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

Abstract

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Fig. 1

Kif-3a-deficient cranial bases and synchondroses are abnormal. (A–B,H–I) Skulls from P7 control (Kif3a^fl/fl) and Kif3a-deficient (Kif3a^fl/fl;Col2-Cre) mice were analyzed by μCT and are shown by bird-eye view at low and high magnification. Location of intrasphenoidal (is), spheno-occipital (so) and intra-occipital (io) synchondroses is indicated in controls; arrows point to defects in mutant specimens. (C–E,J–L) Parasagittal H&E-stained sections of control and Kif3a-deficient cranial bases displaying is and so synchondroses and intervening endochondral bone. Note that mutant synchondroses’ histology and organization are markedly abnormal compared to controls, and distance between the synchondroses is reduced as well (indicated by horizontal line). (F–G,M–N) Immunolocalization of acetylated α-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 for A,H; 2 mm for B,I; 300 μm for C–E and J–L; 100 μm for F,M; and 35 μm for G,N.

Fig. 2

Synchondrosis growth plate organization and chondrocyte proliferation are deranged in Kif3a-deficient cranial bases. (A–D) Parasagittal sections of P7 control spheno-occipital synchondrosis. Note the presence of resting (rz), proliferative (pr), pre-hypertrophic (phz) and hypertrophic (hz) growth plate zones and primary bone spongiosa (arrowheads in D). Boxed colored areas in A are shown at higher magnification in B–D. (E–G) Presence and location of proliferating chondrocytes in control P0, P7 and P15 so synchondroses as revealed by histone 4C gene expression by in situ hybridization. Hybridization signal was given an artificial color for illustration purpose. Note the presence of two well-defined proliferative zones indicated by arrowheads flanking a central resting zone. (H) PTHrP gene expression in control P0 synchondrosis that characterizes resting and proliferative zones. (I–L) Parasagittal sections of P7 Kif3a-deficient so 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 Kif3a-deficient synchondrosis Scale bars: 300 μm for A,I; 40 μm for B,C,J,K; 80 μm for D,L; and 150 μm for E-H and M-P.

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) so synchondroses were processed for in situ hybridization analysis of indicated genes, using radiolabeled riboprobes. Hybridization signal was given artificial colors and images were superimposed to hematoxylin histologic images of corresponding field. Scale bar is 100 μm for A–J.

Fig. 4

Gene expression of cartilage-to-bone-associated genes and formation of primary spongiosa are inhibited in Kif3a-deficient synchondroses. (A,F) Parasagittal serial sections of P7 control (A) and mutant so synchondrosis (F) were stained with fast green/Safranin O to reveal bone tissue. Note the marked reduction of primary bone spongiosa in mutant tissue (F) that is instead quite clear in controls (A, arrowheads). (B–E) and (G–J) Expression of indicated genes as revealed by in situ hybridization with serial sections of control (B–E) and mutant (G–J) tissue. Scale bar is 80 μm for A–J.

Fig. 5

Intramembranous ossification is excessive near Kif3a-deficient synchondroses. Parasagittal serial sections of P7 control (A–D) and mutant so synchondroses were processed for staining with fast green/Safranin O (A,E) or in situ hybridization analysis of indicated genes. Note the presence of intramembranous bone collar adjacent to the pre-hypertrophic and hypertrophic zones in control (A–D, arrowhead) and its absence near the proliferative and resting zones (A–D, arrow) as to be expected. Note instead that intramembranous bone had formed all along the flank of the Kif3a-deficient synchondrosis (E–H). Scale bar is 75 μm for A–H.

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 H&E or processed for in situ hybridization analysis of collagen II expression. Ectopic cartilaginous masses forming in mutant specimens (arrows in D–E) are recognizable by their typical histology and expression of collagen II. Such phenomenon is never observed in control specimens where the chondro-perichondrial boundary is clear and un-violated (A–C). Scale bar is 75 μm for A–F.

Fig. 7

Topography of hedgehog signaling is altered in Kif3a-deficient synchondroses. Serial section of P0 control (A–L) and mutant (M–X) is and so synchondroses were processed for expression analysis of indicated genes. Note in controls that Patched-1 and Gli-1 are expressed in proliferative zone (pz) and perichondrium flanking pre-hypertrophic and hypertrophic zones (C–D, I–J, single arrowhead), but not in perichondrium flanking the resting and proliferative zones (C–D, I–J, double arrowheads). In mutants, however, Patched-1 and Gli-1 are minimally expressed within the growth plates, but are expressed all along the perichondrial tissues (O–P, U–V, single arrowhead). Note also that syndecan-3 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. Scale bar is 150 μm for A–X.

Fig. 8

Conditional postnatal Ihh deficiency causes cranial base abnormalities. (A,E) Skulls from P15 control and Ihh-deficient mice were subjected to μCT analysis and one orthogonal plane through the cranial base is shown here. Note the presence of well defined is and so synchondroses in controls (A, arrows) and the ill-defined synchondroses and reduced antero-posterior length in mutants (E). (B–D, F–H) Sections from P7 and P15 control and mutant so synchondroses processed for collagen X gene expression (B,F) or histological analysis (C–D,G–H). Note that collagen X transcripts are restricted to hypertrophic zones in control (B) but are widespread throughout the mutant synchondrosis (F). Note also the presence of a well-formed intramembranous bone collar flanking the pre-hypertrophic and hypertrophic zones in control (C, arrowheads) that is undetectable in mutant (G, arrowhead). Note also that much of mutant synchondrosis is replaced by endochondral bone by P15 (H). Scale bar is 2 mm for A,E; 150 μm for B,F; 75 μm for C,G; and 250 μm for D,H.