The closure of cranial sutures is a key process during skull morphogenesis. At the edges of the flat bones, which are centers of regulated bone formation, fibrous sutures unite the individual bones. The sutures allow for further growth and movement of individual bones as the brain grows;once the neurocranium is fully grown, the sutures fuse, in early adulthood in humans. Importantly, patency of the sutures and brain development are intimately connected. Premature closure of the sutures, craniosynostosis, results in abnormal head shape and is associated with increased intracranial pressure, impaired cerebral blood flow, impaired vision and hearing, and mental disabilities. Despite their prominent roles in skull growth and brain development, the molecular and cellular mechanisms by which sutures form, how they interact with the skull bones to regulate growth, and the signals that induce suture fusion are not well understood. Much of our current knowledge of the regulation of suture formation comes from the identification of only a few of the many human mutations leading either to craniosynostosis or other defects in suture formation. Here I propose to harness the power of the zebrafish system for genetic and live imaging studies of suture formation. Skull and suture formation are relatively late events in the zebrafish, and there is almost nothing in the literature about these processes. While several large-scale mutant screens have revealed key pathways in patterning of the craniofacial skeleton up to six days of development, the later processes of skull and suture formation, at ~4-6 weeks, have not been assayed in any large-scale screen. Zebrafish mutants with specific defects in late skull and suture formation have not been identified;therefore, we propose a genetic screen to isolate and characterize such mutants. First, we will conduct a forward genetic screen of 1000 ENU-mutagenized genomes by examining F3 offspring of ENU- mutagenized males at 6 weeks, the time when skull closure is being completed in wild-type fish. In an ongoing pilot screen of 330 genomes, we have identified 12 mutants with specific defects in skull and suture formation and other late aspects of skeletal development. Based on these numbers, we anticipate identifying a total of ~30 specific mutants. Second, we will take advantage of a large collection of transgenic lines available in our lab to characterize the mutant phenotypes further, classifying them according to cell types and processes affected. Finally, we will map most if not all of the mutants to intervals of <3 Mb. Based on the phenotypic characterization, a subset of 8-10 mutants will be cloned to identify the mutated gene. Combined, the proposed experiments will establish a collection of well-characterized zebrafish mutations affecting many aspects of later skeletal development, in particular those with very specific defects in skull and suture formation, thereby establishing zebrafish as a powerful model system for the study of these clinically relevant later aspects of skull formation. Importantly, current surgical treatments for craniosynostosis and other skull defects are limited, and a detailed understanding of the underlying biology will lead t better approaches for treatment and prevention.

Public Health Relevance

Craniosynostosis and other defects in formation of cranial sutures represent a significant health problem, and current surgical treatments are imperfect. To better understand the processes leading to abnormal suture development, we will be carrying out a genetic screen in the zebrafish for mutants affecting skull and suture formation.

National Institute of Health (NIH)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Scholnick, Steven
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University of Pennsylvania
Anatomy/Cell Biology
Schools of Medicine
United States
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