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

First published online December 20, 2005
doi: 10.1242/10.1242/dev.02200

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 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 Sasaki, T. Articles by Chai, Y. Search for Related Content PubMed PubMed Citation Articles by Sasaki, T. Articles by Chai, Y. Social Bookmarking                         What's this?

TGFß-mediated FGF signaling is crucial for regulating cranial neural crest cell proliferation during frontal bone development

¹ Center for Craniofacial Molecular Biology School of Dentistry University of Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033, USA.
² Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Gakkocho-dori, Niigata 951-8514, Japan.

View larger version (92K): [in a new window]   Fig. 1. Developmental failure of the frontal bone primordium in Tgfbr2fl/fl;Wnt1-Cre mice. (A,B) E16.5 embryo skeletal preparation reveals severe defects of frontal (fr), parietal (pr) and interparietal (ip) bone in the Tgfbr2fl/fl;Wnt1-Cre mutant. Only the orbital aspect of the frontal bone was developed in the Tgfbr2fl/fl;Wnt1-Cre mutant. (C,D) At E12.5, the CNC-derived mesenchyme begins to form a condensation (arrow) that serves as the template for frontal bone development in both the wild type and the Tgfbr2fl/fl;Wnt1-Cre mutant. (E,F) A well-defined, CNC-derived frontal bone primordium is clearly visible (arrow) in the wild-type sample at E13.5. In the Tgfbr2fl/fl;Wnt1-Cre mutant, the development of the frontal bone primordium is retarded when compared with the wild-type sample (arrow). (G,H) At E14.5, a well-developed bone matrix is visible within the frontal bone primordium of the wild-type sample. In the Tgfbr2fl/fl;Wnt1-Cre mutant, however, bone matrix fails to form within the frontal primordium (arrow), while the orbital aspect of frontal bone is present (arrowhead). (I,J) At E16.5, frontal bone formation is evident in the wild-type sample (arrow). No bone formation is detected in the calvarial aspect (arrow) of the frontal primordium in the Tgfbr2fl/fl;Wnt1-Cre mutant. (K,L) At birth (NB), the frontal bone is well developed to form the roof of bony orbit (arrowhead) and covers the side of skull (arrow). In the Tgfbr2fl/fl;Wnt1-Cre mutant, the frontal bone development is retarded with a rudiment of orbit surface region (arrowhead), whereas there is no development of the calvarial aspect of frontal bone (arrow). Scale bars: 1 mm in A,B; 200 µm in C-L.

View larger version (117K): [in a new window]   Fig. 2. Conditional null mutation of Tgfbr2 in the CNC-derived ectomesenchyme does not adversely affect the neural crest migration during early craniofacial development. (A,B) At E11.5, the CNC-derived mesenchyme has populated the region of the developing frontal primordium (arrow). There is no apparent difference in the number of cells between the wild-type and the Tgfbr2fl/fl;R26R;Wnt1-Cre mutants, suggesting that there is no CNC migration defect prior to frontal bone primordium development. (C,D) Apparent developmental defect of the frontal primordium is observed within the Tgfbr2fl/fl;R26R;Wnt1-Cre mutant sample when compared with the wild-type control at E13.5. There is a diminished number of CNC-derived cells in the frontal primordium (arrow) of the Tgfbr2fl/fl;R26R;Wnt1-Cre mutant. Scale bars: 200 µm.

View larger version (77K): [in a new window]   Fig. 3. Cell proliferation analysis during frontal bone development. (A) At E12.5, the frontal bone primordium (between the broken lines) is well defined. There is active cell proliferation in the CNC-derived frontal bone mesenchyme of the control sample. (B) In Tgfbr2fl/fl;Wnt1-Cre mutants, similar cell proliferative activity is observed as compared with the control. (C,D) Comparable cell proliferative activity between the wild type and Tgfbr2fl/fl;Wnt1-Cre mutant is observed within the CNC-derived frontal bone primordium at E13.5. (E) There is active CNC cell proliferation in both the calvarial (arrow) and the orbital aspect (arrowhead) of the frontal bone primordium in the wild-type sample at E14.5. (F) Significant reduction in CNC cell proliferation is clearly visible in both the calvarial aspect (arrow) and the orbit region of frontal bone mesenchyme (arrowhead) of Tgfbr2fl/fl;Wnt1-Cre mutant. (G) Statistical analysis of cell proliferative activity between the wild type and the Tgfbr2fl/fl;Wnt1-Cre mutant. There is a significant reduction in cell proliferation in the calvarial and orbital aspects of the frontal bone anlagen in the Tgfbr2fl/fl;Wnt1-Cre mutant at E14.5. Scale bars: 100 µm.

View larger version (121K): [in a new window]   Fig. 4. Loss of Tgfbr2 within the CNC-derived mesenchyme does not affect cell survival during frontal bone development. (A) At E13.5, there is no detectable apoptotic activity within the frontal bone primordium in the wild-type sample. (B) In the Tgfbr2fl/fl;Wnt1-Cre mutant sample, there is no increase in cell death compared with wild type within the frontal primordium. (C) Sporadic apoptotic signals are present adjacent to the frontal primordium in the control (arrows). (D) Similarly, a few apoptotic signals (arrows) are seen in the frontal primordium in the Tgfbr2fl/fl;Wnt1-Cre mutant, as seen in C, suggesting that loss of TGFß signaling does not cause a change in apoptotic activity within the CNC-derived frontal mesenchyme. Scale bar: 100 µm.

View larger version (136K): [in a new window]   Fig. 5. Comparison of osteogenic progenitor cell differentiation in the frontal primordium between the wild type and the Tgfbr2fl/fl;Wnt1-Cre mutant samples. (A) At E13.5, definitive osteogenic differentiation marker Runx2 is expressed in the frontal primordium (arrowhead), indicating that these mesenchymal cells have committed to an osteogenic lineage. (B) Runx2 expression is also detected in the frontal bone primordium of Tgfbr2fl/fl;Wnt1-Cre sample (arrowhead). (C,D) Type I collagen (ColI) is detected in the frontal primordium of both wild type and mutant samples. (E,F) Osterix expression is detected in the frontal primordium of both wild-type and Tgfbr2fl/fl;Wnt1-Cre mutant sample (arrowhead). (G,H) Ibsp expression is present in the frontal primordium in both wild-type and Tgfbr2fl/fl;Wnt1-Cre sample (arrowhead). (I) At E14.5, Runx2 is expressed throughout the frontal primordium, on both the calvarial (arrow) and the orbit (arrowhead) aspects. (J) In the E14.5 Tgfbr2fl/fl;Wnt1-Cre sample, Runx2 expression is only detected in the orbital aspect of the frontal bone anlagen, suggesting that proper osteogenic lineage determination on the calvarial aspect of the frontal primordium (arrow) specifically requires TGFß signaling. (K,L) Type I collagen is detected in the frontal primordium (arrow) and the orbit region (arrowhead) of the frontal bone in both wild-type and mutant samples. (M,N) Osteonectin expression pattern is comparable within the frontal primordium (arrow and arrowhead) between the wild type and the Tgfbr2fl/fl;Wnt1-Cre mutant sample. (O,P) Osterix expression is detected in the frontal primordium of the wild-type sample. There is a reduced osterix expression within the calvarial aspect of the frontal primordium (arrow), while normal osterix expression is present within the orbital surface region of the frontal bone in the Tgfbr2fl/fl;Wnt1-Cre mutant sample (arrowhead). (Q-V) Bone matrix protein expression analysis shows that there is a defect in the production of bone matrix proteins, as indicated by reduced expression of osteopontin, Ibsp and osteocalcinin the calvarial aspect of frontal primordium of Tgfbr2fl/fl;Wnt1-Cre mutant sample. Comparable expression of osteopontin, Ibsp and osteocalcin are detected in the orbit region of the frontal bone anlagen (arrowhead). (W,X) Alkaline phosphatase (ALPase) is present in the well-developed frontal bone primordium in the wild type, while its expression is greatly reduced in the Tgfbr2fl/fl;Wnt1-Cre mutant, thus indicating compromised frontal bone matrix maturation. All insets show a low-power image (coronal-section) of the frontal region of the head at E14.5. The arrows in the insets indicate the frontal primordium region. All in situ probes were labeled with 33P. Scale bars: 200 µm.

View larger version (67K): [in a new window]   Fig. 6. Fgfr2, Dlx5, and Twist1 are downstream targets of TGFß signaling in regulating frontal bone development. (A) At E13.5, the expression of Fgfr2 is detected in the frontal primordium (arrow) of the wild type. (B) The expression of Fgfr2 is reduced in the frontal primordium (arrow) of the Tgfbr2fl/fl;Wnt1-Cre mutant. (C) At E14.5, frontal bone matrix begins to form within the frontal primordium of the wild-type sample. The expression of Fgfr2 is detected throughout osteogenic progenitor cells in the frontal bone primordium (arrow) and the orbital surface region of the frontal bone (arrowhead). (D) In the Tgfbr2fl/fl;Wnt1-Cre mutant, there is virtually no Fgfr2 expression in the calvarial aspect of the frontal primordium (arrow), while residual Fgfr2 expression is detected within the orbital aspect of the frontal bone in the Tgfbr2fl/fl;Wnt1-Cre mutant sample (arrowhead). (E) At E13.5, Dlx5 is expressed in the CNC-derived frontal bone primordium (arrow). (F) Comparable Dlx5 expression is present in the frontal primordium in the Tgfbr2fl/fl;Wnt1-Cre mutant (arrow). (G) The frontal bone matrix begins to form at E14.5. Dlx5 is expressed in both the calvarial (arrow) and the orbital (arrowhead) aspects of the frontal bone primordium. (H) The Tgfbr2fl/fl;Wnt1-Cre mutant fails to express Dlx5 in the calvarial aspect of the frontal bone primordium, whereas expression on the orbital aspect (arrowhead) is normal. (I) No Twist1 expression is detected in the frontal primordium in the wild-type sample. (J) Elevated Twist1 expression is detected in the frontal bone primordium (arrow), whereas there is no Twist1 expression in the orbit region of the frontal bone anlagen (arrowhead) in the Tgfbr2fl/fl;Wnt1-Cre mutant. Scale bar: 200 µm.

View larger version (71K): [in a new window]   Fig. 7. TGFß stimulates CNC proliferation while FGF signals downstream of TGFß in mediating cell proliferation during frontal bone development. (A,D) E16.5 calvarial primordium treated with BSA bead for 1 day shows normal growth of the CNC and activity of cell proliferation, as demonstrated by BrdU labeling (D, arrow). (B,E) TGFß1 treatment results in substantial increase in tissue thickness (B, arrow) and cell proliferation (E, arrow) in the E16.5+1 cultured explant. (C,F) Similarly, TGFß2 treatment also results in expansion of frontal bone primordium (C, arrow) and increase in cell proliferation (F, arrow). (G) E14.5 frontal bone primordium treated with BSA beads for 1 day shows normal level of Fgfr2 expression (arrow), as indicated by whole-mount in situ hybridization analysis (dark purple indicates a positive signal). (H) TGFß1 beads induce elevated expression of Fgfr2 (arrows) in the frontal primordium. (I) E14.5 Tgfbr2fl/fl;Wnt1-Cre mutant frontal bone primordium treated with BSA bead shows very little tissue growth (outlined) and reduced cell proliferation activity (BrdU positive cells in red). (J) E14.5 Tgfbr2fl/fl;Wnt1-Cre mutant frontal bone primordium treated with FGF2 bead shows extensive increase in tissue growth (outlined) and restored cell proliferation activity (arrows). (K) E14.5 Tgfbr2fl/fl;Wnt1-Cre mutant parietal bone primordium explant treated with BSA beads (left side) for 1 day shows basal level of Fgfr2 expression, while TGFß beads treatment (right side) shows strongly induced Fgfr2 expression (arrows, dark brown). None of the explants contains dura matter or epithelium. Scale bars: 100 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter