SPINE-GEL: a global gene expression library for the developing nucleus pulposus

Abstract

SPINE-GEL: a global gene expression library for the developing nucleus pulposus Kathryn Cheah1, Tiffany Au.1,Ben Niu1,Danny Chan1 , Michael Q. Zhang2. 1School of Biomedical Sciences, University of Hong Kong, Hong Kong, China; 2 Department of Biological Sciences, Center for Systems Biology, The University of Texas at Dallas, Richardson, TX, USA Introduction: The intervertebral disc (IVD) forms by segmentation of the embryonic axial skeleton which comprises the rod-like notochord (NC) surrounded by the somatic mesenchyme of the sclerotome. The notochord-is the source of signals that are critical for induction and patterning of the sclerotome, which gives rise to the vertebral body. At mid-gestation the notochord is segmented, forming the nucleus pulposus (NP) of the future IVDs. We and others have shown from lineage tracing in mice that the origin of NP cells are the notochord. In adult NP cells persisting notochordal cells (NCCs) are thought to be precursors of (NPCs), perhaps suggesting NCCs are the progenitors of NP cells. Little is known of the molecular changes that occur during the transition from NCC to NPC. We aimed to define the changes in molecular signature of the NCC as they become NPCs at embryonic, fetal and adult. Materials and Methods: We generated mice in which NC and NP cells are marked by GFP by crossing our notochord specific Foxa2 MNE-cre mice to Z/EG mice. We purified NCCs and NPCs by FACS from GFP-tagged NCCs and generated the transcriptomes of embryonic (at E8.5-E10) notochord (NC), E12.5 as the NCC start making the transition to NPC, and at postnatal states (at P2, 4weeks) nucleus pulposus (NP). Results: NCC data compared with the published E8.0-E8.5 notochord microarray expression data showed a high degree of similarity (>0.8 Spearman’s rank correlation coefficient). Comparison of NCC transcriptome data with published data for mouse ES/iPS/EpiS cells showed a much lower correlation consistent with the progressive differentiation from early stages of development to NCCs. For NCCs Wnt signaling was the major pathway, expression of genes involved in oxidative phosphorylation and cell cycle/proliferation was enriched. As expected, the transcription factors characteristic of NCCs were Brachyury and Foxa2. Interestingly we found enrichment for Hox genes. Hox genes control the body plan along the anterior and posterior (A/P) axis but their roles in notochord cell fate is not clear. For NPCs the TGF-beta, FGF, VEGF pathways were dominant. Unlike NCCs, for NPCs, genes involved in ECM-cell interaction (integrin signaling, focal adhesion) and Klf transcription factors dominated. Conclusion: We have created a library of gene expression changes of the developing NP (SPINE-GEL). The data reveal a dynamic picture of changing ECM niches and signaling pathways for NCCs and NPCs. Interestingly many of the genetic risk factors identified for Intervertebral Disc Degeneration (IDD) affect the ECM (e.g. COL9A1, asporin, CHST3, CILP, ACAN). The changing transcriptome landscape correlated well with the known IDD genetic risk factors, suggesting the library of NCC and NPCs molecular signatures will be a rich source of candidate genes for genetic risk factors for IDD.