Plastic Surgery Research Council

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Mechanisms of Disruption in Craniofacial Ossification Caused by fgfr1a Mutations
Yufan Yan, Joanna K. Ledwon, Lauren Kelsey, Arun K. Gosain.
Nothwestern University Feinberg School of Medicine, Ann & Robert H. Lurie Children's Hospital of Chicago Division of Plastic and Reconstructive Surgery, Stanley Manne Children's Research Institute, Chicago, IL, USA.

Purpose: Mutations in the FGFR1 gene have been implicated in craniosynostosis in human patients, though the molecular mechanisms of pathogenesis are not well understood. FGFR1 is highly conserved between humans and zebrafish, allowing these molecular pathways to be studied using zebrafish as an animal model.
Methods: Four fgfr1a zebrafish mutants were generated using the CRISPR/Cas9 system in the same genetic region as the mutation associated with Pfeiffer syndrome. The four mutants are a 12-nucleotide in-frame deletion (fgfr1adel12bp), two frameshift deletions of 13- (fgfr1adel13bp) and 17-nucleotides (fgfr1adel17bp), and a 14-nucleotide rearrangement (fgfr1adel13+1bp). Whole mount staining with Alizarin red was performed to examine ossification of bones. qRT-PCR and RNAscope in situ hybridization analyses were used to identify differentially expressed genes. Pentachrome staining was performed to evaluate cranial cartilage formation and organization of the endochondral growth plate.
Results: Two phenotypic changes are evident in the in-frame deletion mutant (fgfr1adel12bp) - flattening of the skull contour with a smaller distance between the top of the head and eyes, and decreased sutural space along the midline sutures between the two halves of the calvaria (Fig. 1). These findings suggest changes in intramembranous ossification of the skull bones, with likely increased ossification in the mutant calvaria, resulting in more rapid overlap of frontal and parietal bones along midline sutures.

Endochondral ossification is also affected in mutants, as evidenced by qRT-PCR results demonstrating upregulation of the cthrc1b gene in all mutants except one, and of the nppc gene in the in-frame deletion mutant (fgfr1adel12bp) (Fig. 2 A, B). These two genes have been identified as regulators of endochondral ossification in human and mouse models. qRT-PCR results were supported by RNAscope in situ hybridization analysis for cthrc1b. More cells expressed cthrc1b, and intensity of the detected signal was stronger in the palatoquadrate cartilage (a cranial endochondral cartilage) of the fgfr1adel12bp mutant (Fig. 2 D).

Pentachrome staining of the same cartilage in one of the frameshift deletion mutants (fgfr1adel17bp) demonstrates disruption of the neatly organized endochondral growth plate as well as enhanced ossification, indicated by a decreased number of chondrocytes and shorter length of the growth plate (Fig. 3).

Conclusions: Mutations in the fgfr1a gene in zebrafish appear to disrupt normal craniofacial physiology through effects on both intramembranous ossification of the cranial bones and endochondral bone development. Additional analysis will be performed to better characterize the molecular mechanisms leading to the observed phenotype. Because fgfr1a is highly conserved in zebrafish and humans, we believe this knowledge will translate to better understanding of human craniosynostosis.

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