Functional dissection of ultraconserved non-coding regions present near the Hoxd complex in vertebrates

Abstract

Ultraconserved non-coding elements (UCEs) exhibit near-perfect sequence conservation across vertebrate genomes, implying severe evolutionary constraint and essential biological functions. Near the vertebrate Hoxd complex, a master regulator of axial patterning and limb morphogenesis, three Conserved Regions (CR1, CR2, and CR3), each longer than 200 bp, show exceptional sequence conservation and drive robust regulatory activity in transient reporter assays. To investigate their endogenous in vivo functions, we used CRISPR-Cas9 genome editing to generate a series of deletions of varying sizes spanning CR2 and CR3, individually and in combination, within hoxd complex in zebrafish. Screening thousands of founder embryos revealed a clear size and conservation-dependent relationship between deletion extent and viability: complete deletion of core CR2, CR3, or the combined CR2/3 loci could not be transmitted through the germline, whereas the smaller and least-conserved deletion, a 626-bp subregion of CR2, was the only allele to yield a stable, homozygous-viable line. Our results indicate that the core ultraconserved sequences are developmentally indispensable, whereas flanking, less conserved sequences can be removed without consequence. Detailed phenotypic and molecular characterization of the mutant embryos revealed normal primary and secondary body axis formation, intact somite segmentation, and unperturbed craniofacial and pectoral fin development. Expression of hoxd complex genes showed no significant change in mutant embryos by RNA-ISH or RT-qPCR, although RT-qPCR revealed a consistent, modest upward trend at 24 and 48 hpf, raising the possibility that the deleted subregion normally contributes a weak repression of overall hoxd complex genes transcription. While this study reveals some functional aspects of ultraconserved elements, much more remains to be uncovered.