Transformation of resident notochord-descendent nucleus pulposus cells in mouse injury-induced fibrotic intervertebral discs

Abstract

Intervertebral disc degeneration (IDD), a major cause of low back pain, occurs with ageing. The core of the intervertebral disc, the nucleus pulposus (NP), embedded in a proteoglycan-rich and gelatinous matrix, is derived from the embryonic notochord. With IDD, the NP becomes fibrous, containing fewer cells, which are fibroblastic and of unknown origin. Here, we used a lineage tracing strategy to investigate the origin of cells in the NP in injury-induced mouse IDD. We established a Foxa2 notochord-specific enhancer-driven Cre transgenic mouse model (Foxa2mNE-Cre) that acts only in the embryonic to foetal period up to E14.5, to genetically label notochord cells with enhanced green fluorescent protein (EGFP). When this mouse is crossed to one carrying a Cre recombinase reporter, Z/EG, EGFP-labelled NP cells are present even at 2 years of age, consistent with their notochordal origin. We induced tail IDD in Foxa2mNE-Cre; Z/EG mice by annulus puncture and observed the degenerative changes for 12 weeks. Soon after puncture, EGFP-labelled NP cells showed strong Col2a1+ expression unlike uninjured control NP. Later, accompanying fibrotic changes, EGFP-positive NP cells expressed fibroblastic and myofibroblastic markers such as Col1a1, ASMA, FAPA and FSP-1. The number of EGFP+ cells co-expressing the fibroblastic markers increased with time after puncture. Our findings suggest resident NP cells initially upregulate Col2a1+ and later transform into fibroblast-like cells during injury-mediated disc degeneration and remodelling. This important discovery concerning the cellular origin of fibrotic pathology in injury-induced IDD has implications for management in disease and ageing.

Keywords: disc degeneration; fibroblast; fibrosis; notochord; nucleus pulposus.

Figure 1

Lineage tracing in Foxa2mNE‐Cre;Z/EG mice. (a) Foxa2 minimal notochord element (Foxa2mNE) and a Sox9 minimal promoter‐driven Cre recombinase (Cre) and lacZ via an IRES sequence. (b) β‐gal staining of Foxa2mNE‐Cre transgenic foetus at E12.5 in whole‐mount (i); mid‐sagittal sections showing notochord specificity and absence of staining in surrounding tissue (ii‐iv); mid‐cross section showing developing limbs (v,*). (c) Cre immunofluorescence in the developing notochord at E9.5 (i) and E12.5 (ii), and in (iii) rostral, (iv) trunk developing nucleus pulposus and (v) caudal notochord at E14.5 (outlined). (d) Cre expression and activity (EGFP) in Foxa2mNE‐Cre;Z/EG compound heterozygotes at (i) foetal stages and quantification of EGFP+ cells in the developing rostral (anterior to hindlimb) and caudal (posterior to hindlimb) nucleus pulposus (mean ± SEM < 1%); (ii) Tracking at neonatal (P0), adolescent (2 week, 1 month) and adult stage (3 months, 6 months, 1 year) by native EGFP signal detection in P0 and 1 year old tails, or EGFP immunostaining. nd, notochord; np, nucleus pulposus; vb: vertebrae; ivd: intervertebral disc; white dashed line encircled developing nucleus pulposus region

Figure 2

Resident NP cell transformation into fibroblast‐like cells in induced disc degeneration. (a) Combined EGFP immunofluorescence and Col2a1 in‐situ hybridization at 1 wpp and 12 wpp. (b) Combined EGFP immunofluorescence and Col1a1 in‐situ hybridization at 1 wpp and 12 wpp. NP region encircled. GP: growth plate; IAF: inner annulus fibrosus; EP: endplate; scale bar: 200 µm. Co‐immunolocalization of EGFP with ASMA (c), FAPA (d) and FSP‐1 (e) at 4 wpp and 12 wpp (f–h). (i) Quantification of NP cells positive for EGFP or the fibroblastic markers (>3 mice, 6 sections each timepoint). (j) Percentage of EGFP+ cells co‐expressing the fibroblastic markers. (k) Percentage of EGFP—cells co‐expressing the fibroblastic markers. *p < 0.05; **p < 0.01; ****p < 0.0001; ns: p > 0.05. wpp: weeks post‐puncture. Scale bar in (i & iii): 25 μm; 50 μm (ii & iv). For single staining images, see Figures S5 and S6