacZ cells could
respond, and to ensure that our assay system was sensitive enough to detect a
small amount of hedgehog protein in the environment, we conducted another
series of experiments in which we placed beads soaked in SHH-N protein next to
the Ihh-/-;PtchlacZ elements. After 48 hours in
the renal capsule, Ptch expression was strongly up regulated in both
wild-type and mutant elements in response to the exogenous hedgehog protein
(Fig. 7C,D,H,I; n=6
wild type and n=4 Ihh-/-).
|
Formation of the perichondrium is impaired in Ihh-/- limbs
We next focused our attention on how the loss of IHH affected morphogenesis
of the perichondrium. During endochondral ossification, the majority of
osteoblasts are derived from the perichondrium
(Colnot et al., 2004) and
although osteoblast precursors were present in Ihh-/-
perichondrium (Fig. 6), the
organization of the tissue was clearly affected. The first reported evidence
of an Ihh-/- skeletal defect is at E13.5
(St-Jacques et al., 1999
), but
we decided to re-examine the mutant skeletal elements beginning at the stage
when Ihh is first expressed, at E11.5 in the central chondrogenic
condensation (Bitgood and McMahon,
1995
). Within 24 hours of the initial induction of Ihh
transcription (i.e. E12.5) we found that in mutants,
Ihh-/- perichondrial cells failed to align and condense
around the cartilage core like their wild-type counterparts
(Fig. 8A,F). The reason for
this became clearer when we mapped the expression of Ptch and
Gli1 to cells that surrounded the wild-type condensation, in the
region of the putative perichondrium (Fig.
8B, and data not shown). These two hedgehog target genes were
absent from Ihh-/- limbs
(Fig. 8G, and data not shown),
correlating with the thin and disorganized perichondrium
(Fig. 8F). Thus, an
Ihh-dependent perichondrial defect was present well before the advent
of the pre-hypertrophic and hypertrophic chondrocyte defects that have been
reported previously (St-Jacques et al.,
1999
).
|
The Ihh-/- vasculature was also affected at this very early stage. Vessels surrounding the chondrogenic condensation normally reside within a Ptch-, Gli1-positive domain (Fig. 8B, and data not shown) but in Ihh-/- elements neither of these hedgehog target genes was expressed, which markedly changed the local environment of the Ihh-/- endothelial cells (Fig. 8G). So although endothelial cells still surrounded the mutant cartilage condensations, the lack of hedgehog signaling adversely affected their subsequent development. For example, while PECAM-positive endothelial cells normally reside within the inner layer of the perichondria (Fig. 8E, arrows) the same cells were restricted to regions outside the Ihh-/- perichondria (Fig. 8J, arrows). Although blood vessels eventually infiltrated Ihh-/- perichondria (data not shown) and reached the hypertrophic cartilage, they did not persist in the putative ossification center (Fig. 3). We therefore concluded that the inability of Ihh-/- endothelial cells to respond properly to angiogenic signals in and around the ossification center at E14.5 was probably due to improper programming at E11.5, when Ihh is first expressed and its target genes are expressed in this cell type (Fig. 8B, and data not shown).
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Discussion |
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Our results also indicate a role for IHH beyond its potential function as a
regulator of osteoblast differentiation. Long before osteoblasts are called
upon to differentiate, the Ihh-/- phenotype is evident.
Ihh-/- cells surrounding the mutant cartilage condensation
are disorganized by E12.5 (Fig.
8). Consequently, Ihh-/- perichondria are
thinner and since this tissue is the primary source of osteoblasts during
endochondral ossification (Colnot et al.,
2004), this provides a probable explanation for the lack of a bone
collar in the Ihh-/- appendicular skeleton.
There is another, earlier defect that contributes to the lack of a bony collar and that is in the recruitment of cells to the perichondrium. The hedgehog-responding domain (marked by Ptch and Gli1 expression) initially extends some distance from the edge of the cartilage condensation; with time, the domain becomes restricted to the perichondrium proper (Fig. 8, and data not shown). The consolidation of hedgehog-responsive cells corresponds to the consolidation of Col1, from a broad expression domain to a tightly localized one, surrounding the cartilage condensation (Fig. 8, and data not shown). In Ihh-/- elements the hedgehog-responding domain is lost and the Col1 expression boundary fails to sharpen in a timely manner (Figs 6 and 8). Thus, the Ihh-/- osteoblast phenotype is exacerbated because IHH is probably required for the proper segregation of cells to a perichondrial lineage, and later, for osteoblast differentiation.
Does IHH convey an identity to blood vessels?
The Ihh-/- vascular defect also arises in the early
stages of skeletogenesis. First, Ihh-/- blood vessels do
not position themselves properly with respect to the perichondrium
(Fig. 8), and second, they do
not persist after an initial invasion of hypertrophic cartilage
(Fig. 2). These
skeletal-associated blood vessels normally develop in a field of active IHH
signaling, and since Ihh-/- vessels develop in the absence
of this morphogen, we theorized that the cells might be less mature than
wild-type vessels. In comparing the vessels within the
Ihh-/- vascular islands to those present at the time of
vascular invasion in wild-type elements, however, we could find no clear
difference in their maturity. Both sets of vessels were positive for markers
of early (PECAM) and late (COL4) endothelial cell maturation and devoid of
SMA-positive pericytes (Fig.
4). Therefore, since SMA-positive pericytes are not present at the
time of vascular invasion in wild-type tissues, it is unlikely that a smooth
muscle defect contributes to the Ihh-/- vascular defect
(i.e. failure of vessels to expand into a true ossification center and their
subsequent disappearance). This is in spite of the fact that pericytes are
plainly responsive to hedgehog proteins in the marrow cavity (data not shown)
and in adjacent tissues (Fig.
4F,G). Although we were unable to identify a difference in the
expression of vascular markers between wild-type and
Ihh-/- blood vessels, these results still leave open the
possibility that IHH may act on local vessels in more subtle ways than were
detectable by our immunological assays.
Some hints as to these kinds of vascular defects came from the ex vivo
environment, where we found that even though Ihh-/-
endothelial cells can invade (Fig.
3), and become associated with pericytes
(Fig. 4L), they eventually
disappear and leave behind vessels composed entirely of wild-type endothelial
cells (Fig. 3). Because
wild-type and Ihh-/- endothelial cells in the ex vivo
setting are exposed to the same extracellular environment, the eventual
disappearance of Ihh-/- endothelial cells cannot be
attributed to an absence of some trophic signal in the surrounding
environment. Neither can the disappearance of mutant endothelial cells be
explained by evoking their inability to produce IHH, since wild-type
endothelial cells do not normally produce it. They do, however, respond to the
morphogen. So while one report refutes the idea that hedgehog proteins act
directly on endothelial cells (Pola et
al., 2001), others provide evidence that hedgehog proteins
directly induce endothelial cells to form capillaries
(Kanda et al., 2003
;
Vokes et al., 2004
). The
altered functionality of Ihh-/- endothelial cells
associated with the endochondral skeleton would not necessarily manifest
itself until the cells were challenged to perform a specific task, such as
persisting and expanding within the ossification center (see
Fig. 2). Neither would an
altered functionality necessarily register on in vitro assays (e.g. migration,
proliferation, or differentiation assays), as their function may be context
dependent. A subtle effect of hedgehogs on endothelial cell identity and
function is not without precedent, as sonic hedgehog is required to impart
arterial over venous identity in the zebrafish
(Lawson et al., 2002
). One
enticing possibility then is that IHH modulates the ability of endothelial
cells to respond to local environmental cues that direct organ-specific
angiogenesis.
Overall, the data shown here and those of others indicate that IHH is
involved in multiple steps and acts through multiple mechanisms in the program
of skeletogenesis, ranging from a direct role in cartilage and perichondrial
differentiation to a role in determining osteoblast and endothelial cell fate.
While previous studies have demonstrated that IHH regulates chondrocyte
proliferation and maturation (Chung et al.,
2001; St-Jacques et al.,
1999
; Vortkamp et al.,
1996
), we have here begun to uncover the earliest manifestations
of the Ihh-/- phenotype and to understand how these early
defects may contribute to the full-blown defects characteristic of late-stage
Ihh-/- embryos.
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ACKNOWLEDGMENTS |
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