The establishment and growth of the arterial endothelium requires the coordinated expression of numerous genes. However, regulation of this process is not yet fully understood. Here, we combined in silico analysis with transgenic mice and zebrafish models to characterize arterial-specific enhancers associated with eight key arterial identity genes (Acvrl1/Alk1, Cxcr4, Cxcl12, Efnb2, Gja4/Cx37, Gja5/Cx40, Nrp1 and Unc5b). Next, to elucidate the regulatory pathways upstream of arterial gene transcription, we investigated the transcription factors binding each arterial enhancer compared to a similar assessment of non-arterial endothelial enhancers. These results found that binding of SOXF and ETS factors was a common occurrence at both arterial and pan-endothelial enhancers, suggesting neither are sufficient to direct arterial specificity. Conversely, FOX motifs independent of ETS motifs were over-represented at arterial enhancers. Further, MEF2 and RBPJ binding was enriched but not ubiquitous at arterial enhancers, potentially linked to specific patterns of behaviour within the arterial endothelium. Lastly, there was no shared or arterial-specific signature for WNT-associated TCF/LEF, TGFβ/BMP-associated SMAD1/5 and SMAD2/3, shear stress-associated KLF4 or venous-enriched NR2F2. This cohort of well characterized and in vivo-verified enhancers can now provide a platform for future studies into the interaction of different transcriptional and signalling pathways with arterial gene expression.

Heritable fragile bone disorders (FBDs), ranging from multifactorial to rare monogenic conditions, are characterized by an elevated fracture risk. Validating causative genes and understanding their mechanisms remain challenging. We assessed a semi-high throughput zebrafish screening platform for rapid in vivo functional testing of candidate FBD genes. Six genes linked to severe recessive osteogenesis imperfecta (OI) and four associated with bone mineral density (BMD) from genome-wide association studies were analyzed using CRISPR/Cas9-based crispant screening in F0 mosaic founder zebrafish. Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, and 90 days post-fertilization (dpf) using microscopy, Alizarin Red S staining, and microCT was performed. Larval crispants showed variable osteoblast and mineralization phenotypes, while adult crispants displayed consistent skeletal defects, including malformed neural and haemal arches, vertebral fractures and fusions, and altered bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR revealed differential expression of osteogenic markers bglap and col1a1a, highlighting their biomarker potential. Our results establish zebrafish crispant screening as a robust tool for FBD gene validation, combining skeletal and molecular analyses across developmental stages to uncover novel insights into gene functions in bone biology.