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First published online 10 December 2003
doi: 10.1242/dev.00940

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Skeletal development is regulated by fibroblast growth factor receptor 1 signalling dynamics

Mohammad K. Hajihosseini^1,*,, Maria D. Lalioti^1,,, Sandrine Arthaud¹, Helen R. Burgar¹, Jill M. Brown², Stephen R. F. Twigg², Andrew O. M. Wilkie² and John K. Heath^1,

¹ School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
² Weatherall Institute of Molecular Medicine, The John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK

View larger version (58K): [in a new window]   Fig. 1. Generation and analysis of mutant BAC transgenes. (A) Schematic drawing of Fgfr1, harbouring three extracellular immunoglobulin (Ig)-like domains, with relative contributions of exons 6-9 indicated (dashed arrows). The grey region in the C-terminal half of the third Ig loop (III) shows the position of alternative splicing between the IIIb and IIIc isoforms, generated by the use of exons 8 or 9, respectively. Dashed lines expand and highlight the nucleotide sequence of the linker region where the Pro252Arg activating mutation (BAC16) and Leu257Leu silent mutation (BAC15) were introduced by base substitutions. Note that the modifications also introduce MspI-sensitive (BAC16) or PstI-sensitive (BAC15) sites (underlined). (B) FISH analysis, using Fgfr1 probes on metaphase chromosomes, shows integration of BAC transgene into chromosome 4 (arrows). This particular sample was derived from a 4C-transgene animal (see text) and, accordingly, the signal from the transgene is twice as intense as that emanating from the endogenous allele on chromosome 8 (arrowheads). (C) Reverse-transcriptase PCR (RT-PCR) products generated by primers from exons 6 and 8, exposed to MspI enzyme and resolved on a 3% agarose gel. Left and right lanes, products derived from wild-type and BAC16 mouse liver RNA, respectively. Note that the level of transgene expression (303 bp) is equal in intensity to that of the wild-type (343 bp). (D) Resolution of PCR products amplified from tail genomic DNA and digested with MspI. In the left lane (2C), the intensity of the mutant bands is equal to that of the wild type, whereas in the right lane, the mutant band is almost twice as bright. Measurements of these intensities using NIH-imaging software confirm a 2:2 ratio for the 2C, and greater ratios for the 4C mutants (values not shown).

View larger version (75K): [in a new window]   Fig. 2. Cranial and sternal defects in 2C- and 4C-BAC16 mutants. (A,B) Dorsal views of E18.5 skulls, showing presence of boney islands (double arrows) and a more advanced medial growth/ossification of frontal bones (f; distance between arrowheads) in 4C (B) compared with 2C (A) mutants. Apposition of parietal bones (P) also appears to be marginally advanced. (C,D) Lateral view of zygomatic arch bones at the lower rim of the left eye, showing advanced/premature ossification of sutures (arrows) separating the zygomatic branch of maxilla and the jugal bones in a 2C mutant (D) compared with wild type (C). (E-G) Dorsal views of older wild-type (E), 2C (F) and 4C (G) mutant skulls, showing growth and persistence of boney islands (double arrowheads) within the metopic sutures, as well as precocious synostosis of frontal bones (arrows in F). 2C mutant (F) also shows the slight bending of the face that becomes more severe in 4C mutants (G). (H-J) Ventral views of the palate from a wild-type (H), a 2C (I) and a 4C (J) BAC16 mouse. Arrow (I) indicates fusion of the joint separating the left premaxilla (pm) and maxillary (m) bones. Note that the joint on the right is patent. (J) The left pm appears less developed and the incisors have overgrown/ingrown (arrow). (K-M) Frontal views of the sternum stained for both Alizarin Red and Alcian Blue. (K) Wild type; (L) 2C mutants; (M) 4C mutants. 2C mutant (L) shows fusion of the fourth and fifth sternebrae (arrow) through ectopic ossification of their intervening cartilage. (M) Precocious ossification is more advanced in 4C mutants, and the xiphoid process is also bifurcated (arrows). Scale bars: 1 mm in C,K,L,M; 2mm in A; 2.5 mm in H; 3 mm in F,J; 4 mm in E; 5 mm in G.

View larger version (84K): [in a new window]   Fig. 3. Homeotic transformations within the vertebral column. (A,B) Ventral views of the cervical and upper thoracic vertebrae from P7 wild-type (A) and a 4C-BAC16 mutant (B) littermates. The clavicle, first sternebrae (manubrium) and ribs have been removed to expose the vertebrae. (B) The left side of the sixth cervical vertebrae, C6, has lost its anterior tuberculum (white arrow), whereas C7 has acquired a rudimentary process (black arrow). (C,D) Left lateral views of the same region in two other 4C animals. (C) Partial loss of C6 anterior tuberculum (white arrow) accompanied by fusion of ribs 1 and 2, and their lack of articulation with the sternum. Note that this lack of articulation results in precocious ossification in the joint separating the manubrium and second sternebrae. (D) The left part of C7 behaves like T1, by extending a rudimentary bone (black arrow) that articulates abnormally with the first rib. (E,F) Ventral views of lower lumbar and upper sacral vertebra of wild-type (E) and 4C-BAC16 (F) littermates. (F) The sixth lumbar vertebrae has acquired an S1 fate and begins to articulate with the adjacent pelvic bones. Scale bars: 2 mm in A,C,D; 3 mm in E,F.

View larger version (70K): [in a new window]   Fig. 4. Range of pre-axial polydactylies observed in the hind limbs of 4C-BAC16 mutants. (A,C,E,G,I) Polydactylous limbs prior to skeletal preparation/staining. (B,D,F,H,J) The corresponding skeletal structure after staining with Alcian Blue and Alizarin Red. (A,B) Dorsal view of postnatal day 6 wild-type right hind limb, showing the biphalangeal nature of digit I (toe) and triphalangeal nature of digits II-V. (C,D,F-J) Dorsal views of postnatal day 7 mutant limbs; (E) ventral view. (C,D) Left hind limb; (E-J) right hind limbs; C-F show limbs from the same animal. Note the triphalangeal nature of mutant digit I in F,H,J. The most frequent form of polydactyly observed (n=7/18) was that shown in F, and in seven out of eight bilateral polydactylous limbs, this type of defect was accompanied by the type shown in D. Note that there is no abnormality in shape, size, length or rate of ossification of the remaining digits. Scale bars: 2 mm in B,D,F,H,I,J; 3 mm in A,C,E,G,I.

View larger version (77K): [in a new window]   Fig. 5. Gene expression and patterns of cell death in developing 4C-BAC16 limbs. (A) Identical pattern of Shh expression in 4C-BAC16 mutant and wild-type limb buds. White arrow indicates the anterior part of mutant hind limb. (B-E) Expression patterns of Hoxd13 in the right hind limbs of 2C (B,D) and 4C (C,E) BAC16 mutants. 2C-BAC16 expression patterns resemble wild type (not shown). Dashed lines (B,C) represent the expected normal posterioranterior limit of Hoxd13 expression in developing hind limb buds; limb anterior is towards the bottom of photograph. (F) Medial and (G,H) anterior views of 2C (G) and 4C (H) E11.5 hind limb buds, showing a reduction in Dkk1 expression in the anterior two-thirds of the AER (region between the open arrowhead and black arrowhead) in the 4C-BAC16 limb. The 2C pattern resembles wild type (not shown) and `I' marks the anterior side of each limb corresponding to where digit I develops. Arrowheads point to the ANZ, where Dkk1 expression is completely quenched in the 4C limb bud. (I) Dorsal view of left and right hind limbs (LHL and RHL) from a 4C-BAC16 mutant stained with Neutral Red. The cell death pattern in this 4C-LHL resembles that of wild type (not shown). `I' marks the position of developing digit I condensates and the arrow indicates the absence of Neutral Red-positive cells in the abnormal (larger digit I) right hind limb, compared with the presence of such cells in the corresponding region in the normal left hind limb. Also note the similar, unperturbed, patterns of Neutral Red staining at the posterior margins of each limb. (J) Comparison of Wnt5a expression in wild-type and 4C-mutant embryos shows a higher Wnt5a signal in both fore and hind limbs of 4C-mutants, when compared with the wild type, particularly in the region generating digit I in the hind limb buds (arrows). Scale bars: 400 µm in F,I; 600 µm in B,D; 1.0 mm in J; 1.2 mm in A.