An Essential Role for B1 Integrin Mediated Cell-Matrix Interactions in the Development and Maintenance of the Nucleus Pulposus

Abstract

Introduction In humans, the almost total disappearance of notochordal cells in the nucleus pulposus (NP) correlates with early degenerative changes in the intervertebral disk which include changes in the extracellular matrix (ECM). Recent cell lineage studies in mice suggest cells within the NP are derived from the early embryonic notochord. Since notochordal cells are surrounded by a rich ECM, the functions of the notochord and the NP are likely mediated via cell-matrix interactions. Integrin heterodimers are transmembrane proteins and are the major adhesion receptors that mediate cell-matrix interactions. They connect the ECM to the intracellular cytoskeleton and transduce bidirectional signals between the extracellular matrix and intracellular compartments. At least 24 integrin heterodimers have been identified, and among them receptors containing b 1 integrin comprise the largest integrin-subfamily. Materials and Methods Here, we used Cre recombinase to specifically inactivate b 1 integrin gene in the notochord in mice, to understand the role of b 1 integrin-mediated cell-matrix interactions in the development and maintenance of the notochord and NP. The development and fate of the notochordal cells were analyzed at embryonic stages E8.5-E10.5 using key expression markers such as Shh, Brachyury, and Noto and cell apoptotic assay. NP formation was studied by histological analysis. The function of b 1 integrin in cell-matrix interactions in notochord cells isolated by FACS were analyzed using a functional blocking antibody specific to b 1 integrin. Fibronectin matrix and Cadherin-2 expression pattern were investigated by immunofluorescence and confocal microscopy. Results Mice lacking b 1 integrin subunit in notochordal cells display abnormal vertebral column development with reduced size or total absence of the notochord descendents and NP in the intervertebral disks (IVD). This abnormality is accompanied with disorganized kinked patterning of the tail vertebrae and hemivertebrae. These defects are related to defects in the development and maintenance of the notochord at early embryonic stages from E8.25. Abnormalities in localization of the notochord were found, such as displacement from D-V or L-R midline axes, and failure to separate from the foregut endoderm. Assembly of ECM components was also impaired. Impaired cell matrix assembly and cell-cell contacts found in the mutants may possibly contribute to the notochord mallocalization. Increased cell apoptosis together with impaired cell-cell adhesion may potentially contribute to the defective notochord tissue integrity. b 1 integrin antibody blocking assays on notochordal cells display impaired cell adhesion, delayed maturation of focal adhesion complexes and decreased migration ability on matrix substrates. Conclusion Maintenance of b 1 integrin mediated cell-matrix interactions is essential for the proper development and maintenance of the notochord and NP, and later the proper patterning of the axial skeleton. Impaired cell-matrix interactions which affect cell survival, cell polarity, and migration may contribute to the progressive loss of NP cells during disk degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared