Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Impact of koliber Mutation on Patency of Cranial Suture in Zebrafish
Jolanta M. Topczewska, PhD; Rupa Mirmira, BS; Joanna P. Tomaszewski, MS; Rebecca Anderson, PhD candidate; Jacek Topczewski, PhD; Arun Gosain, MD
Northwestern Feinberg School of Medicine, Chicago, IL

PURPOSE:
Craniosynostosis (CS) is a human disorder that results from premature fusion of cranial bones. It leads to skull malformation and aversely constricts the growing brain. Although great progress has been made in recognizing the mutations associated with some types of CS, the molecular mechanisms of these genetic regulations are not fully understood. Recently, significant technological advancement has been made in the genetic studies. Successful and economical modeling of human diseases in zebrafish becomes available. Koliber is a recessive, yet to be identified, Mendelian mutation. Adult koliber mutants display a misshapen skull and body, and reduced body length. The first morphological signs of koliber abnormalities become visible in juvenile fish and the pathology progresses with age. In contrast to humans, normal zebrafish cranial sutures remain patent throughout the organism’s life. The Koliber skull is laterally widened and dorsally flattened, suggesting compromised function of the cranial sutures. Accordingly, in this study we focused on koliber interfrontal suture development and patency.
METHODS:
Animals aged between 5 weeks and 6 months were genotyped and analyzed in pairs of siblings composed of a wild type control and homozygote mutant.
To analyze gene expression pattern within the sutural tissue, and to identify the presence of essential genes involved in suture development in mammals, we established RNAscope in situ hybridization on paraffin sections.
The birefringence of collagen fibers in polarized light using Picro Sirius Red staining of paraffin sections allowed us to analyze the fibers’ organization; H&E and Movat’s Pentachrome histological methods were used to evaluate sutural tissue.
All results were concluded based on three independent experiments. IACUC committee approved zebrafish husbandry and experimental methods.
RESULTS:
Our studies found that the zebrafish koliber mutant exhibits pathologically fusing frontal bones. These changes do not involve obliteration of the sutural tissue but rather insular bone fusions. Histological analysis exposed an increased amount of fibrillar collagen in sutural tissue of the mutant homozygote as compare to wild type sibling. RNA in situ hybridization also confirmed an increased amount of col1a transcripts in the interfrontal suture of the mutant. Interestingly, the penetrance of the koliber mutation seems to be unequal between siblings, as apparent gradation of phenotypes can be detected, both on the molecular level and morphologically. This observation suggests involvement of compensatory mechanisms that prevents cranial bone fusion in zebrafish.
CONCLUSION:
The presented data demonstrate localized fusion of frontal bones and compromised functionality of the interfrontal suture in koliber mutant. The zebrafish model has enormous capacity for genetic manipulations and our results provide promising advances not only in understanding vertebrate cranium development but also in the feasibility of the zebrafish model for CS studies.


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