An Objective Description of Zebrafish Cranial Suture Development Ramy A. Shoela, MBBCh; Joanna P. Tomaszewski, MS; Jolanta M. Topczewska, PHD; Arun K. Gosain, MD Abstract Background: The zebrafish is a promising model used to better understand the developmental processes involved in the craniofacial skeleton formation and their genetic controls. This study was performed to analyze the natural history of calvarial bone and cranial suture development in zebrafish. We evaluated the cranial vault ossification through development, which brings the calvarial bones from wide mesenchymal separation to the stage when the bones. Thus we provide a reference for normal vault development, which can be used in future studies to evaluate abnormal calvariae observed in transgenic lines carrying mutations that mimic human craniosynostosis syndromes. Methods: Seventy-two wild type zebrafish were collected at different developmental stages according to IACUC protocol and double stained with Alizarin Red for bones and Alcian blue for cartilage [1]. Standard length (snout to caudal peduncle) was used to characterize fish development given that it is a better indicator of the skeletal development than age [2]. The cranial vault was dissected and photographed using a Zeiss stereomicroscope and Axiovision software. The area of each calvarial bone was calculated in addition to the area of the mesenchymal tissue between the bones. These measurements were analyzed using Excel and Statplus. In addition, histological analysis of cranial sutures was conducted. Results: The following bones were described: anterior frontal, posterior frontal, parietal and supraoccipital bones, and their corresponding cranial sutures: anterior interfrontal, posterior interfrontal, sagittal and coronal. A representative calvaria dissected from an 8.1-millimeter zebrafish is presented (Figure1). Based on the regression analysis, the development of cranial bones and cranial vault dimensions demonstrate a strong correlation with standard length. Figure 2 shows the correlation of standard length with the cranial length.

Figure 1. Dorsal view of an 8.1 mm standard length zebrafish

Figure 2. Correlation of standard length with cranial length. Standard Error= 0.11042. P Value<0.000001 Conclusions: Our data supports the hypothesis that the development of cranial sutures in zebrafish is analogous to that in mammals. This study provides quantitative description of the normal development of zebrafish cranium thus stipulating guidelines to aid in detecting abnormal development. Hence, we lay the groundwork for further use of zebrafish to elucidate genetic mechanisms involved in craniosynostosis. We propose that zebrafish can be used as an alternative model to study cranial bones and suture development. References 1. Parichy DM, Elizondo MR, Mills Mg, Gordon TN, Engeszer RE: Normal Table of Postembryonic Zebrafish

Development: Staging by Externally Visible Anatomy of the Living Fish; Dev Dyn; 238(12); 2975-3015; 2009. 2. Walker MB, Kimmel CB: A Two-Color Acid-Free Cartilage and Bone Stain for Zebrafish Larvae; Biotech

Histochem; 82(1); 23-8; 2007. Disclosure/Financial Support Supported by the internal grant from Surgery Department at Lurie Children’s Hospital Research Center. None of the authors have any financial interest in any of the products or devices mentioned.