QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online November 3, 2003
doi: 10.1242/10.1242/dev.00793

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ishii, M. Articles by Maxson, R. E. Search for Related Content PubMed PubMed Citation Articles by Ishii, M. Articles by Maxson, R. E., Jr Social Bookmarking                         What's this?

Msx2 and Twist cooperatively control the development of the neural crest-derived skeletogenic mesenchyme of the murine skull vault

¹ Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center and Hospital, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176, USA
² Department of Morphology, Faculty of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel
³ Department of Craniofacial Development, King's College, London, UK
⁴ Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176, USA

View larger version (40K): [in a new window]   Fig. 1. Osteoblast and osteoclast marker expression in postnatal Msx2 mutant mice. (A,B) Skull vaults of postnatal day 4 mice were stained with Alizarin Red S. Note the large foramen in the frontal bone (fb) of the Msx2–/– skull (arrow) and increased parietal foramen (pf, open arrowhead). (C-F) Cross sections, indicated by lines in A and B, at the level of the defect, stained for alkaline phosphatase (ALP) activity. The rectangles in C and D demarcate areas shown at higher magnification in E and F. The osteogenic fronts of Msx2–/– mutants (F) are smaller than their wild-type counterparts (E). (G,H) Autoradiograms of in situ hybridization with a radiolabeled (35S) probe against bone sialoprotein. The autoradiographic signal has been transformed to red. (I,J) TRAP stains of osteoclasts (red color, arrowheads). There was no discernible difference in numbers of osteoclasts between mutant (J) and wild type (I). cs, coronal suture; e, eye; fb, frontal bone; fs, frontal suture; ls, lambdoid suture; of, osteogenic front; pb, parietal bone; pf, parietal foramen; sk, skin; ss, sagittal suture. Scale bars: 2 mm in B; 200 µm in D,J; 100 µm in F; 80 µm in H.

View larger version (83K): [in a new window]   Fig. 2. Restricted domains of alkaline phosphatase and Runx2 expression in Msx2 mutant embryos. We examined alkaline phosphatase (ALP) activity and Runx2 expression in the developing frontal bones of Msx2 mutant and control embryos. (A-L) Whole-mount ALP stains of heads at the indicated stages. (A-D) Dorsal views; (E-L) Lateral views. Boxed areas are shown at higher magnification in the panels below. Note the reduced area of ALP staining in mutant embryos (C,D arrowheads; H, arrowhead, broken line). The arrow in K indicates a halo of ALP-positive cells which is not present in the Msx2 mutant (L). (M,N) Cross-sections at the levels indicated by lines in K and L. The area of ALP staining in three mutant and three wild-type individuals was measured by quantitating pixels (O). Error bars indicate one standard deviation. The wild-type and Msx2–/– data sets were compared using Student's t-test. (P-S) In situ hybridization analysis of Runx2 expression in wild type (P,R) and Msx2 mutant (Q,S) embryos. Tissue sections through the prospective frontal bone of E12.5 (P,Q) and E11.5 (R,S) embryos were incubated with a 33P-labeled Runx2 antisense probe. The sections were subjected to autoradiography and photographed under dark field. Schematics depicting key anatomical features of the sections are shown on the right. Note that Runx2 expression is first detectable between E11.5 and E12.5. Note also that the hybridization signal is reduced in the frontal bone rudiment of the Msx2 mutant compared with the wild type. ch, cerebral hemisphere; cs, coronal suture; e, eye; fb, frontal bone rudiment; pb, parietal bone rudiment. Scale bars: 1 mm in B,F; 500 µm in D,H,J; 200 µm in L,N,Q.

View larger version (135K): [in a new window]   Fig. 3. Normal levels of apoptosis in frontal bone rudiments of Msx2 mutant embryos. We performed TUNEL stains on cross-sections through the presumptive frontal bone of Msx2–/– and wild-type littermate embryos at E11.5 (A,B) and E12.5 (C-F). The broken lines indicate the approximate area occupied by the frontal bone skeletogenic mesenchyme. The squares in C,D demarcate areas shown at higher magnification in E and F. Note that at E11.5, no TUNEL-positive cells (green) were evident in the presumptive frontal bones of either mutant or wild-type embryos. A few positive cells were evident elsewhere in the section (e.g. arrowhead). At E12.5, there were near-equal numbers of apoptotic cells in frontal bone mesenchyme (stained green, arrowheads) of mutant and wild-type embryos. ep, epithelium; ch, cerebral hemisphere. Scale bars: 200 µm in B,D; 50 µm in F.

View larger version (57K): [in a new window]   Fig. 4. Normal distribution of neural crest cells in Msx2 mutant mice. Mice carrying the R26R lacZ allele were crossed with mice bearing a Wnt1-Cre transgene, thus marking neural crest cells with lacZ. Embryos at the indicated developmental stages were stained for ß-galactosidase activity and the stain visualized in wholemount or cross section. (E,F) Frozen sections, (G-J,M,N) paraffin sections. Enlarged views of boxed areas in G and H are shown in I and J. Lines in K and L indicate the level of sections shown in M and N. No significant difference in neural crest distribution was evident at E9.5 (A,B). At both E11.5 and E12.5, there was no difference in the density or distribution of lacZ-positive cells the area of the frontal bone rudiment (C,D, arrowheads; E,F, arrows). In some E12.5 embryos there was a delay in the migration of neural crest dorsally (compare G and I with H and J). By E16.5, no difference was apparent (compare K and M with L and N). br1, first branchial arch; bv, blood vessel; ch, cerebral hemisphere; e, eye; ep, epithelium; fb, frontal bone; fn, fronto-nasal process; pb, parietal bone; sm, sutural mesenchyme. Scale bars: 200 µm in B,F,H,N; 1 mm in D,L; 50 µm in J.

View larger version (89K): [in a new window]   Fig. 5. Reduced proliferation of osteogenic cells in the frontal bone rudiment of Msx2 mutant mice. An antibody against serine 10-phosphorylated histone H3 (P-H3) was used to stain mitotic cells in sections of embryos in the area of the frontal bone rudiment at E12.5 (A-D) and E14.5 (F-I). Adjacent sections were stained for alkaline phosphatase activity. (A,C,F,H) Alkaline phosphatase stain; (B,D,G,I) P-H3 stain. P-H3-positive cells within the ALP stained area (arrowheads) were counted, and the counts normalized to the area of the ALP-stained region. (E,J) Plots of these normalized values. Error bars are standard deviations derived from three independent experiments. Note that at E12.5, P-H3 labeling indices of mutant and wild-type embryos were similar. At E14.5, a statistically significant (Student's t-test) reduction of labeling index was evident in mutant embryos. ch, cerebral hemisphere; fb, frontal bone; ep, epithelium. Scale bars: 100 µm.

View larger version (73K): [in a new window]   Fig. 6. Msx2 and Twist cooperatively control the patterning of the frontal bone. (A-F) Skulls of mice with the indicated genotypes were taken at postnatal day 4. Mineralized bone was stained with Alizarin Red S. (G) Measurements of the areas of frontal foramina obtained from several individuals of each genotype. Note that the defect (arrows, B-F) is more severe in Msx2-Twist double heterozygotes than in either individual heterozygote, indicating a cooperative interaction between Msx2 and Twist in the control of frontal bone development. cs, coronal suture; fb, frontal bone; fs, frontal suture; ls, lambdoid suture; pb, parietal bone; pf, parietal foramen; ss, sagittal suture. Scale bar: 2 mm.

View larger version (57K): [in a new window]   Fig. 7. Msx2 and Twist cooperatively regulate the differentiation and proliferation of skeletogenic mesenchyme. Cross-sections through the frontal bone rudiments of E12.5 (A-F) embryos of the indicated genotypes were tested for alkaline phosphatase activity (A,B), Runx2 expression (C,D) or Wnt1-Cre-driven-R26R-lacZ expression (E,F). Note reduced expression of ALP and Runx2, but unchanged expression of lacZ in Msx2+/–; Twist+/– embryos compared with Msx2+/– embryos. (G-J) Cross-sections of E14.5 frontal bones stained for ALP activity (G,I) and adjacent sections stained for 10-phosphorylated histone H3 (H,J) to mark proliferating cells. P-H3-positive cells (arrowheads) were counted in the regions of developing frontal bones shown in H and J. The resultant counts were normalized to the area of ALP-staining shown in G and I, and are plotted in K. Error bars represent standard deviations derived from three independent experiments. Note the statistically significant (Student's t-test) reduction of labeling index in Msx2+/–; Twist+/– mutant embryos compared with Msx2+/– embryos. ch, cerebral hemisphere; ep, epithelium; fb, frontal bone. Scale bars: 100 µm.

View larger version (91K): [in a new window]   Fig. 8. Analysis of regulatory interactions between Msx2 and Twist in the developing frontal bone. In situ hybridization was used to assess Msx2 expression in Twist+/– mutant embryos and Twist expression in Msx2–/– embryos. (A-F) Msx2 probe hybridized with wild-type and Twist mutant heads. (G-L) Twist probe hybridized with wild-type and Msx2 mutant heads. (A-D,G-J) Embryo wholemounts at E10.5 and E11.5 hybridized with digoxigenin-labeled probes. Arrowheads point to the site of the frontal bone primordium. (E,F,K,L) In situ hybridization of radiolabeled (33P) probes with tissue sections through frontal bone rudiments at E12.5. Note apparent lack of change in Msx2 expression in Twist mutant, and Twist expression in Msx2 mutant at E10.5, E11.5 and E12.5. ch, cerebral hemisphere; e, eye; fn, frontonasal process. Scale bars: 200 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter