Abstract
There are a number of human genetic conditions of abnormal skeletal development associated with disrupted DNA replication, with several linked to mutations in RECQL4. While several mouse models targeting Recql4 have been developed, however these models have not substantially replicated skeletal phenotypes observed in the limbs of human patients.
To induce skeletal phenotypes mimicking those observed in Rothmund-Thomson syndrome (RTS), RAPADILINO syndrome and Baller Gerold syndrome (BGS) in the model organism Xenopus laevis, CRISPR/Cas9 mediated gene knockout was employed. Mutations were induced in either exons 11 or 16 creating a variety of mutations in the helicase domain of Recql4. A variety of genotyping methods were established, including MiSeq, which revealed tadpoles were highly mosaic.
Tadpoles presented with a range of skeletal phenotypes, the most severe of which was loss of forelimb or hindlimb. Many of the phenotypes were consistent with patient skeletal features. Investigation of tadpoles with alcian blue (cartilage) and alizarin red (bone) stains revealed delayed ossification primarily in endochondrally ossified bones providing further evidence that endochondral ossification is more sensitive to recql4 disruption. Unexpectedly tadpoles developed microphthalmia, facial asymmetry, and in extreme cases lacked formation of part of the frontoparietal bone indicating that phenotypes observed mimicked severe RTS.
Successful induction of skeletal phenotypes in X. laevis that successfully replicate those observed in human RECQL4 patients, combined with the accepted functions of Recql4 in X. laevis in DNA replication initiation suggests further refinement of the disrupted processes leading to abnormal skeletal development - namely, the role of Recql4 in the CMG helicase formation is not instrumental for the development of skeletal phenotype. Delayed ossification phenotypes observed in this project provide a unique avenue for continued investigation into the mechanism by which DNA replication-related skeletal abnormalities arise during development.