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First published online February 6, 2009
doi: 10.1242/10.1242/dev.028605

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EphA4 as an effector of Twist1 in the guidance of osteogenic precursor cells during calvarial bone growth and in craniosynostosis

Department of Biochemistry and Molecular Biology, Norris Cancer Hospital, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176, USA.

View larger version (52K): [in this window] [in a new window]   Fig. 1. Fusion of coronal sutures in EphA4 mutant mice. Skulls of animals at P21 were stained with Alizarin Red S and photographed. The diagram (left) shows the area depicted in the photographs. Note open coronal sutures in control (A), EphA4+/- (D) and Efna2-/- (E); note partially fused sutures in Twist1+/- (B) and EphA4-/- (C), and little or no influence of Efna2 genotype on suture fusion in EphA4-/- mutant (F). cs, coronal suture; fb, frontal bone; ms, metopic suture; pb, parietal bone; ss, sagittal suture.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Increased number of alkaline phosphatase-expressing cells and defect in the neural crest-mesoderm boundary in coronal sutures of EphA4-/- embryos. Heads of wild-type Wnt1-Cre; R26R, Twist1+/-; Wnt1-Cre; R26R, and EphA4-/-; Wnt1-Cre; R26R embryos at E14.5, E16.5 were sectioned in the plane indicated and alternate sections were stained either for alkaline phosphatase (A-C and G-I) or lacZ expression (D-F and J-L). Pups at P0 were examined only for lacZ expression (M-O). Schematics depicting key results are shown below each image (A'-O'). Note widening of ALP domain (A-C, brackets) and expansion of ALP expression into suture (G-I, arrows). Also note lacZ-positive (neural crest) cells located ectopically in prospective coronal suture and parietal bone (D-F,J-L,M-O, arrows). cs, coronal suture; fb, frontal bone; pb, parietal bone.

View larger version (93K): [in this window] [in a new window]   Fig. 3. Altered expression of EphA4 mRNA in Twist1 mutant suture. Probes against EphA4 (A-B') or Twist1 (E-F') mRNA were incubated with sections of wild-type, Twist1+/- or EphA4-/- embryos at E 14.5. Alternate sections were stained for ALP activity (C,D,G,H). Note co-expression of EphA4 and Twist1 in ectocranial mesenchyme (arrows). Also note reduced expression of EphA4 in Twist1+/- mutant but unchanged expression of Twist1 in EphA4-/- mutant. Plane of section was as in Fig. 2. fb, frontal bone; pb, parietal bone.

View larger version (104K): [in this window] [in a new window]   Fig. 4. Genetic interaction between Twist1 and EphA4. Heads of wild-type, Twist1+/- and Twist1+/-; EphA4+/- embryos were examined for the expression of ALP and the osteoblast determinant, Runx2. ALP was assessed by histochemistry, Runx2 by immunostaining. Skulls of P21 animals were stained with Alizarin Red (J-L). Note progressive loss of ALP-free zone of coronal suture with reduction of Twist1 and EphA4 dosage (A-C). Also note increased staining of Runx2 and ALP in sections of heads at E14.5 (D-I) and increased coronal suture fusion in skulls of P21 mice (J-L). cs, coronal suture; fb, frontal bone; pb, parietal bone; ss, sagittal suture.

View larger version (106K): [in this window] [in a new window]   Fig. 5. Co-regulation of Erk1/2 phosphorylation in developing coronal suture by Twist1 and EphA4. (A-H) Heads of E14.5 embryos with the indicated genotypes were sectioned as in Fig. 2 and stained either for P-Erk1/2 or ALP. (I-L) Sections were stained for P-Erk1/2 (I,K) or total Erk (J,L). Note progressive reduction in P-Erk1/2 staining in ectocranial layer (arrows) as dosage of Twist1 and EphA4 are reduced. Note also that total Erk1/2 is unaffected in Twist1+/-; EphA4+/- embryos whereas P-Erk1/2 is substantially reduced. Thus Twist1 and EphA4 regulate Erk1/2 phosphorylation specifically. cs, coronal suture; fb, frontal bone; pb, parietal bone.

View larger version (109K): [in this window] [in a new window]   Fig. 6. Altered distribution of P-Smad1/5/8-expressing cells in coronal sutures of Twist1 and EphA4 individual and combination mutant embryos. Heads of E14.5 embryos were sectioned as in Fig. 2 and stained for P-Smad1/5/8 activity. Alternate sections were stained for ALP. Right panels show enlargements of dashed squares in left panels. Note concentration of stained nuclei in osteogenic fronts of wild-type embryos (A-D). In mutant embryos, note scattered stained nuclei and loss of concentration of stained nuclei in osteogenic fronts (E-P). fb, frontal bone; pb, parietal bone.

View larger version (74K): [in this window] [in a new window]   Fig. 7. Cooperative control of neural crest-mesoderm boundary at coronal suture by Twist1 and EphA4. Either the neural crest marker Wnt1-Cre; R26R or the mesoderm marker Mesp1-Cre; R26R was crossed into mice with the indicated genotypes. Heads of E16.5 embryos were sectioned as in Fig. 2 and stained for lacZ. Schematics depicting key results are shown below each image. Note sharp mesoderm-neural crest boundary in wild-type embryo (A,A',D,D'). Note both neural crest-derived cells and mesoderm-derived cells crossing boundary in mutant embryos. Also note increased severity of boundary defect in Twist1+/-; EphA4+/- embryo (C,C',F,F') compared with Twist1+/- embryo (B,B',E,E').

View larger version (71K): [in this window] [in a new window]   Fig. 8. Mis-migration of osteogenic precursor cells in Twist1+/-; EphA4+/- mutant embryos. We injected DiI into parietal bone rudiments of E13.5 wild-type (A-D,I-K,O-Q) and mutant (E-H,L-N,R-T) embryos, in vivo. Embryos were allowed to develop exo utero to E16.5 (A-N) or E15.5 (O-T), when they were examined for the distribution of DiI by epifluorescence microscopy. Sections shown in I,K,L,N,O,Q,R and T were also stained for ALP activity. E16.5 (A-N): embryos shown in whole mount in A-H were sectioned; images of the sections are shown in I-N. (A,E) Brightfield images of whole mounts. Note location of coronal sutures (arrowheads). (B,F) Epifluorescence images, with dotted lines showing location of sutures. (C,G) Merged brightfield and epifluorescence images. Area within dashed square is magnified in D,H. Labeled osteogenic precursor cells migrate apically (B-D) and insert into the growing bone (J,K). Note DiI-labeled cells are excluded from coronal sutures of wild-type (B,D, dotted lines) but not mutant embryos (G,H arrows). This is also clearly evident in sections: compare J,K with M,N. E15.5 (O-T): images are from coronal sections of E15.5 embryos (plane of section indicated). (O,R) Brightfield images stained for ALP; (P,S) epifluorescence images; (Q,T) combined brightfield and epifluorescence images. Note that in wild-type embryos, labeled cells are located largely in layer flanking (ectocranial to) the osteogenic layer (arrows, `ec'). In mutant embryos, labeled cells are located diffusely in and around osteogenic layer of parietal bone (arrowheads, `pb'). Closely similar results were obtained in more than ten injected wild-type embryos and three injected mutant embryos.

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