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Characterization Of The Koliber Mutant: A New Model For Craniosynostosis Research
Yen-Yen S. Gee, MMus1, Ramy A. Shoela, MD2, Rebecca Anderson, PhD3, Jacek Topczewski, PhD3, Arun Gosain, MD2, Jolanta M. Topczewska, PhD2.
1Lurie Children's Hospital Research Center, Chicago, IL, USA, 2Lurie Children's Hospital Research Center, Department of Surgery, Chicago, IL, USA, 3Lurie Children's Hospital Research Center, Department of Pediatrics, Chicago, IL, USA.
Zebrafish (Danio rerio) is an established model for craniofacial development studies. Here, we investigated cranium development in a recently identified kolibernu7 mutant. The kolibernu7 is characterized by a misshapen body, reduced length, and malformed skull as a result of hyperossified endochondral bones, bone fusions, and loss of cartilage (Anderson, in preparation). In this project, we investigated development of intramembranous calvaria bones and cranial sutures in kolibernu7 mutant and wildtype siblings. Our lab has established baseline biostatistical data for cranial vault development through progressive developmental stages (Shoela, in preparation). Here we expand on this method by using landmark-based morphometric analysis, with an aim to quantify the growth of individual bones of the cranial vault, the cranium as a whole, and the asymmetrical positioning of bilateral structures in kolibernu7 mutants.
Wildtype (WT) (n=25) and kolibernu7 (n=23) zebrafish were collected at different developmental stages and double-stained with Alizarin Red and Alcian Blue to visualize bone and cartilaginous structures, respectively. Images of whole fish and isolated cranial vaults were taken using standard light microscopy (Figure 1. Dorsal view of wildtype (A) and kolibernu7 (B) cranial vaults.). Following initial measurements of cranial width and length, total area, and overlapping areas of the anterior frontal, posterior frontal, parietal, and supraoccipital bones, the calvaria were enzymatically cleared using a trypsin solution with sodium borate. Individual bones were separated and photographed and similar measurements to those described above were taken. Morphometric analysis was conducted using landmarks and allowed for the creation of deformation grids of wildtype versus kolibernu calvaria. Similar analysis was conducted for individual cranial bones.
The ossification pattern and developmental schedule of cranial sutures are noticeably different between wildtype and kolibernu7 mutant. In contrast to the wildtype, where we observed a strong linear correlation between size of individual calvaria bones, total calvaria size, and standard length, in the kolibernu7 mutant such correlation was lost. Interestingly, we observed a loss of coordinated growth resulting in asymmetric bone shapes as well as accelerated growth of frontal and parietal bones toward the midline in the kolibernu7 mutants.
Our research indicates that kolibernu7 is an interesting model to study membranous bone overgrowth and cranial sutures malformations.
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