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Updated: May 31, 2026
Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone
Published on: May 10, 2019
Molecular analysis of coronal perisutural tissues in a craniosynostotic rabbit model using polymerase chain reaction
James J Cray1, Phillip H Gallo, Emily L Durham
1Pittsburgh and Philadelphia, Pa. From the Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Pittsburgh and Pediatric Craniofacial Biology Laboratory, Children's Hospital of Pittsburgh; the Center for Genomic Sciences, Allegheny-Singer Research Institute, West Penn Allegheny Health Systems; the Departments of Anthropology, Orthodontics, Oral Biology, and Bioengineering, University of Pittsburgh; the Division of Plastic Surgery, Allegheny General Hospital of Pittsburgh; and the Department of Microbiology and Immunology, Drexel University College of Medicine.
Insights
Craniosynostosis gene expression differs between affected rabbits and controls. This study identified key genes involved in bone formation and resorption pathways, offering insights into craniosynostosis pathogenesis.
Area of Science:
- Genetics
- Developmental Biology
- Craniofacial Surgery
Background:
- Craniosynostosis, affecting 1 in 2000-3000 births, involves premature suture fusion, leading to head shape deformities and potential developmental issues.
- A heritable rabbit model of coronal suture synostosis is crucial for studying craniofacial growth but lacks molecular tools.
- Understanding the molecular basis of craniosynostosis pathogenesis is essential for developing targeted therapies.
Purpose of the Study:
- To compare gene expression profiles in perisutural tissues of wild-type and craniosynostotic rabbits.
- To identify genes differentially expressed in a naturally occurring craniosynostosis model.
- To elucidate the molecular mechanisms underlying craniosynostosis.
Main Methods:
- Utilized suppression subtractive hybridization polymerase chain reaction (SSH-PCR) on RNA from rabbit calvariae.
- Compared gene expression between 10-day-old wild-type and craniosynostotic rabbits.
- Confirmed differential gene expression using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).
Main Results:
- Identified approximately 140 cDNA clones overexpressed and 130 underexpressed in craniosynostotic tissues.
- Confirmed overexpression of HBB, SPP1, SPARC, and CTSK in craniosynostotic sutural tissue.
- Confirmed underexpression of COL3A1 and RNF12 in craniosynostotic samples.
Conclusions:
- Differential gene expression in this model suggests alterations in the bone formation/resorption pathway.
- Identified specific genes (HBB, SPP1, SPARC, CTSK, COL3A1, RNF12) implicated in craniosynostosis.
- Findings provide a foundation for further molecular investigation into craniosynostosis.
Background:
In the United States, the incidence of craniosynostosis (premature fusion of the sutures of the cranial vault) is one in 2000 to 3000 live births. The condition can cause increased intracranial pressure, severely altered head shape, and mental retardation. The authors have previously described a colony of rabbits with heritable coronal suture synostosis. This model has been instrumental in describing the postsurgical craniofacial growth associated with craniosynostosis. The molecular analysis of this model has been limited by the lack of molecular tools for use in rabbits. To understand the pathogenesis of craniosynostosis, the authors compared gene expression in perisutural tissues between wild-type and craniosynostotic rabbits using polymerase chain reaction suppression subtractive hybridization.
Methods:
Suppression subtractive hybridization polymerase chain reaction was performed on RNA derived from pooled samples of calvariae from 10-day-old wild-type (n = 3) and craniosynostotic (n = 3) rabbits to obtain cDNA clones enriched in either wild-type tissues (underexpressed in craniosynostotic tissue) or craniosynostotic tissues (overexpressed in craniosynostotic compared with wild-type).
Results:
Differential expression was identified for approximately 140 recovered cDNA clones up-regulated in craniosynostotic tissues and 130 recovered clones for wild-type tissues. Of these, four genes were confirmed by quantitative reverse-transcriptase polymerase chain reaction as being overexpressed in craniosynostotic sutural tissue: β-globin (HBB), osteopontin (SPP1), osteonectin (SPARC), and cathepsin K (CTSK). Two genes were confirmed to be underexpressed in the craniosynostotic samples: collagen 3, alpha 1 (COL3A1) and ring finger protein 12 (RNF12).
Conclusion:
The differential expression of these gene products in our naturally occurring craniosynostotic model appears to be the result of differences in the normal bone formation/resorption pathway.
