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First published online July 27, 2007
doi: 10.1242/10.1242/dev.007542

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Evc is a positive mediator of Ihh-regulated bone growth that localises at the base of chondrocyte cilia

Victor L. Ruiz-Perez^1,2,*,,, Helen J. Blair^1,*, M. Elena Rodriguez-Andres², Maria Jose Blanco³, Amy Wilson¹, Yu-Ning Liu¹, Colin Miles¹, Heiko Peters¹ and Judith A. Goodship^1,

¹ Institute of Human Genetics, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK.
² Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
³ Department of Human Anatomy and Embryology, Faculty of Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Generation of Evc-/- mice. (A) Wild-type Evc allele and the targeting vector. The length of homologous sequence flanking the lacZ and neomycin resistance cassettes in the targeting vector is indicated; the two loxP sites are shown as triangles. (B) Southern blot analysis confirming homologous recombination at the Evc locus. DNA from wild-type and Evc+/- mice was digested with HindIII (left) or EcoRI (right) and hybridised, respectively, with probes 1 and 2 (see A). Asterisks designate hybridisation signals corresponding to the targeted allele. (C) PCR products from tail-tip genotyping. (D) RT-PCR showing total absence of Evc transcript in Evc-/- mice and unaffected transcription of Crmp1. Three different primer pairs were used to amplify Evc cDNA (NM_021292) downstream of exon1 and Crmp1 cDNA (NM_007765). Hprt was used as a control.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Evc localises to the basal body and is not required for ciliogenesis. (A) Acetylated tubulin and -tubulin immunofluorescence (green) distinguish the ciliary axoneme and the two centrioles, respectively, in mIMCD3 cells. Anti-Evc (red) reveals Evc to be localised at the base of the cilium. Arrowheads, centrioles. (B) -tubulin (green) and Evc (red) immunofluorescence show that Evc signal overlaps with one of the two centrioles. (C) In situ Evc immunostaining of wild-type and Evc-/- chondrocytes of P0 proximal tibia demonstrate Evc localisation, the presence of cilia in the mutant and the specificity of the antibody. Scale bar: 2 µm.

View larger version (140K): [in this window] [in a new window]   Fig. 3. Evc expression pattern. ß-gal activity at E11.5 (A,B), E12.5 (C), E13.5 (D), E15.5 (E-J) and in newborn (K-N) mice demonstrating expression in the mouth and tooth-forming areas and in the developing skeleton. The ossified centres of the long bones are not ß-gal-positive. E is a coronal vibratome section of the head of an embryo. H,I,J are sagittal paraffin sections of the head, lower jaw and vibrissae. (K,L) Absence of endogenous ß-gal activity in control (K) and expression of lacZ in the proximal nail bed in Evc-/- (L, arrow) mice. (M,N) Vibratome sections showing growth plates of distal (M) and proximal (N) tibia of newborn mice. The blue signal in the bone marrow is background, as it was also detected in equivalent wild-type sections. Evc expression has the same distribution in heterozygotes and mutants from E12.5 to birth. All images relate to Evc+/- embryos with the exception of newborn mice (L-N), which were Evc-/-, and K which was wild-type. 1, lateral nasal process; 2, maxillary process; 3, mandibular process; e, eye; ns, nasal septum; nc, nasal capsule; jo, Jacobsos organ; ul, upper lip; oc, oral cavitity; ui, upper incisor; mx, maxilla; ma, mandible; mc, Meckel's cartilage; tb, temporal bone; li, lower incisor; dp, dermal papillae. Growth plate chondrocytes labelling: R, resting; P, proliferative; PH, prehypertrophic; H, hypertrophic; B, bone.

View larger version (86K): [in this window] [in a new window]   Fig. 4. Phenotype of Evc-/- mice. (A) Radiographs at P18. (B) Skeletal staining of the vertebral column, basiocciput, ribs, forelimb, hindlimb, forepaw and hindpaw of P6 mice. There is premature mineralisation across vertebral synchondroses in the mutant (arrows), an irregular margin between bone and cartilage in the basiocciput and costochondral junctions, shortening of the radius and ulna, tibia, metacarpals and metatarsals and a modelling defect at the distal ulna. (C) Mutants (upper panels) showed small dysplastic incisors and (lower panels) conical lower molars, size reduction of the first molar and an enamel defect.

View larger version (69K): [in this window] [in a new window]   Fig. 5. The growth plate in Evc-/- mice. In all panels, the wild type is shown on the left, Evc-/- mutant on the right. (A) Haematoxylin-Eosin staining of the upper end of the tibia of E17.5 embryos. The proliferative and hypertrophic zones are indicated by arrows. The mutant epiphysis lacks the normal convex shape. (B) von-Kossa staining of the upper end of tibia from E17.5 embryos. Arrows indicate absent perichondrial mineralisation. (C) Top, trichromic staining of the knee joint at P16 showing delay of secondary ossification and a defect in the shape of the tibial epiphysis. Below, magnification of the P16 growth plates from panels above, showing disorganised growth plate structure and fewer cells in the columns of proliferative chondrocytes.

View larger version (60K): [in this window] [in a new window]   Fig. 6. Evc is required for Ihh signalling. (A) Tissue in situ hybridisation analysis from E16.5 embryos showing indistinguishable Ihh expression between wild type and mutant, but markedly decreased hybridisation signals of Ptch1 and Gli1 in Evc-/- embryos. Bright-field (left) and dark-field (right) hybridisation pictures from proximal tibia are shown. (B) RT-PCR analysis of Ptch1 expression in wild-type and two different Evc-/- MEF cultures grown with (+) or without (-) purmorphamine. Hprt expression is used as a control. (C,D) Ptch1and Gli1 quantitative RT-PCR analysis in chondrocytes cultured with (+) or without (-) purmorphamine. ß-actin was used as control for both quantitative PCR experiments. (E) Western blot showing unchanged levels of full-length Gli3 (FL, 190 kDa) and Gli3R (R, 83 kDa) in limb protein extracts of E14.5 wild-type and Evc-/- mice. Ratios of the ß-catenin control band:Gli3R band were 1.47 and 1.41 for wild-type and Evc-/-, respectively.

View larger version (86K): [in this window] [in a new window]   Fig. 7. Evc-deficient mice show premature hypertrophic differentiation of proliferating chondrocytes. (A) Analysis of chondrocyte proliferation. On the left, E17.5 wild-type and mutant proximal tibia sections, stained with BrdU immunhistochemistry; the rectangles indicate the areas of columnar chondrocytes that were counted. On the right, bar chart showing that the percentage of BrdU-positive cells in the proliferating zone does not significantly differ between wild type and mutant (ANOVA, P=0.2374). (B) Expression of differentiation markers Col2a1, Col10a1, Pthrp and PPR in E16.5 wild-type and mutant embryos. The region of Pthrp expression is outlined by the dashed line. Note the absence of PPR signal in the perichondrium adjacent to late proliferating and early prehypertrophic chondrocytes in Evc-/- sections (arrowheads). (C) Comparative analysis of Fgfr3 and Fgfr1 expression pattern in wild-type and mutant E16.5 distal tibiae. There is no difference in Fgfr1 expression, but marked shortening of the Fgfr3 expression domain. The boxed areas of the left-hand panels are shown at high mignification in the middle panels for Fgfr3 and in the right-hand panels for Fgfr1.