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First published online 13 December 2006
doi: 10.1242/dev.02732

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Intraflagellar transport is essential for endochondral bone formation

Courtney J. Haycraft^1,*, Qihong Zhang^1,*,, Buer Song², Walker S. Jackson^3,, Peter J. Detloff³, Rosa Serra¹ and Bradley K. Yoder^1,

¹ Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
² Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
³ Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Generation of a conditional allele of Ift88. (A) Schematic representation of the targeting construct and the Ift88 genomic region, showing the location of the exons and the loxP sites. The deletion allele (Ift88n) was generated by leaky germline Cre activity in prx1cre females. The diagram is not drawn to scale. (B) Wild-type, floxed and null alleles (generated from the floxed allele) of Ift88 were distinguished by PCR using genomic DNA isolated from tail biopsies of mice. The approximate locations of the primers used for genotyping are indicated in A. (C) At E11.5, mice heterozygous for the (Ift88n) deleted allele and the previously described targeted Ift882-3ß-gal null allele of Ift88 (right) exhibited phenotypes characteristic of the congenic Ift882-3ß-gal null mice, including defects in the neural tube when compared with a wild-type littermate (left).

View larger version (99K): [in this window] [in a new window]   Fig. 2. Cre-mediated recombination of the conditional allele results in loss of cilia and Ift88 protein. (A-C) Cilia were identified in frozen sections of E11.5 wild-type (A) and prx1cre;Ift88fl/n conditional mutant (B,C) limbs by immunofluorescence staining with antibodies to detect acetylated -tubulin (green) and Ift88 (red). Nuclei are shown in blue. In wild-type samples (A), cilia were visible on most cells in the mesenchyme. By contrast, cilia were rarely observed on cells in the mesenchyme of conditional mutants (B). The presence of cilia on the ectoderm of prx1cre;Ift88fl/n mutant limbs (C) was not affected. (D) Protein lysate from conditional mutant limbs at E11.5 (prx1cre) showed a dramatic reduction in the amount of Ift88 expressed when compared with lysate from a control littermate (WT). Samples were simultaneously blotted for ß-tubulin as a loading control. Approximate molecular weights are shown to the left of the blot. (E,F) At E11.5, cilia are apparent in the dorsal ectoderm of the hindlimb in msx2cre;Ift88fl/n mice (E) by immunofluorescence staining for acetylated -tubulin (green) and Ift88 (red), but are absent from the ventral ectoderm of the same limb (F).

View larger version (69K): [in this window] [in a new window]   Fig. 3. msx2cre;Ift88fl/n mice show no phenotype but prx1cre;Ift88fl/n mice develop polydactyly. (A-D) Images of the dorsal (A,B) and ventral (C,D) surfaces of the hindlimbs of wild-type (A,C) and msx2cre;Ift88fl/n conditional mutant (B,D) adult mice. No defects in limb patterning were visible in the conditional mutants. (E-H) Alizarin Red and Alcian Blue staining of forelimbs (E,F) and hindlimbs (G,H) from wild-type (E,G) and prx1cre;Ift887fl/n conditional mutant (F,H) mice at E18.5. Forelimbs of prx1cre;Ift88fl/n (F) develop eight or more nonpatterned digits. While the forelimb exhibited extensive polydactyly, the hindlimb (H) typically showed a duplication of digit I (asterisk). (I) Alizarin Red-stained forelimb of wild-type (bottom) and prx1cre;Ift88fl/n (top) conditional mutant at postnatal day 11. The proximodistal length of the limbs is shortened in conditional mutants, but the patterning of the long bones is largely unaffected. Anterior is up in all panels.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Expression of Gli1 and gremlin is altered in the forelimbs of prx1cre;Ift88fl/n conditional mutants. Whole-mount in situ hybridization analysis of Gli1 (A-D) and gremlin (E-H) expression in wild-type (A,C,E,G) and prx1cre;Ift88fl/n (B,D,F,H) forelimbs at E10.5 (A,B,E,F) and E11.5 (C,D,G,H). At E10.5, Gli1 expression is similar in conditional mutants (B) and wild-type controls (A). At E11.5, the expression of Gli1 is retained in wild-type controls (C) but is almost completely absent in conditional mutants (D), except small patches of cells expressing Gli1 can occasionally be seen in prx1cre;Ift88fl/n mutants (arrowhead). Gremlin expression is restricted to the posterior mesenchyme at E10.5 in wild-type forelimbs (E) but is expanded into the anterior mesenchyme in prx1cre;Ift88fl/n conditional mutants (arrowhead in F). Expression of gremlin is minimal in wild-type forelimbs at E11.5 (G). By contrast, in prx1cre conditional mutant forelimbs (H), ectopic anterior expression of gremlin is maintained (arrowhead in H). Anterior is up in all panels.

View larger version (89K): [in this window] [in a new window]   Fig. 5. Endochondral bones in prx1cre;Iftfl/n conditional mutant limbs are shorter than in wild-type littermates. Sections of hindlimbs from E18.5 wild-type (A,B), prx1cre;Ift88fl/n (C,D), and prx1cre;Kif3afl/n (E,F) conditional mutant embryos were stained with H&E to analyze bone formation. Frozen sections through the tibia are shown. The overall size of the growth region in conditional mutants is reduced (double-headed arrows in B,D,F). Vascularization is evident in conditional mutant tibiae (asterisks in D,F), although the marrow cavity is poorly formed. (G,H) Cilia, as visualized by localization of acetylated -tubulin (green) and Ift88 (red), are apparent on the chondrocytes in control animals (G). By contrast, few chondrocytes in the developing tibia of prx1cre;Ift88fl/n embryos (H) expressed a cilium.

View larger version (96K): [in this window] [in a new window]   Fig. 6. Ihh signaling is disrupted in the long bones of prx1cre;Ift88fl/n conditional mutants. (A-D) Radioactive in situ hybridization analysis of Ihh expression in the fibula of wild-type (C) and conditional mutant (D) mice at E18.5. Strong expression of Ihh is seen in the prehypertrophic chondrocytes in the wild-type sample (C). Ihh expression is also evident in the prehypertrophic chondrocytes of conditional mutants (D) although at reduced levels. (E-L) Analysis of Ptch1 (E-H) and Gli1 (I-L) expression in wild-type (G,K) and conditional mutant (H,L) tibiae. In wild-type samples, Ptch1 and Gli1 expression is increased in the proliferating and prehypertrophic regions (asterisks) in addition to the perichondrium (arrowhead). By contrast, no significant expression of Ptch1 or Gli1 is seen in conditional mutant samples. (M-P) Expression of PTHrP in wild-type (M,O) and conditional mutant (N,P) radii at E14.5. PTHrP is expressed in both wild-type (O) and conditional mutant (P) radii at E14.5. Bright field images of each section (A,B,E,F,I,J,M,N) show histology.

View larger version (157K): [in this window] [in a new window]   Fig. 7. Perichondrial architecture and bone collar development are affected in prx1cre;Ift88fl/n conditional mutant mice. (A-D) H&E-stained sections from E18.5 wild-type (A,C) and conditional mutant (B,D) tibiae. The outer and inner edges of the perichondrium are marked by green lines. The perichondrium in wild-type samples (A) is a compact layer flanking the bone anlagen. By contrast, the cells adjacent to the diaphysis in conditional mutant samples (B) are disorganized. The perichondrium varies in thickness along the length of the bone and is flanked by cells that more closely resemble chondrocytes than osteoblasts (green arrowheads in B). Metaphysis and perichondrium of tibia sections stained with H&E from E18.5 wild-type (C) and prx1cre;Ift88fl/n conditional mutant (D) tibiae, show loss of bone collar formation in conditional mutants. (E,F) Sections of wild-type (E) and conditional mutant (F) tibiae at E18.5 stained for alkaline phosphatase activity (blue) to identify the bone collar (arrow). Sections were counterstained with Nuclear Fast Red. Alkaline phosphatase expression is visible in the cells of the bone collar flanking the proliferating and prehypertrophic chondrocytes in wild-type samples (E), but is absent in the perichondrial region of conditional mutants (F). Insets in E and F show higher magnification views of the regions indicated by the arrows. The distal end of the tibia is left in all panels. HC, hypertrophic chondrocytes.

View larger version (102K): [in this window] [in a new window]   Fig. 8. Chondrocytes develop in the perichondrium of prx1cre;Ift88fl/n conditional mutant tibiae. (A,B) In sections of wild-type E18.5 tibia (A), cartilage matrix, identified with Safranin O staining (red), is only seen in the growth region of the long bone, whereas bone, identified with Fast Green staining (blue-green), is present along the diaphysis. In conditional mutants (B), regions of cartilage matrix are evident along the diaphysis of the tibia (arrowheads) and appear to be derived from the overlying perichondrium. (C,D) In sections of the wild-type tibia (C) at E18.5, aggrecan is localized to the hypertrophic region and excluded from the perichondrium by immunofluorescence. Aggrecan expression is found ectopically in the perichondrium of the conditional mutant tibia (asterisk in D). Nuclei are stained blue and the epiphysis is up in both panels. (E) An adjacent section of the conditional mutant tibia shown in D was stained with Safranin O and Fast Green to confirm the presence of cartilage matrix in the perichondrium of the mutant sample.