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

In order to study FGFR3's role in cranial vault development, we generated the first fgfr3 loss-of-function zebrafish (fgfr3lof1/lof1). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. In order to define cellular and molecular processes responsible for this phenotype, we performed single-cell RNA sequencing of cranial vault cells from fgfr3lof1/lof1 and fgfr3+/+ zebrafish. Our data allow us to define transcriptomic profile of each osteogenic subpopulation involved in cranial vault formation and we revealed a defect in osteoblast differentiation associated with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis and we conclude that changes in the extracellular matrix within growing bone impair cell-cell communication, mineralization, and new osteoblast recruitment. Overall design: Using cells isolated from cranial vaults from two fgfr3lof1/lof1 fish and two fgfr3+/+ zebrafish (fgfr3lof/lof: zebrafish line was obtained using CrisPR/Cas9 technology with a stop codon at position 377), we profiled the transcriptome of nearly 28000 single cells using the Chromium system (10x Genomics). Cells suspensions were obtained using both collagenase digestion and cell sorting to eliminate death cells. Cells of the cranial vault is a mix of several cell types (osteogenic cells, chondrogenic cells, immune cells epidermal cells, endothelial cells). The scRNA-seq libraries were generated with a Chromium Single Cell 3' Library & Gel Bead Kit v.3 (10x Genomics). The purified libraries were sequenced on a Novaseq 6000 (Illumina) on paired end strands (read 1: 28bp; read 2: 91bp), with a mean read depth of 25000 reads per cell.