Mini-Spine: an Ex-vivo Mouse Model System for Functional Studies on the Notochord and Nucleus Pulposus

Abstract

Mini-Spine: an Ex-vivo Mouse Model System for Functional Studies on the Notochord and Nucleus Pulposus Tiffany Y.K. Au1, Ron M.H. Wu1, Kathryn S.E. Cheah1 School of Biomedical Sciences, The University of Hong Kong. Abstract The intervertebral disc (IVD) is composed of three components: the nucleus pulposus (NP), annulus fibrosus (AF) and cartilagionous endplate (CEP). In the making of an IVD, which involves the formation of the notochord, its descendants to become NP at the centre of the IVD signaling to guide the formation of spine, whereas the vertebra between the IVDs originate from sclerotomes, the ventromedial region of somites. To understand the formation of the intervertebral disc, we begin with notochord formation. In the mouse, fate-mapping studies have shown the embryonic notochord (NC) originates from three distinct regions, including prechordal plate, head process and node. The notochord is then forming the primitive anterior/posterior axis of the embryo, which acts as the early axial skeleton of the embryo until the vertebrae form. From early fetal stage onwards (around E12.5 in the mouse), the notochord undergoes remodeling, in which segmentation of notochord/notochord transition into nucleus pulposus begins to take place from anterior to posterior. The segmentation/transition of the notochord into nucleus pulposus will be completed along the whole vertebral column right before birth. However, little is known of the molecular control/changes that occur during the transition from NC to NP. Towards that goal, we developed a double transgenic mouse model Foxa2mNE-Cre/Z/EG to tag NC cells, by which we traced and followed embryonic NC and its descendants to NP in vivo. Furthermore, we have established an initial protocol for isolation of fetal notochord and ex-vivo model system in mouse for functional studies on IVD development. In which, we isolated unsegmented mouse notochord at E12.5 and monitored the development of the notochord and the surrounding mesenchyme including sclerotome cells in vitro. After 24-36hrs, the isolated notochord segments (without surrounding mesenchyme) can develop into NP-like structure (bulge/outgrowth). Over a 21-day period, the isolated notochord segments further develop into pre-vertebrate-like tissue with differentiating chondrocytes and intervertebral regions encapsulating a NP-like structure. In addition, isolated E12.5 notochordal cells formed spheroids in culture within 24 hours that can be maintained and expanded in culture for over a month. These notochord spheroids (organoids) express notochord markers (SHH, COL2A1, FOXA2 and T), indicating fetal notochord cells can self-assemble to produce a niche for maintenance in culture. Our results suggest this ex-vivo model system (Mini-Spine) recapitulate the segmentation of notochord and its development into NP. Furthermore the isolated notochord with/ without the surrounding mesenchyme enable us to dissect how the two tissues differentiate to form the different compartments in the future IVD and vertebrae.