An Objective Description of Zebrafish Cranial Suture Development

Introduction: Zebrafish is a promising model to better understand the developmental biology of the craniofacial skeleton, specifically relating to the genetic aspects of this development.  However, there is very little objective data to describe the development of the cranial vault in zebrafish. This study was performed to analyze the natural history of cranial suture development in zebrafish. We aim to provide an alternative model to study fusion of cranial bones leading to craniosynostosis. We intend to evaluate the ossification of zebrafish calvarial bones that transform the sutural region from wide mesenchymal separation to overlap of the cranial bones with skull development.

Methods: Thirty 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. Standard length was used to characterize fish development since it is a better indicator of the skeletal development than age [1]. 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. In addition, histological analysis of cranial sutures was conducted.

Results: The following cranial sutures were identified: anterior interfrontal, posterior interfrontal, sagittal and coronal sutures. Figure 1 shows the dissected calvarium of an 8.1-millimeter zebrafish with appropriate labels. The suture mesenchyme size was highly correlated with standard length in juvenile zebrafish, with correlation coefficients ranging from -0.84 to -0.90 (p ≤ 0.05). Figure 2 shows the correlation of the anterior interfrontal mesenchymal area with standard length; negative values represent suture overlap. We also observed that the anterior interfrontal suture forms first, followed by the posterior interfrontal and sagittal sutures.

Conclusions: Our data supports the hypothesis that the development of cranial sutures in zebrafish is analogous to that in mammals. We propose that zebrafish can be used to better understand the biology and genetic regulation of cranial sutures. This study lays the groundwork for further use of zebrafish to elucidate genetic mechanisms involved in   craniosynostosis.

Figure  SEQ Figure \* ARABIC 1

Figure  SEQ Figure \* ARABIC 2