The Morphogenesis of Cranial Sutures in Zebrafish

Abstract

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

FGFR3 is a positive regulator of osteoblast expansion and differentiation during zebrafish skull vault development

“…In the wild type, fgfr3 appeared to the fgfr gene most strongly expressed by osteoblasts and chondrogenic cells ( Figure 7A-C); this observation was in line with the literature data (Ledwon et al, 2018;Topczewska et al, 2016). fgfr3 was expressed in 40% of the OP5 late osteoprogenitors and in 75% of the osteoblasts.…”

“…On the fertile side of the sutures, FGFR3 is expressed less strongly than FGFR2 and FGFR1 (Delezoide et al, 1998). In the zebrafish, previous studies and our present single-cell transcriptomic analysis revealed that expression levels of fgfr3 are higher than those of fgfr1a, fgfr1b and fgfr2 in (i) late osteoprogenitors and osteoblasts during cranial vault development and (ii) the osteogenic cells lining the suture's mineralization front (Topczewska et al, 2016). The situation is different in the rodent; Fgfr3c is almost completely absent from developing cranial bones, and is more weakly expressed than Fgfr2c and Fgfr1c in osteogenic fronts and sutures (Molténi et al, 1999;Rice et al, 2003).…”

“…Hence, FGFR3's specific role in skull vault formation has yet not been determined. In the zebrafish, Fgfr3 is highly conserved, and is expressed in mature osteoblasts within the osteogenic fronts and along the frontal bone (Topczewska et al 2016, Ledwon 2018. The late development and accessibility of the cranial vault in the juvenile zebrafish provides a unique opportunity to study each of the steps in osteogenesis during skull bone formation (Cornille et al, 2019;Topczewska et al, 2016).…”

“…In the zebrafish, Fgfr3 is highly conserved, and is expressed in mature osteoblasts within the osteogenic fronts and along the frontal bone (Topczewska et al 2016, Ledwon 2018. The late development and accessibility of the cranial vault in the juvenile zebrafish provides a unique opportunity to study each of the steps in osteogenesis during skull bone formation (Cornille et al, 2019;Topczewska et al, 2016). In the present report, we describe strong skull vault anomalies in an Fgfr3 loss-of-function mutant zebrafish model (fgfr3 lof/lof ) with abnormal frontal and parietal bones, the presence of ectopic bones, and a lack of suture formation.…”

Altered bone growth dynamics prefigure craniosynostosis in a zebrafish model of Saethre-Chotzen syndrome

“…Less is known, however, about how potential defects in the embryonic progenitors that grow the skull bones may prefigure suture loss, although a few reports describe altered bone formation during embryonic stages in craniosynostosis mutants ( Merrill et al, 2006 ). The ex utero development and transparency of zebrafish provide a unique opportunity to track the progenitors that form the skull bones, and to better understand how defects in the dynamics of bone growth relate to a later ability to form and maintain sutures once the bones come together ( Quarto and Longaker, 2005 ; Laue et al, 2011 ; Kague et al, 2016 ; Topczewska et al, 2016 ).…”