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First published online May 16, 2007
doi: 10.1242/10.1242/dev.001586

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Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis

Eiki Koyama^1,, Blanche Young¹, Motohiko Nagayama¹, Yoshihiro Shibukawa^1,*, Motomi Enomoto-Iwamoto¹, Masahiro Iwamoto¹, Yukiko Maeda², Beate Lanske², Buer Song^3,, Rosa Serra³ and Maurizio Pacifici^1,

¹ Department of Orthopaedic Surgery, Thomas Jefferson University College of Medicine, Philadelphia, PA 19107, USA.
² Department of Developmental Biology, Harvard School of Dental Medicine, Boston, MA 02138, USA.
³ Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

View larger version (82K): [in this window] [in a new window]   Fig. 1. Kif3a-deficient cranial bases and synchondroses are abnormal. (A,B,H,I) Skulls from P7 control (Kif3afl/fl; A,B) and Kif3a-deficient (Kif3afl/fl;Col2-Cre; H,I) mice were analyzed by micro-computed tomography (µCT) and are shown by birds-eye view at low (A,H) and high (B,I) magnification. The location of intrasphenoidal (is), spheno-occipital (so) and intra-occipital (io) synchondroses is indicated in controls (B); arrows point to defects in mutant specimens (I). (C-E,J-L) Parasagittal hematoxylin and Eosin (H&E)-stained sections of control (C-E) and Kif3a-deficient (J-L) cranial bases at P0, P7 and P15 displaying is and so synchondroses as well as intervening endochondral bone. Notice that the histology and organization of mutant synchondroses are markedly abnormal compared with controls, and that the distance between the synchondroses is also reduced (indicated by double-headed arrow). Arrowheads indicate ectopic bone formation. (F,G,M,N) Immunolocalization of acetylated -tubulin in primary cilia (arrowheads) in P0 control (F,G) and Kif3a-deficient (M,N) synchondrosis growth plate and associated perichondrium. Scale bars: 5 mm in H for A,H; 2 mm in I for B,I; 300 µm in J for C-E and J-L; 100 µm in M for F,M; and 35 µm in N for G,N. is, intrasphenoidal; io, intra-occipital; P, postnatal day; so, spheno-occipital.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Synchondrosis growth plate organization and chondrocyte proliferation are deranged in Kif3a-deficient cranial bases. (A-D) Parasagittal sections of a P7 control spheno-occipital synchondrosis. Notice the presence of resting (rz), proliferative (pr), pre-hypertrophic (phz) and hypertrophic (hz) growth plate zones and primary bone spongiosa (arrowheads in D). Areas in colored frames in A are shown at higher magnification in B-D. (E-G) Presence and location of proliferating chondrocytes in control P0 (E), P7 (F) and P15 (G) spheno-occipital synchondroses as revealed by histone 4C gene expression by in situ hybridization. The hybridization signal was given an artificial color for illustration purpose. Notice the presence of two well-defined proliferative zones (indicated by arrowheads) flanking a central resting zone. (H) Pthrp gene expression in a control P0 synchondrosis, which characterizes the resting and proliferative zones. (I-L) Parasagittal sections of a P7 Kif3a-deficient spheno-occipital synchondrosis showing that the growth plate zone structure is totally abnormal (J,K) and that there is a near absence of primary spongiosa (L). (M-O) Near absence of histone 4C-expressing proliferating chondrocytes in Kif3a-deficient synchondroses. (P) Pthrp gene expression in a Kif3a-deficient synchondrosis. Scale bars: 300 µm in I for A,I; 40 µm in J for B,C,J,K; 80 µm in L for D,L; and 150 µm in M for E-H and M-P. P, postnatal day; rz/pr/phz/hz, resting/proliferative/pre-hypertrophic/hypertrophic growth plate zones.

View larger version (83K): [in this window] [in a new window]   Fig. 3. Gene expression of chondrocyte-maturation-associated genes is depressed in Kif3a-deficient synchondroses. Serial sections from the medial portion of P7 control (A-E) and Kif3a-deficient (F-J) spheno-occipital synchondroses were processed for in situ hybridization analysis of the indicated genes, using radiolabeled riboprobes. The hybridization signal was given artificial colors and images were superimposed with hematoxylin histologic images of the corresponding field. Scale bar: 100 µm in J for for A-J.

View larger version (88K): [in this window] [in a new window]   Fig. 4. Gene expression of cartilage-to-bone-associated genes and the formation of primary spongiosa are inhibited in Kif3a-deficient synchondroses. (A,F) Parasagittal serial sections of P7 control (A) and mutant (F) spheno-occipital synchondrosis were stained with fast green/Safranin O to reveal bone tissue (dark blue). Notice the marked reduction of primary bone spongiosa in mutant tissue (F), which is highly visible in controls (A, arrowheads). (B-E,G-J) Expression of the indicated genes as revealed by in situ hybridization with serial sections of control (B-E) and mutant (G-J) tissue. The hybridization signal was given artificial colors and images were superimposed with hematoxylin histologic images of the corresponding field. Scale bar: 80 µm in E for A-J.

View larger version (143K): [in this window] [in a new window]   Fig. 5. Intramembranous ossification is excessive near Kif3a-deficient synchondroses. Parasagittal serial sections of P7 control (A-D) and mutant (E-H) spheno-occipital synchondroses were processed for staining with fast green/Safranin O (A,E) or in situ hybridization analysis of the indicated genes (B-D,F-H). (A-D) In controls, notice the presence of the intramembranous bone collar adjacent to the pre-hypertrophic and hypertrophic zones (arrowheads) and its absence near the proliferative and resting zones (arrows), as is to be expected. Notice instead that the intramembranous bone had formed all along the flank of the Kif3a-deficient synchondrosis (E-H). Scale bar: 75 µm in H for A-H.

View larger version (172K): [in this window] [in a new window]   Fig. 6. Presence of ectopic cartilage masses near Kif3a-deficient synchondroses. Sections of P7 and P15 control (A-C) and mutant (D-F) synchondroses were stained with hematoxylin and Eosin (H&E) or processed for in situ hybridization analysis of collagen II expression. Ectopic cartilaginous masses forming in mutant specimens (arrows in D-F) are recognizable by their typical histology and expression of collagen II. Such a phenomenon is never observed in control specimens, in which the chondro-perichondrial boundary is clear and unviolated (A-C). Scale bar: 75 µm in D for A-F.

View larger version (128K): [in this window] [in a new window]   Fig. 7. Topography of hedgehog signaling is altered in Kif3a-deficient synchondroses. Serial sections of P0 control (A-L) and mutant (M-X) intrasphenoidal (is) and spheno-occipital (so) synchondroses were processed for expression analysis of the indicated genes. (C,D,I,J) Notice that, in controls, Patched 1 (C,I) and Gli1 (D,J) were expressed in the proliferative zone (pz) and in the perichondrium flanking the pre-hypertrophic and hypertrophic zones (single arrowheads), but not in the perichondrium flanking the resting and proliferative zones (double arrowheads). (O,P,U,V) In mutants, however, Patched 1 (O,U) and Gli1 (P,V) are minimally expressed within the growth plates, but are expressed all along the perichondrial tissues (arrowheads). (F,L,R,X) Notice also that syndecan 3 gene expression is mainly restricted to the proliferative zone in controls (F,L), but is extremely low in mutants (R,X). Expression of Ihh and smoothened was similar in control (B,H,E,K) and mutant (N,T,Q,W) tissues at this stage. is, intrasphenoidal; pz, proliferative zone; so, spheno-occipital. Scale bar: 150 µm in A for A-X.

View larger version (81K): [in this window] [in a new window]   Fig. 8. Ihh distribution is altered in Kif3a-deficient synchondroses. (A-D) Serial sections of P0 control (A,B) and Kif3a-deficient (C,D) spheno-occipital synchondroses were processed for immunohistochemistry using Ab80 rabbit hedgehog antibodies. Sections were reacted with secondary fluorescent antibodies and counterstained with the nuclear dye DAPI (blue). Positive immunosignal is orange in color. (A,B) In controls, Ihh is present in the pre-hypertrophic zone (phz) and in adjacent proliferative chondrocytes and inner perichondrium (B, single arrowhead), but is undetectable in the upper growth plate zones and flanking perichondrium (B, double arrowhead). (C,D) However, in Kif3a-deficient specimens, Ihh is present in a more extensive and expansive gradient form throughout much of growth plate and all along perichondrium (D, single and double arrowheads). (E,F) Notice that the hedgehog antibodies produced no detectable signal with sections from Ihh-null synchondroses, attesting to their specificity. (B,D,F) Higher-magnification images of the boxed area in A,C and E, respectively. rz/pr/phz, resting/proliferative/pre-hypertrophic growth plate zones. Scale bar: 65 µm in F for B,D,F.

View larger version (128K): [in this window] [in a new window]   Fig. 9. Conditional postnatal Ihh deficiency causes cranial base abnormalities. (A,E) Skulls from P15 control (A) and Ihh-deficient (E) mice were subjected to micro-computed tomography (µCT) analysis; one orthogonal plane through the cranial base of each is shown here. Notice the presence of well-defined intrasphenoidal (is) and spheno-occipital (so) synchondroses in controls (A), and the ill-defined synchondroses and reduced anteroposterior length in mutants (E). (B-D,F-H) Sections from P7 and P15 control (B-D) and mutant (F-H) so synchondroses processed for collagen X gene expression (B,F) or for histological analysis (C,D,G,H). Notice that collagen X transcripts (red) are restricted to hypertrophic zones in controls (B) but are widespread throughout the mutant synchondrosis (F). Notice also the presence of a well-formed intramembranous bone collar flanking the pre-hypertrophic and hypertrophic zones in controls (C, arrowheads), which is undetectable in mutants (G, arrowhead). (D,H) Additionally, in mutants, much of the synchondrosis is replaced by endochondral bone by P15 (H). is, intrasphenoidal; so, spheno-occipital. Scale bar: 2 mm in E for A,E; 150 µm in F for B,F; 75 µm in G for C,G; and 250 µm in H for D,H.

View larger version (61K): [in this window] [in a new window]   Fig. 10. Model illustrating the phenotypic consequences of Kif3a deficiency. In a wild-type (WT) synchondrosis growth plate, Ihh would produce a physiologically restricted gradient (red) from the pre-hypertrophic zone (phz) into the flanking perichondrium and the preceding proliferative zone (pz). The upper limit of this restricted field of action would be set by Patched 1 and syndecan 3 (and other heparan sulfate proteoglycans), and would allow the normal proliferation of chondrocytes and the normal formation of intramembranous bone. In Kif3a-deficient (Kif3a-/-) growth plates, however, the Ihh distribution gradient would be expanded because of the marked reduction of syndecan 3 and Patched 1 expression. As a consequence, the topography of hedgehog signaling and action would be altered as well. Within the growth plate, Ihh signaling would be feeble, causing abnormal behavior of chondrocytes. In perichondrial tissues, Ihh signaling would be abnormally high and widespread, triggering excessive intramembranous bone deposition and ectopic cartilage formation. phz, pre-hypertrophic zone; pz, proliferative zone; WT, wild type.

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