Slater BJ et al. (2009), Cranial osteogenesis and suture morphology in X...

XB-ART-39022

PLoS One 2009 Jan 01;41:e3914. doi: 10.1371/journal.pone.0003914.

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Cranial osteogenesis and suture morphology in Xenopus laevis: a unique model system for studying craniofacial development.

Slater BJ , Liu KJ , Kwan MD , Quarto N , Longaker MT .

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BACKGROUND: The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied. METHODOLOGY/PRINCIPAL FINDINGS: We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (microCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13. CONCLUSION: A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process.

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Species referenced: Xenopus laevis
Genes referenced: mmp1 mmp13 mmp13l

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	Figure 1. Development of the bony skull during pre-metamophosis (stages 52–58).A–D. Dorsal view of unstained Xenopus cranium demonstrates gross morphology, 0.7× magnification. E–H. Alizarin red (bone) and Alcian blue (cartilage) whole-mount staining of skull. Dashed yellow lines denote region of frontoparietal bone. I–L. μCT scans delineate ossified portions of the cranium. Note ossification proceeding from the base of the skull to the anterior aspect. Ossification of the frontoparietal bone begins at Stage 52 initially appearing laterally (arrow heads in panel I).
	Figure 2. Development of the bony skull during metamorphosis (stages 59–61).A′–C′. Dorsal view of unstained Xenopus cranium demonstrates gross morphology, 0.7× magnification. D′–F′. Alizarin red (bone) and Alcian blue (cartilage) whole-mount staining of cranial skull. G′–I′. μCT scans delineate ossified portions of the cranium. Note ossification of the frontoparietal bone proceeding from the lateral aspect of the bone corresponding to the Alizarin red staining.
	Figure 3. Development of skull after metamorphosis (stages 63 - frog).A″–C″. Dorsal view of unstained Xenopus cranium demonstrates gross morphology, 0.7× magnification. D″–F″. Alizarin red and Alcian blue whole-mount staining of cranial skull. G″–I″. μCT scans delineate ossified portions of cranium. Ossification is complete by Stage 64–66. Labelling based on Trueb and Hanken, FP: frontoparietal.
	Figure 4. Frontoparietal sections.A–H. Coronal sections through the frontoparietal bone stained with Movat's pentachrome, 20× magnification. Ossification begins at Stage 52–54 and is completed by Stage 58–60. (yellow = bone, blue = cartilage, red = cytoplasm, dark red = osteoid) FP: frontoparietal.
	Figure 5. Anatomy of the midline suture.A–C. Movat's pentachrome stained coronal sections through the frontoparietal bone of A. Stage 54, B. Stage 57, and C. Stage 59, 40× magnification demonstrating midline, abutting sutures. Fusion of the suture occurs at Stage 58–60. OF: osteogenic front, FP: frontoparietal bone. The yellow dots indicate the margins of the osteogenic fronts and ossified bone.
	Figure 6. Morphology of the posterior suture.A. Dry skull preparation of adult Xenopus demonstrating the frontonasal and posterior sutures (outlined in blue dashed lines). B. Dorsal view of posterior suture at 1.25× magnification. C–E. Pentachrome stained coronal sections of an adult skull (5× magnification). The location of the planes of section are notated by red dashed lines in the dry skull prep in A. C′–E′. 20× magnification of suture. There are no midline sutures evident but at the posterior aspect, lateral overlapping sutures are present (Arrow in E′ note overlapping pattern). The skull base ventral to the brain has a cartilaginous layer throughout (arrowheads). F. Dry skull preparation of adult mouse demonstrating the lambdoid suture. G–I. Pentachrome stained parasagittal sections of adult mouse calvaria. Note the overlapping pattern of the suture (arrows).
	Figure 7. Proliferation during ossification of the skull.A–C. Phospho-Histone H3 staining of the frontoparietal bone. A. Stage 52, B. 56, and C. 60, 40× magnification. There is positive staining in the lateral aspects of the frontoparietal bone and surrounding cartilages at Stages 52 and 56 (white arrow heads); no staining is observed at Stage 60 once the frontoparietal bone has completely ossified.
	Figure 8. Expression of matrix metalloproteinases MMP-1 and MMP-13.A–B. semi-quantitative quantitative PCR. A. MMP-1 and -13 gene expression in the frontoparietal region. B. MMP-1 and -13 gene expression in the surrounding cartilage. Tissues were dissected at the indicated stages and semi-quantitative RT-PCR for MMP-1 and -13 was performed at indicated stages and normalized to expression of the housekeeping gene ornithine decarboxylase (odc). Note the peaks of expression correlating with stages with abundant tissue turnover. -rt: no reverse transcriptase control. C–D. Stage-specific gelatinase activity. Tissue samples from frontoparietal bone (C) and surrounding cartilage (D) were dissected at the indicated stages, concentrated and subjected to gelatin zymography. The clear bands represent proteinase activity. Major bands at 41 and 48 kDA correspond to the molecular weight of MMP-1 and MMP-13 respectively. Note the peak of lytic activity appears at a later stage than the peak of gene expression with PCR.
	Figure 9. mRNA expression of MMP-1 and MMP-13.A–D. MMP-1 In situ hybridization of sections of frontoparietal bone from various stages (antisense probes), 20× magnification. A′–D′. 40× magnification of same area. E. Representative sense probe of stage 60 to demonstrate lack of non-specific staining. Brown staining represents positive localization, blue represent hematoxylin counterstaining. F–I. MMP-13 In situ hybridization of sections of frontoparietal bone from various stages (antisense probes), 20× magnification. F′–I′. 40× magnification of same area. J. Representative sense probe of stage 60. Note increased brown staining in Stage 56 and 60 with both probes.

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