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First published online 18 February 2009
doi: 10.1242/dev.023820
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Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA.
* Author for correspondence (e-mail: a-dudley{at}northwestern.edu)
Accepted 28 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Fzd signaling, Polarity, Chondrocyte, Planar cell polarity, Morphogenesis, Skeleton
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Resting
chondrocytes comprise a population of progenitor cells that reside at the ends
of the bones and exhibit a low level of cell cycle activation. Maturation
begins with the formation of discoid proliferative chondrocytes that display
increased cell cycle activity. After many rounds of division, the cell cycle
is downregulated and the cells change shape (prehypertrophic chondrocytes).
Subsequently, prehypertrophic chondrocytes enlarge to form hypertrophic
chondrocytes that secrete the extracellular matrix protein collagen X and
undergo cell death. Thus, the rate of long-bone growth is regulated via
changes in cell number and cell volume
(Breur et al., 1991;
Hunziker et al., 1987).
The acquisition of unique morphogenetic properties is one remarkable
characteristic of the maturation of resting chondrocytes into proliferative
chondrocytes (Dodds, 1930).
During the transition, individual resting chondrocytes, which are round and
dispersed in the cartilage matrix, give rise to large single columns of
tightly packed discoid proliferative chondrocytes. Whether the precise
arrangement of chondrocytes depends on physical or mechanical properties of
the extracellular environment (Aszodi et
al., 2003; Ham,
1932), or is regulated by secreted signaling molecules
(Abad et al., 2002), is
unknown. Here we show that proliferative chondrocytes undergo oriented cell
divisions that occur orthogonal to the direction of growth and then
intercalate to form columns in a process that appears highly similar to
convergent extension by cell intercalation
(Keller et al., 1989;
Keller et al., 2000). Both the
division plane and cell orientation depend on noncanonical frizzled (Fzd)
signaling. Disruption of Fzd signaling additionally alters polarized growth of
the long bones, resulting in skeletal elements that are shorter and wider than
wild-type bones. Together, these results suggest a model in which regulation
of cell polarity by noncanonical Fzd signaling plays a crucial role in
controlling the three-dimensional morphogenesis of skeletal elements.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Construction of recombinant retrovirus
Retrovirus constructs were generated in RCAS(A)
(Hughes, 2004) using the
Slax21 shuttle vector and standard methods
(Logan and Tabin, 1998).
Fzd7 and dnFzd7 were subcloned as previously described
(Hartmann and Tabin, 2000).
Other sequences were amplified from stage-25 chick embryo cDNA by PCR
(reaction details and primer sequences are available upon request).
RCAS-Dkk, and RCAS-dnLef1 and RCAS-daCamKII were
gifts from C. M. Chuong (University of Southern California) and C. J. Tabin
(Harvard Medical School), respectively. RISAP and RCAS(A)-daβ-catenin
were provided by C. L. Cepko (Harvard Medical School) and Matsuhiro Iwamoto
(Thomas Jefferson University College of Medicine), respectively. Concentrated
virus stocks (108-109 pfu/ml) were prepared by standard
procedures (Logan and Tabin,
1998), except for RISAP, RCAS(A)-D2, RCAS(A)-D2KM and
RCAS(A)-dnRock, which were pseudotyped with VSV-G and produced by transient
expression to 106-107 pfu/ml
(Chen et al., 1999).
Processing of chick embryos
Fertilized chicken eggs (Phil's Fresh Eggs, Forreston, IL, USA) were grown
at 37.5°C and staged (HH) as described
(Hamburger and Hamilton,
1992). Concentrated retrovirus stocks were injected into the right
forelimb at HH 19-21. For mosaic analysis, stocks were diluted to
107 pfu/ml in DMEM+0.1% FBS. After incubation, the humerus and ulna
were dissected and fixed in 4% paraformaldehyde in PBS (pH 7.2) at 4°C.
Micromass cultures were prepared and analyzed as described
(Cottrill et al., 1987;
Tufan et al., 2002).
Histology
To analyze cell morphology, tissue was fixed overnight, dehydrated through
an ethanol series, embedded in paraffin (Paraplast X-tra, Tyco-Healthcare) and
sectioned. Hematoxylin and Eosin (H&E) staining was performed using a
standard protocol. For quantification of bone dimensions, the length and width
(halfway between the articular surface and the mineralized region) were
determined from the center section of a cartilage element (n=4 for
each condition). For lineage analysis, harvested tissue was fixed for 4 hours,
washed in PBS, heat-inactivated at 65°C for 30 minutes, and stained for
alkaline phosphatase activity as described
(Chen et al., 1999).
To analyze cell proliferation, S-phase cells were labeled with the nucleotide analog bromodeoxyuridine (BrdU) for 4 hours prior to tissue harvest. Sections of BrdU-labeled tissue were digested in trypsin and depurinated in hydrochloric acid. Incorporated BrdU was detected using a mouse anti-BrdU monoclonal antibody (G3G4, S. J. Kaufman, Developmental Studies Hybridoma Bank, Iowa, USA) and Cy2-labeled donkey anti-mouse antibody (Jackson ImmunoResearch, Pennsylvania, USA). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The BrdU labeling index was calculated as the percentage of total nuclei (DAPI-stained) that were BrdU-positive.
Gene expression analysis
Transcripts of Fzd pathway components were detected by PCR (reaction
details and primer sequences are available upon request). Whole-embryo RNA
from pools of HH 16-23 chick embryos was used as a positive control (data not
shown). Bands were visible on ethidium bromide-stained agarose gels after
27-29 cycles, whereas background bands in -RT samples were only observed after
more than 35 cycles. For in situ hybridization analysis, tissue sections were
hybridized to RNA probes labeled with digoxigenin (Roche) as described
(http://genepath.med.harvard.edu/~cepko/protocol).
Analysis of cell division orientation and cell arrangement
We adapted previously described methods to analyze cell division in
sections of fixed tissue (Gong et al.,
2004; Tibber et al.,
2004). Dissected cartilage was fixed overnight, equilibrated in
30% sucrose in PBS, and frozen in O.C.T. Compound (Tissue-Tek). Tissue was
cryosectioned at 20 µm, mounted on Superfrost Plus slides (VWR), and
briefly air-dried. Sections were washed in PBS, permeabilized in 1% Triton
X-100, and incubated with rhodamine-phalloidin (1:100; Molecular Probes) and
DAPI (0.1 µg/ml; Sigma) to label the contractile ring and DNA,
respectively. Three-dimensional images were generated from a z-series
of optical sections collected on a Zeiss apotome deconvolution microscope. The
orientation of cell division was determined by mathematical modeling in which
a cube was drawn with daughter nuclei occupying diagonal corners on opposite
faces (see Fig. 2D). In this
model, line segment `b' is set parallel to the long axis of the cartilage
(equivalent to the distance between the nuclei in the y-axis) and
line segment `a' is the distance between nuclei in the x-axis. Line
segments `a' and `b' were measured using Zeiss Axiovision software. Line
segment `c' (the distance between daughter nuclei in the z-axis) was
determined by counting the number of 1 µm optical sections spanning the
centers of the daughter nuclei. The angle (
) generated by the
intersection of line segment `b' and a line segment connecting the daughter
nuclei was determined using the formula shown in
Fig. 2D. Calculated angles were
categorized into bins of 10° and the percentage of the total cells in each
bin was plotted as a bar chart. Cells in metaphase were imaged using
anti-
-tubulin (1:1500; Sigma), secondary anti-mouse Cy3 (1:500; Jackson
ImmunoResearch) and DAPI (0.1 µg/ml). Spindle orientation at metaphase was
calculated by applying the same mathematical model as above except that the
cube was drawn with apposing centrosomes at opposite corners.
The orientation of the long axis of the cell was determined from section in
situ hybridizations. cell is defined by the intersection of
a line drawn through the longitudinal axis of the cell profile and a line
drawn parallel to the long axis of the cartilage.
Statistical methods
Data for each manipulation were obtained from at least six limbs (a minimum
of three limbs on each of two days) except where noted in the text and see
Table S1 in the supplementary material. The Kolmogorov-Smirnov test was used
to determine whether the distributions of were distinct for different
experimental conditions (Herrick,
1965; Ong and LeClare,
1968). For this analysis, P>0.05 demonstrates
similarity between compared distributions. Tests for arbitrary angles compared
the observed values to a uniform distribution over =0-90°.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). To
determine whether the unique organization of proliferative chondrocytes
depends on physical restriction by the extracellular matrix, we examined cell
behavior in chick long bones at early developmental stages. At embryonic day 9
(E9; HH 33-35), proliferative chondrocytes are elongated but not organized
into obvious columns (Fig. 1C).
However, progeny of individual chondroprogenitor cells labeled by infection
with the replication-defective avian retrovirus, RISAP
(Chen et al., 1999), displayed
a zone-specific arrangement. Thus, resting chondrocytes gave rise to clones
displaying radial symmetry (25/25) (Fig.
1D), whereas 80% of clones (39/48) in the proliferating zone
formed single columns (Fig.
1E). A minority of clones (data not shown) appeared either as two
adjacent columns (7/48) or as large groups of labeled cells (2/48). Thus,
columnar organization is an intrinsic behavior of proliferative chondrocyte
clones at all stages of development.
Although columns of proliferative chondrocytes grow by clonal expansion,
cell division is biased against orientations that result in daughter cells
aligned parallel to the column (Dodds,
1930). We asked whether the division plane, like column
organization, is regulated prior to the thickening of the extracellular
matrix. In sections of E9 long bones (Fig.
2B), cells in telophase were identified by the presence of an
actin-rich contractile ring between two future daughter cells
(Fig. 2C,E). An angle of cell
division, termed , was determined from three-dimensional images
(Fig. 2D) (see Materials and
methods), such that 0° defines a cleavage plane that generates daughter
cells arranged in a column, and 90° divisions displace daughters laterally
with respect to the forming column. Wild-type resting chondrocytes divided at
arbitrary angles, whereas wild-type and GFP-expressing proliferative
chondrocytes exhibited a strong bias for telophase =81-90°
(Fig. 2F; see Fig. S1 in the
supplementary material). Bias in telophase was observed in E6 (HH 27)
(Fig. 2A,F) proliferative
chondrocytes and in E9 prehypertrophic chondrocytes
(Fig. 2F). Thus, collectively,
and consistent with our lineage data, resting and proliferative chondrocytes
display distinct behaviors that remain substantially unaffected by
developmental changes in the microenvironment.
What, then, restricts the plane of division in proliferative chondrocytes?
One possibility consistent with Hertwig's Rule
(Wilson, 1900) is that the
discoid cell shape constrains the mitotic apparatus to lateral orientations
(Carreira-Barbosa et al.,
2003; Toyoshima and Nishida,
2007). If this is the case in proliferative chondrocytes,
orientation of the mitotic spindle at metaphase
(Fig. 2G) should be similar to
telophase . Unexpectedly, metaphase
(Fig. 2H) was significantly
different from telophase (Fig.
2F) (P<0.0001), demonstrating that spindle formation
is not restricted to a specific axis. Furthermore, metaphase was
highly similar in the morphologically distinct resting and proliferative
chondrocytes (Fig. 2H)
(P=0.555), indicating that the position at which the spindle forms is
not strongly influenced by chondrocyte shape.
Decreased Fzd signaling interferes with oriented cell divisions
Spindle rotation during asymmetric cell divisions in Drosophila
(Adler and Taylor, 2001;
Roegiers et al., 2001) and in
Caenorhabditis elegans (Wu and
Herman, 2006a) requires Fzd function. Although Fzd
signaling is important for growth plate function
(Hartmann and Tabin, 2000), a
role for Fzd in regulating the division plane in chondrocytes has not
been reported. To test this possibility, we used replication-competent avian
retrovirus (RCAS) (Hughes,
2004) to express a C-terminal truncated form of chicken frizzled 7
(Fzd7-C) (Hartmann and Tabin,
2000) in the chick forelimb. Chicken Fzd7-C is
analogous to a molecule that inhibits Fzd signaling by a dominant-negative
mechanism in zebrafish (Nasevicius et al.,
1998). E9 humeri uniformly expressing high levels of
Fzd7-C are significantly shorter and wider than those of
uninfected controls (Fig.
3A,C), but cell proliferation was not affected
(Fig. 4). In these limbs,
infected resting chondrocytes appeared normal
(Fig. 4C), whereas
proliferative chondrocytes displayed aberrant cell morphology and the long
cell axes were not aligned (Fig.
4I). Nonetheless, infected cells flattened at a position
consistent with the normal interface between resting chondrocytes and
proliferative chondrocytes (data not shown), suggesting that downregulation of
Fzd signaling neither interferes with cell morphology in general, nor disrupts
global organization of the growth plate.
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).
Although Ihh expression is decreased,
Fzd7-C-expressing resting and proliferative chondrocytes
express parathyroid hormone related peptide (Pthlh) and patched
(Ptch), respectively (Hartmann
and Tabin, 2000). Pthlh and Ptch are two
Ihh-responsive genes that are also components of a key feedback loop that
regulates proliferative chondrocyte formation (reviewed by
Kronenberg, 2003;
Yang et al., 2003). Together,
these data suggest that chondrocytes expressing Fzd7-C
undergo maturation and participate in long-range signaling interactions
between the resting and prehypertrophic chondrocytes that are crucial to
maintain growth plate architecture and function.
We next examined the division plane in infected E9 chondrocytes. Expression
of Fzd7-C did not affect telophase of resting
chondrocytes, whereas telophase for infected proliferative
chondrocytes was uniformly distributed from 0-90° (P=0.133)
(Fig. 3B). In agreement with
previous studies that demonstrated similar phenotypes in limbs expressing
Fzd7-C and wild-type Fzd7
(Hartmann and Tabin, 2000), we
found that Fzd7 expression resulted in a telophase
indistinguishable from that of Fzd7-C-expressing
proliferative chondrocytes (Fig.
3B) (P=0.738). Collectively, our data suggest that
chondrocyte polarity is highly sensitive to Fzd signaling levels.
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;
Hinoi et al., 2006), a tissue
that lines the developing cartilage. Thus, effects of
Fzd7/Fzd7-C expression on chondrocyte morphogenesis might
result from defects in PC/PO function. To test this possibility, we asked
whether infection of the PC/PO is sufficient to produce the observed
phenotype. This test is possible because we found that soft tissues of the
limb, including the PC/PO, are more easily infected with replication-competent
virus than cells of the cartilage. Thus, injection of lower titer virus stocks
results in highly infected PC/PO and few infected chondrocytes
(Fig. 3C; see Fig. S3 in the
supplementary material). Moreover, these data demonstrate that in situ
hybridization can be used to identify infected cells in tissue sections
because the inserted sequence is expressed at higher levels than the
endogenous locus. In this context, limbs infected with low titer virus are
similar to wild-type limbs in cartilage length and chondrocyte morphology
(Fig. 3C; see Fig. S3 in the
supplementary material). These data strongly suggest that Fzd signaling in the
PC/PO does not regulate growth plate architecture or the growth properties of
cartilage elements.
We next asked whether the organization of the proliferative chondrocytes
depends on cell-autonomous Fzd function. We created mosaic embryos by
injecting diluted virus to generate small patches of infected chondrocytes
surrounded by large numbers of wild-type cells. Cells within patches
expressing Fzd7-C displayed a range of morphological
defects, but most cells had clearly defined long and short axes
(Fig. 3E). Quantification of
cell orientation (cell; see Materials and methods) revealed
that wild-type cells adjacent to infected patches displayed normal orientation
(Fig. 3D,E,E'). By
contrast, expression of Fzd7-C randomized the cell
orientation, even when infected chondrocytes were surrounded by wild-type
cells that displayed normal morphology
(Fig. 3D,E,E'). To
confirm that altered morphology results from cell-autonomous defects in Fzd
function, we asked whether a similar phenotype would result from
downregulation of dishevelled (Dsh, also known as Dvl), an intracellular
signaling mediator that directly interacts with Fzd via a PDZ domain
(Wallingford and Habas, 2005).
PDZ-domain deletions of Dsh [D2 construct in Rothbacher et al.
(Rothbacher et al., 2000)]
abrogate Fzd signaling in a dominant-negative manner
(Axelrod et al., 1998). Cells
expressing D2 had a measurable long axis that was misaligned
(Fig. 3D,F,F'). Thus,
Fzd function autonomously regulates cell polarity and cell
arrangement in the proliferative chondrocytes.
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). To
test whether reduced β-catenin function accounts for the observed defects
in polarity, we expressed dickkopf 1 (Dkk)
(Suksaweang et al., 2004), an
extracellular antagonist that blocks activation of the
β-catenin-dependent pathway, and a putative dominant-negative mutant form
of Lef1 (dnLef1)
(Kengaku et al., 1998), a key
component of the β-catenin transcriptional complex
(Fig. 5A-C). Consistent with
their predicted activities, expression of these inhibitory molecules enhanced
cartilage formation in micromass cultures of chick limb mesenchyme
(Fig. 5D). In vivo,
chondrocytes expressing dnLef1 and Dkk displayed minor
morphological changes (Fig.
5B,C), but telophase was similar to that of wild-type
proliferative chondrocytes (Fig.
5I). As a further test of the requirement to regulate
β-catenin activity, we expressed a truncated form of β-catenin that
lacks the N-terminal domain that confers instability and is therefore
constitutively activated (daβ-cat)
(Rubinfeld et al., 1997). In
mosaic cartilage, groups of morphologically normal
daβ-cat-expressing cells were readily observed in both
resting and proliferative chondrocytes
(Fig. 5F,G). Collectively,
these data suggest that proliferative chondrocyte morphogenesis is not
sensitive to β-catenin levels. Consistent with this, proliferative
chondrocytes display normal organization in β-catenin mutant mice
(Hill et al., 2005;
Mak et al., 2006).
Noncanonical Fzd signaling regulates the plane of cell division
Since interfering with the canonical pathway did not alter the division
plane, we next addressed whether the polarity defects observed in
Fzd7-C-expressing chondrocytes could result from altered
noncanonical signaling. Gene expression analysis revealed the presence of
multiple isoforms of calcium-calmodulin dependent protein kinase II (CamKII),
which are components of the noncanonical Fzd/Ca2+ pathway in chick
growth plate cartilage (Kuhl et al.,
2000). Because putative dominant-negative forms of CamKII are weak
inhibitors of the pathway, we tested the role of Fzd/Ca2+ signaling
by ectopically expressing a constitutively active form of CamKII
(daCamKII) (Abzhanov et al.,
2006; Kuhl et al.,
2000). Expression of daCamKII altered cell morphology
(Fig. 5H) and shifted the plane
of cell division from telophase =81-90°
(Fig. 5I). These data are
consistent with a potential role for noncanonical Fzd signaling in orienting
the plane of cell division, but do not fully account for the uniform telophase
in cells expressing Fzd7-C.
Our gene expression analysis also showed that some components of the
noncanonical planar cell polarity (PCP) pathway
(Klein and Mlodzik, 2005) are
expressed in the developing growth plate
(Fig. 5J). The possibility that
a PCP-like pathway functions in proliferative chondrocytes is further
supported by the similar phenotypes in Fzd loss- and gain-of-function
experiments (Fig. 3B)
(Krasnow and Adler, 1994) and
the fact that D2 has a stronger inhibitory effect on the PCP pathway
than on the β-catenin-dependent pathway
(Axelrod et al., 1998;
Krasnow and Adler, 1994;
Rothbacher et al., 2000).
Since PCP signaling requires the DEP domain in Dsh
(Rothbacher et al., 2000;
Wallingford and Harland, 2002;
Wallingford et al., 2000), we
first tested whether the effect of D2 on cell orientation requires
DEP function. We introduced a missense mutation into D2 (K441M;
D2KM), analogous to the dsh1 mutation in
Drosophila that specifically abrogates DEP-dependent PCP signaling by
dishevelled but permits normal activation of β-catenin-dependent
signaling (Axelrod et al.,
1998; Boutros et al.,
1998). The K441M mutation blocked the ability of D2 to
interfere with chondrocyte morphogenesis
(Fig. 3D,G,G'). These
data suggest that D2 function depends on interaction with effectors
of PCP signaling.
In the Drosophila wing, localization of Van Gogh (Strabismus;
vertebrate Vangl), a four-pass transmembrane regulator of PCP signaling that
interacts with Dsh (Park and Moon,
2002), to the plasma membrane on one face of the cell confers
polarity that aligns hairs in the plane of the epithelium
(Klein and Mlodzik, 2005). As
such, both gain- and loss-of-function Vangl mutations disrupt PCP in
Drosophila and vertebrates
(Krasnow and Adler, 1994). We
expressed chick Vangl2 bearing a C-terminal hemagglutinin epitope tag
(Vangl2HA) in the wing cartilage and confirmed, by
immunofluorescence, that the protein is secreted to the membrane
(Fig. 6B,B'). Like
Fzd7-C, expression of Vangl2 generated short,
wide cartilage elements (Fig.
6A) containing disorganized proliferative chondrocytes
(Fig. 4K) that displayed normal
cell cycle characteristics (Fig.
4) but reduced zones of mature chondrocytes (see Fig. S2 in the
supplementary material). Similar to Fzd7-C, expression of
Vangl2 resulted in a uniform telophase
(Fig. 6D) (P=0.103).
Expression of the related gene, Vangl1, produced results
indistinguishable from those of Vangl2 by histology
(Fig. 4J) and telophase
(Fig. 6D) (P=0.75). In
addition, Vangl2 expression randomized the orientation of
proliferative chondrocytes in a cell-autonomous manner
(Fig. 6C,C',E).
Expression of a truncated form lacking the C-terminal PDZ-binding motif
(Vangl2-C) that is required for interaction with the PCP
pathway but not the canonical pathway
(Park and Moon, 2002), did not
affect cartilage growth (Fig.
6A) or the division plane (Fig.
6D), suggesting that, as with D2, the effect of
Vangl2 expression is dependent on interaction with downstream
effectors of the PCP pathway. Potential downstream effectors of this pathway
include the Rho GTPases and Rho-associated kinase 2 (Rock2)
(Kim and Han, 2005;
Phillips et al., 2005). We
determined the effect of altered Rho pathway function by expressing a putative
dominant-negative fragment of chicken Rock2 (Rock2-N)
(Leung et al., 1996) in the
developing wing. Expression of Rock2-N had a marked effect on
proliferative chondrocytes, resulting in changes in cell morphology
(Fig. 4L) and in the division
plane (Fig. 6D) that were
highly similar to those of chondrocytes expressing Fzd7-C
and Vangl2. Collectively, these data suggest the possibility that the
regulation of Rho by noncanonical Fzd signaling controls the plane of cell
division and cell polarity in proliferative chondrocytes.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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=81-90° in proliferative
chondrocytes, but remains unrefined in resting chondrocytes. Collectively, our
data suggest that noncanonical (β-catenin-independent) Fzd signaling
regulates the refinement of spindle orientation in proliferative chondrocytes
(Fig. 7). One possible
mechanism is that Fzd signaling directly affects spindle alignment by
regulating the localization of proteins that position the centrosomes.
Alternatively, noncanonical Fzd signaling might regulate cell flattening
during mitosis that in turn constrains the mitotic spindle in telophase. Both
models are consistent with known functions of the noncanonical pathways, but
the latter model is unlikely to act predominately because the division plane
is largely unchanged in daCamKII-expressing chondrocytes that do not
flatten. That the division plane and cell shape are not tightly linked is
further supported by the fact that neighboring affected cells often display
different axial polarities. Instead, this suggests that, as in the neural tube
(Ciruna et al., 2006),
noncanonical Fzd signaling additionally functions to re-establish polarity in
chondrocytes following cell division.
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;
Keller et al., 1989). Daughter
cells with different polarities might be unable to undergo the coordinated
cell movements necessary for intercalation. However, one important difference
with other examples of convergent extension is that chondrocyte columns derive
from clonal expansion and not from cell recruitment, which generates mixed
lineage columns in terminal filaments (TFs) of the Drosophila
ovariole (Godt and Laski,
1995) and in the vertebrate notochord
(Keller et al., 1989). In this
context, noncanonical Fzd signaling might act by maintaining a functional
interface between daughter cells or by promoting complementary shape changes
in interacting cells.
A key question is how polarity is coordinated both locally and globally in
the proliferative chondrocytes. Tissue polarity has been modeled by gradients
of secreted factors or cell adhesion. A ligand-dependent signaling model is
consistent with our data and the observation that Wnt5a functions in
proliferative chondrocyte morphogenesis in mouse
(Yang et al., 2003), in
convergent extension movements in zebrafish
(Topczewski et al., 2001;
Zhu et al., 2006), and in the
polarity of stereocilia of the inner ear
(Qian et al., 2007). However,
it is not known whether cells determine polarity by interpreting a Wnt ligand
gradient or whether Wnt signaling is required for competence to respond to
other graded signals (Witze et al.,
2008), and a gradient of Wnt proteins in the growth plate has not
been demonstrated. One test of this model could come from understanding how
heparan sulfate proteoglycans of the glypican family regulate cell
intercalation via noncanonical Fzd signaling
(Ohkawara et al., 2003) and
from determination of whether ligand-dependent mechanisms are responsible for
abnormal cartilage architecture in Ext1
(Koziel et al., 2004) or
Gpc3 (Viviano et al.,
2005) hypomorphic mice and knypek (gpc4)-null
fish (Topczewski et al.,
2001).
In cartilage, cell-cell contact is unlikely to globally coordinate cell
polarity because cartilage structure is not altered in mature growth plates in
which thick extracellular matrix separates individual columns
(Dodds, 1930). Nonetheless,
order within clones (columns) could result from high-fidelity
adhesion-dependent local propagation of polarity initially conferred upon the
mother row cell that established the column. For example, cell-cell contact
through adherens junctions defines polarity in some epithelial stem cell
populations (Song et al.,
2002). Alternatively, cell-matrix interactions can orient matrix
fibrils and determine polarized cell movement
(Davidson et al., 2006;
Goto and Keller, 2002;
Marsden and DeSimone, 2003)
via integrin receptor function (Yu et al.,
2005; Yu et al.,
2008; Zhou and Kramer,
2005), or provide boundary functions that promote cell
intercalation (Veeman et al.,
2008). A potential role for cell-matrix adhesion is further
supported by altered growth plate structure in mouse mutants of cell-matrix
interaction molecules (see Table S2 in the supplementary material). However,
of these mutants, only loss-of-function of integrin β1
(Aszodi et al., 2003) and
ColIX (Blumbach et al.,
2008; Dreier et al.,
2008) display substantial loss of proliferative chondrocyte
structure. Despite similarities to the phenotypes we describe, differences
such as decreased cell proliferation and hypocellularity in cell adhesion
mutants suggest that the polarity defects described in the present studies
might not be solely due to altered cell-matrix interactions. It remains to be
determined whether Fzd signaling directly regulates integrin/matrix production
or localization, or modulates integrin function by crosstalk via a
Rock-dependent pathway (Fig.
7). Regardless, noncanonical Fzd signaling might help maintain
cell polarity and clone identity by regulating the expression and/or
localization of cell adhesion complexes on proliferative chondrocytes.
Is PCP the noncanonical pathway?
Three findings from our analysis above are consistent with the possibility
that a PCP-like pathway regulates the polarity of proliferative chondrocytes.
First, the similar phenotypes we observe in Fzd loss- and gain-of-function
experiments are a hallmark of PCP signaling
(Krasnow and Adler, 1994).
Second, Vangl gain-of-function phenocopies Fzd
loss-of-function, an effect that depends on the presence of the C-terminus of
Vangl, which interacts with PCP proteins. Third, the cell-autonomous effects
of Fzd are replicated by D2 only when DEP function is maintained.
Further support is provided by microarray analysis that shows specific
regulation of the core PCP gene Prickle1 during chondrocyte
maturation (Belluoccio et al.,
2008).
Although our data strongly point to a role for a PCP-like pathway in growth
plate morphogenesis, direct genetic support for this model is lacking. If not
the PCP pathway, which noncanonical effector pathway acts in proliferative
chondrocytes? One thread that connects pathways that regulate cell polarity
during convergent extension, PCP in the inner ear, and the columnar
organization of proliferative chondrocytes with neurite extension
(Carreira-Barbosa et al.,
2003; Jessen et al.,
2002; Nambiar et al.,
2007) and with one specific asymmetric cell division in the
Caenorhabditis elegans embryo (Wu
and Herman, 2006b) is that each process is consistently and
predictably affected by altered activity of PCP proteins. These underlying
commonalities suggest the presence of a core polarity module
(Lawrence et al., 2007) that
regulates cytoskeletal structure via Rock activity and cell adhesion modules
(Yu et al., 2005;
Yu et al., 2008). Thus,
context-dependent regulators might confer emergent properties on a core
pathway to generate distinct mechanisms for the interpretation and propagation
of cell polarity in diverse tissues.
Column formation and cartilage morphogenesis
One potential physiological role for polarized cell behaviors is the
regulation of cartilage growth. In particular, cell divisions oriented to
produce daughter cells aligned with the axis of growth are characteristic of
growing tissues (Baena-Lopez et al.,
2005; Fischer et al.,
2006). Curiously, proliferative chondrocytes divide and flatten
orthogonal to expectations, requiring the intercalation of daughter cells
following cell division. Subsequent hypertrophy of the chondrocytes, not cell
intercalation or cell proliferation, drives the elongation of cartilage
(Breur et al., 1991) and of
the frog notochord (Adams et al.,
1990). This two-step process could ensure high-density packing of
cells in the longitudinal axis to maximize the growth potential of hypertrophy
while minimizing disruption of column structure during mitosis. In our
studies, conditions that result in column loss produced bones that displayed
decreased longitudinal growth and increased lateral expansion. Consistent with
this observation, a strong correlation between long-bone dimensions and
proliferative chondrocyte structure exists in the literature
(Aszodi et al., 2003;
Blumbach et al., 2008;
Dreier et al., 2008;
Viviano et al., 2005;
Yang et al., 2003). Thus,
chondrocyte columns appear to orient vectors of growth and, therefore,
regulated cell polarity could provide vertebrates with a powerful system for
sculpting the diverse morphologies of bone that are required to generate an
articulated skeleton.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1083/DC1
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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