Interplay between CYYR1 gene and Sonic hedgehog pathway: from zebrafish myogenesis to a potential role in rhabdomyosarcoma disease

CYYR1 (Cysteine/tyrosine-rich 1) cloned on human chromosome 21 defines a family of highly conserved vertebrate-specific genes. The human locus is characterized by a multitranscript-system including at least six alternative spliced isoforms and one ncRNA gene overlapping CYYR1 in antisense orientation (CYYR1-AS1). To date, the function of the CYYR1 product is still unknown even if original results suggest its possible involvement in myogenesis differentiation with a putative role in the tumorigenic process related to the Hh pathway. Zebrafish cyyr1 orthologue is present in single copy and the predicted protein maintains almost 58% of identity with human protein. By WISH approach, we show a cyyr1 broad expression in central nervous system (CNS), somites and muscles during development. The protein seems to localize in plasma membrane and even in nuclear envelope. We perform cyyr1 knock-down with two different approaches: microinjection of morpholino oligos targeting the ATG and the first splice-site of the transcript, and the generation of cyyr1 null fish through CRISPr/Cas9 technique. Defects in heart and muscle development with a significant rescue in embryo co-injected with cyyr1 mRNA, were observed in morphants, while cyyr1 mutants analyses confirmed morphological and molecular weakness in heart. Dysregulation of Nodal and/or Hh pathways in zebrafish decreased cyyr1 expression and the injection of cyyr1 mRNA was able to partially rescue Hh-defective phenotype in embryos. In order to verify a putative role of CYYR1 in process caused by dysfunction of cell differentiation we performed experiments in rhabdomyosarcoma cell lines showing an inverse correlation between CYYR1 expression and the range of differentiating capabilities of these cells. Interestingly, treatments with inhibitors of Shh allow us to confirm a correlation between CYYR1 and this pathway.