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First published online 18 February 2009
doi: 10.1242/dev.023820

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Noncanonical frizzled signaling regulates cell polarity of growth plate chondrocytes

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA.

View larger version (54K): [in this window] [in a new window]   Fig. 1. Resting and proliferative chondrocytes display distinct behaviors. (A) The growth plate contains distinct zones composed of resting, proliferative, prehypertrophic and hypertrophic chondrocytes. (B,C) H&E-stained paraffin sections of E9 chick cartilage reveal the transition from round disordered cells to ordered discoid cells that accompanies the maturation of resting chondrocytes (B) into proliferative chondrocytes (C). (D,E) To determine the relationship between clonal expansion and tissue structure, E3 limb buds were injected with RISAP virus, incubated to E9, and stained for alkaline phosphatase activity (purple). Resting chondrocytes give rise to clones that expand with radial symmetry (D). By contrast, proliferative chondrocyte clones form columns (E). Scale bar: 50 µm in B-E.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Oriented cell divisions in proliferative chondrocytes. (A,B) The structure of the proliferative zone changes as chick cartilage matures between E6 and E9 from a dense packing to an ordered array of individual cells. (C,E) To test whether order in the proliferative chondrocytes involves oriented cell divisions, the plane of cell division was visualized in tissue sections using DAPI to label the nuclei and rhodamine-phalloidin to label the cleavage furrow or contractile ring (arrow in E). (D) The orientation of cell division () relative to the long axis of the cartilage (parallel to `b') was calculated from measurements obtained from 3D images of cells in telophase using the equation provided (see Materials and methods for details). (F) This method revealed that resting chondrocyte progenitors divide at arbitrary angles that are nonuniformly distributed (P<0.005), whereas proliferative chondrocytes and prehypertrophic chondrocytes display divisions aligned at =81-90° (distinct from resting cells at P<0.0001). Aligned planes of cell division are characteristic of proliferative chondrocytes as early as E6 (P<0.0001 versus resting cells). (G) Orientation of the mitotic spindle at metaphase was determined by a similar method, except that the hypotenuse in D is a line that connects the two -tubulin containing centrosomes (arrows) flanking condensed chromatin (blue) at the metaphase plate. (H) When assessed by this method, resting and proliferative chondrocytes display similar distributions of at metaphase (P=0.555). Scale bars: 25 µm in B for A,B; 6.4 µm in E; 3 µm in G.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Cell-autonomous Fzd signaling regulates the polarity of proliferative chondrocytes. (A) Infection of the chick limb with RCAS(A)-Fzd7-C results in long bones that are shorter and wider than wild-type elements. Images oriented with the shoulder joint at the top. (B) Proliferative chondrocytes infected with RCAS(A)-Fzd7-C display a uniform distribution of (P=0.133) that is distinct from that of uninfected (WT) cells (P<0.0001), whereas telophase for RCAS(A)-Fzd7-C-infected resting chondroctyes is non-uniform (P<0.001) as in wild-type resting cells. Telophase of Fzd7-expressing proliferative chondrocytes is indistinguishable from that of cells expressing Fzd7-C (P=0.738). (C) Cartilage growth defects are dependent on infection of chondrocytes. Humeri from uninjected (WT) limbs, and limbs injected with low or high titer RCAS(A)-Fzd7-C virus were mounted on the same slide and hybridized with a riboprobe complementary to chicken Fzd7. WT limbs show low levels of signal in the perichondrium (black arrow) but not in chondrocytes (red arrow). Injection of low titer virus results in a strong infection in the perichondrium and weak to no infection of the cartilage, with no discernable effect on cartilage length. High titer virus results in ubiquitous infection of the chondrocytes and produces growth defects that include shortening (689.0±124.4 µm versus 1494.0±165.3 µm in WT; n=4 humeri per condition) and widening (258.5±49.3 µm versus 137.5±12.8 µm in WT) of the humerus. (E-G') Mosaic cartilage was generated by infection with low titer virus encoding Fzd7-C, D2 or D2KM. Infected cells were detected by in situ hybridization for the mutant molecule expressed from the virus (purple) and visualized by differential interference contrast microscopy. The red wedges define an angle cell that describes the relationship between the orientation of the long axis of the adjacent infected cell and the longitudinal axis of the cartilage (black arrow in E). E'-G' are schematic representations of the orientation of the long axes of the wild-type (blue bars) and infected (red bars) cells in E-G. (E,E') Disorder of Fzd7-C-expressing proliferative chondrocytes is readily apparent by comparing the arrangement of small patches of infected cells with their wild-type neighbors (unstained cells). (F,F') Similar effects are observed in cells expressing D2 (note the abnormal cell division, which is nearly parallel to the long axis of the cartilage; white arrow), but not in cells expressing D2KM (G,G'), which is deficient in blocking noncanonical Fzd signaling. (D) Quantitative image analysis (see E-G') reveals that wild-type and D2KM cells are aligned orthogonal to the cartilage axis, whereas cell for chondrocytes expressing Fzd7-C or D2 is uniformly distributed from 0-90° (P<0.001), demonstrating that inhibition of Fzd signaling results in cell-autonomous defects in chondrocyte polarity. Scale bars: 500 µm in A; 400 µm in C; 50 µm in E-G.

View larger version (88K): [in this window] [in a new window]   Fig. 4. Disruption of Fzd signaling alters the morphology of proliferative chondrocytes. Uninfected wild-type (WT) chick cartilage (A,G,M) and elements expressing Fzd7 (B,H,N), Fzd7-C (C,I,O), Vangl1 (D,J,P), Vangl2 (E,K,Q) or Rock2-N (F,L,R) were stained with H&E. Images of the resting chondrocytes (RC), proliferative chondrocytes (PC) and hypertrophic chondrocytes (HC) demonstrate that defects are first observed in the proliferative chondroctyes. Cell proliferation (%BrdU) is not affected by expression of Fzd-C or Vangl2. n=3 limbs per condition. N.D., not determined. Scale bar: 50 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 5. The orientation of cell division in proliferative chondrocytes is insensitive to canonical Fzd signaling but is partly dependent on Wnt/Ca2+ signaling. (A-C) Paraffin sections of E9 chick long bones stained with H&E. Wild-type (WT) cells are discoid (A), whereas expression of the canonical pathway antagonists dnLef1 (B) and Dkk1 (C) results in cells that display abnormal morphology. (D,E) Controls for expressed protein activity. In micromass cultures, limb mesenchyme cells infected with RCAS(A)-GFP form numerous cartilage nodules (blue) that are similar in size and spacing. By contrast, expression of dnLef1 or Dkk promotes increased chondrogenesis as determined by the decrease in spacing between individual nodules. (E) Widespread expression of a stabilized mutant of β-catenin, daβ-catenin, suppresses cartilage formation. Note the absence of the radius (R) and poor formation of the digits (D; compare with wild-type limbs in Fig. 3A). (I) Despite the change in shape, cell division in infected proliferative chondrocytes is indistinguishable from wild type (P=0.976 and 0.968 for dnLef1 and Dkk, respectively). (F,G) Expression of daβ-catenin (daβ-cat; purple, arrows) does not alter the morphology of resting (n=25/25, 4 limbs) or proliferative (n=29/30, 4 limbs) chondrocytes. The arrows in G indicate infected proliferative chondrocytes dividing in the correct plane. (H) By contrast, constitutive activation of the Wnt/Ca2+ pathway by expression of activated CamKIIa (daCamKII) produces round cells (arrows) that display telophase (see I) that is distinct from both resting (P<0.001) and proliferative (P<0.001) chondroctyes. (J) Gene expression analysis was performed on Fzd signaling pathway core components [Fzd receptors and Dsh (Dvl)], PCP components (Vangl, Prickle/Pk, Daam and Celsr) and Ca2+ regulators (CamKII). cDNA synthesized from total growth plate RNA was analyzed by PCR (+RT) as described in Materials and methods (and see Table S1 in the supplementary material). Control samples using RNA not subjected to reverse transcription (-RT, negative control) and total cDNA from HH 25 embryos (positive control, not shown) were run in parallel. Signal was judged as present (+), weak (w) or absent (-) after 28-30 cycles based on analysis of ethidium bromide-stained agarose gels. Signal was detected in the -RT control after 38 cycles. Scale bars: in A, 50 µm for A-C,F,G and 100 µm for H; 2 mm in D,E.

View larger version (68K): [in this window] [in a new window]   Fig. 6. Disruption of PCP signaling affects the polarity of proliferative chondrocytes. (A) Expression of the PCP signaling component Vangl2, but not the PCP-defective mutant Vangl2-C, results in decreased length and increased width of chick long bones. (B,B') Mosaic tissue expressing an epitope-tagged Vangl2 (green, white arrows) and counterstained with phalloidin (red, red arrows) shows proper secretion of the protein to the cell membrane in both resting (B) and proliferative (B') chondrocytes. In these mosaic tissues, Vangl2-expressing proliferative chondrocytes exhibit defects in the orientation of the long axis, whereas Vangl2-expressing resting chondrocytes are normal. (C,C') As with immunofluorescence, in situ hybridization supports the hypothesis that Vangl2 functions cell-autonomously, as shown by the disorder of infected chondrocytes (purple in C, red bars in C') as compared with neighboring wild-type cells (unstained cells in C, blue bars in C'). Asterisks denote cells for which a long axis could not be determined. (D) Expression of Vangl2 results in a uniform distribution of telophase (P=0.102), whereas expression of Rock2-Nalters the distribution of telphase distinct from wild-type proliferative chondrocytes (P<0.001). Telophase for Vangl1-expressing chondrocytes is indistinguishable from that of chondrocytes expressing Vangl2 (P=0.75). Note that the ectopic expression of either Vangl1 or Vangl2 produces a similar phenotype to the expression of Fzd7-C, whereas the phenotype of Rock2-N is weaker with respect to the orientation of the plane of cell division. Chondrocytes expressing Vangl2-C behave as wild type (P=0.4). (E) Quantification of the orientation of the long axis of proliferative chondrocytes shows that cell for Vangl2 expression is uniformly distributed from 0-90°, whereas uninfected neighboring cells display wild-type alignment. Scale bars: 500 µm in A; 50 µm in C.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Model for regulation of chondrocyte morphogenesis by Fzd signaling. Maturation of chondrocytes involves conversion from progenitor (resting) cells that divide in arbitrary planes (1, multiply oriented arrows) to proliferative chondrocytes that display aligned planes of cell division (2, bidirectional arrow). Cytokinesis in proliferative chondrocytes results in daughter cells that are initially displaced laterally relative to the column axis (3). Subsequently, these cells intercalate (4) to form a single column composed of progeny from a single progenitor cell (5). The canonical Fzd signaling pathway is important for cartilage growth and chondrogenesis, and might play a role in regulating the resting chondrocytes. However, the morphogenetic properties of proliferative chondrocytes are regulated via a noncanonical pathway. At a minimum, this pathway regulates cell polarity that is required for the proper alignment of cell divisions and orientation of the cell body. It remains to be determined whether these effects result from Fzd-dependent regulation of the extracellular matrix or cell-matrix adhesion molecules.

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