Role of cadherin 2 in intervertebral disc maturation

Abstract

N-­cadherin/ Cadherin 2 (Cdh2) is a cell adhesion glycoprotein. Besides its function in cell-­cell interaction, it is crucial for tissue integrity and can regulate mechano-­transduction events. In vertebrae development, notochord differentiates into nucleus pulposus (NP) of intervertebral discs (IVD). Cdh2 is expressed in notochordal cells (NCC), but depleted in adult mature NP cells. Our transcriptome profiling analysis also indicated that Cdh2 is specifically expressed in primitive rat NP in comparison with annulus fibrosus (AF) and hyaline cartilage. Recently, Cdh2 was reported to have a function in maintaining NP cell phenotype. However, the role of Cdh2 in IVD in vivo has not been elucidated to date. We hypothesized that Cdh2 has a function in maintaining NCCs in IVD development and may interact with mechanical factors in disc homeostasis. We studied prenatal and postnatal spine phenotype in a Foxa2-­Cre driven notochord-­specific Cdh2 knockout model (Foxa2-­Cre;; Cdh2flox/flox). A loss of notochord-­ specific marker T and NP progenitor marker Tie2 was observed in the knockout mice. Disappearance of vacuoles, a feature of notochordal cells, and reduced NP-­specific matrix expression was observed in postnatal NP of the mutants. By transmission electron microscopy, we further observed a disruption of connection between the NP cells and abnormal vacuolation in postnatal heterozygous mutants. Interestingly, the NP of lower but not upper lumbar or the coccygeal discs of the heterozygous mutants displayed premature fibrocartilaginous transformation, resembling mature human discs. By applying static bending to the mutant coccygeal discs in a tail looping model, fibrocartilaginous transformation of NP was evidenced in the motion segments with the highest compression angle. On the contrary, such changes were not observed in looped control littermates. By studying the mutant in a background of reporter (Foxa2-­Cre;; Cdh2flox/+;; Z/EG) that permanently activates GFP expression in notochord and hence enables tracing of the notochord descendent cells, GFP positive cells were identified in the fibrocartilaginous NP, suggesting a participation of resident notochordal cells in the transformation process. Furthermore, by depleting CDH2 expression in a human chordoma cell line (JHC7) via CRISPR-­Cas9 gene manipulation technology, we observed a downregulation of T and up-regulation of SOX9 gene expression. Chondrogenic micromass culture could significantly induce COL2A1 gene expression in the mutant cells compared to controls in which COL1A1 gene expression was increased. Altogether, these findings proposed a key role of cadherin 2 in maintenance of NCCs as well as regulating the maturation of IVD through an interplay with mechanical factors. In conclusion, Cdh2 may be involved in the process of NCCs being replaced by chondrocyte-­like cells during disc maturation.