Numerous animal models with spontaneous disc degeneration have been described. In these animals, the mutation is not artificially induced as the animals develop the pathological condition naturally.

The sand rat and the pintail mouse are the first spontaneous models that have been reported (Singh et al. 2005). As was discussed in Chap.​ 20, in the sand rat (Psammomys obesus), cysts and tears in the annulus fibrosus and even herniation of the nucleus pulposus are evident. These conditions resemble the pathological aspects of the degenerate human intervertebral discs. This phenotype may be related to aging but is mainly attributed to an altered metabolism. In fact, diabetic sand rats show a decrease in disc hydration, which leads to less advantageous biomechanical properties and eventually to degeneration of the disc (Silberberg et al. 1979; Ziv et al. 1992).

Another spontaneous disc degeneration mouse model is the pintail mouse (Anas acuta). The nuclei pulposi in the subcervical region have a low mucopolysaccharide content as in the degenerate human discs. This phenotype is stronger in homozygotes. This mouse model was the first evidence that deterioration of the human disc could indeed have a genetic correlate (Berry 1961).

Transcription factors of the Pax family play a major role in intervertebral disc development. Pax-1 is a transcription factor critically involved in various aspects of mammalian organogenesis (Chalepakis et al. 1992). It is expressed in the sclerotome and is implicated in the formation of ventral vertebral structures. Pax-1 is a critical modulator of the crosstalk between notochord and sclerotome in early development (Wallin et al. 1994). For further information on this gene, please see Chap.​ 3.

Mice carrying a naturally occurring mutation of the Pax-1 gene demonstrate impaired development of both the vertebral bodies and intervertebral discs (Wallin et al. 1994). In particular, both vertebral bodies and intervertebral discs may be either absent or severely deformed. The defects are especially evident in the lumbar region and in the tail. The malformations appear at an early embryonic stage and they affect both the sclerotome and the notochord. However, this phenotype most likely involves additional genes, as a targeted null mutation of Pax-1 causes a less severe phenotype (see below).

Watanabe et al. (1997) and, later, Wai et al. (1998) reported that the “cmd” (cartilage matrix deficiency) mouse carries an autosomal recessive mutation in the gene encoding aggrecan, which is expressed in both the nucleus pulposus and the annulus fibrosus. Homozygous mice for this mutation display dwarfism and cleft palate, and they die shortly after birth, whereas modest dwarfism, age-associated hyperlordosis, and disc herniation are typically found in heterozygous mice. Aggrecan is thus likely to be an extremely important molecule in the development and homeostasis of the intervertebral disc.

Li et al. (2004) analyzed instead a spontaneous loss-of-function mutation of growth differentiation factor-5 (GDF-5) in mice. This growth factor has been shown to play a role in a variety of musculoskeletal processes, including joint formation, endochondral ossification, and tendon and ligament maintenance and repair (Francis-West et al. 1999). Mutant mice carrying a spontaneous loss of function of GDF-5 are characterized by short limbs, abnormalities of the joints, and a reduction in the number of phalanges in the second through fifth digits (Merino et al. 1999). More importantly, these mice demonstrate abnormalities of both the annulus fibrosus and the nucleus pulposus, resembling changes seen in some animal models of disc degeneration. In particular, young adult mice displayed decreased water content of the nucleus pulposus as indicated by MRI analysis. Moreover, the mutant nucleus pulposus is smaller and the glycosaminoglycan content of the disc is diminished, although the amount of total collagen is not altered. In addition, the annulus fibrosus in these mutant mice is characterized by loss of the lamellar organization. All of these abnormalities can be corrected by treatment with recombinant GDF-5. Consistent with these data, conditional knockout (see subsequent paragraph) of GDF-5 further highlighted the importance of this growth factor in the biology of the nucleus pulposus.

Sahlman et al. (2001) reported that mice heterozygous for a Col2a1 null allele develop abnormalities in the vertebral bodies and in the disc. In particular, their vertebral end plates undergo premature ossification, which is associated with a mild grade of degeneration of the disc and with a significantly reduced ability to run, likely secondary to the discomfort caused by the anatomical abnormalities. Of note, collagen II is poorly expressed in the nucleus pulposus, though it is a classical marker of the fibrocartilage that forms the annulus fibrosus. Mice homozygous for a null mutation of Col2a1 show abnormalities in both vertebral bodies and intervertebral discs: the vertebral bodies enlarge gradually and they never initiate endochondral ossification; moreover, the notochord persists, leading to a failure in the development of the intervertebral disc (Aszódi et al. 1998).

Along these lines, Sarver and Elliott (2004) created a transgenic mouse with reduced collagen I expression (Mov13 strain). Collagen I is particularly abundant in the matrix of the outer annulus fibrosus. Mechanical testing provided ­evidence that the disc in Mov13 mutant mice is mechanically inferior to controls when subjected to compression and torsion tests. This finding suggests that collagen I in the intervertebral disc is particularly important in absorbing torsional loads.

Another essential matrix protein of the annulus fibrosus is collagen IX. This collagen acts as a bridge between the fibrillar collagens and the other components of the matrix; together with collagens II and XI, it forms a heterofibril, which stabilizes the cartilaginous tissue. Nakata et al. (1993) reported that mice in which a gene construct has been inserted to generate a truncated α1 chain of collagen IX develop chondrodysplasia, osteoarthritis, and corneal abnormalities. In particular, homozygous mice exhibit spine deformities characterized by shortening of the vertebrae, matrix ­disorganization within the annulus fibrosus, and end-plate irregularities (Kimura et al. 1996).

More recently, another study demonstrated that mice homozygous for a loss-of-function mutation of the Col9a1 gene have early-onset osteoarthritis around 6 months of age, secondary to degeneration both in the annulus fibrosus and in the end plate. Interestingly, in this mouse model, the changes in the intervertebral disc are already clearly detectable at 3 months of age and thus precede the degeneration of the vertebral end plate. Notably, disc degeneration occurs in the annulus fibrosus and it is mainly characterized by tears of this structure, whereas the nucleus pulposus is virtually unaffected (Boyd et al. 2008; Allen et al. 2009).

Furukawa et al. (2009) analyzed the function of another component of the extracellular matrix in the intervertebral disc by knocking out biglycan, which is a member of the family of small leucine repeat proteoglycans (SLRPs); SLRPs bind to TGF-βs, collagens, and other matrix proteins. In humans, biglycan is mostly expressed in the outer layer of the annulus fibrosus, while its concentration is very low in the nucleus pulposus. Advancing age leads to an increase in biglycan content in the early stages of degeneration and then, eventually, to a progressive decrease (Cs-Szabo et al. 2002). Previous studies showed that biglycan-deficient mice developed premature osteoarthritis (Ameye et al. 2002). In this study, the authors provide evidence that the absence of biglycan leads to an early degeneration of the intervertebral disc. Using morphometrical and histological analyses, the authors reported that the size of the nucleus pulposus decreases with age in the mutant mice and that, notably, at 6 months of age, mice display an abnormal proliferation of chondrocyte-like cells within the nucleus pulposus. Later, both the nucleus pulposus and the annulus fibrosus in biglycan-deficient mice are characterized by tears associated with “mucous degeneration” of the nucleus. Therefore, biglycan is likely to be an important factor in the maintenance of the intervertebral disc. There are different mechanisms to explain why loss of biglycan accelerates disc degeneration: its loss might cause instability of the extracellular matrix, or it might increase the mechanical stress on the intervertebral disc. The absence of biglycan may also inhibit TGF-β signaling, and this could suppress the damage repair response in the extracellular matrix.

In recent years, numerous mouse mutants with abnormalities of the intervertebral disc have been generated by deleting genes of interest by homologous recombination. This knockout strategy has allowed researchers to define the role of different genes in disc development.

For instance, transcription factors important in chondrogenesis have also been shown to play a critical role in disc development and homeostasis. In particular, Smits and Lefebvre (2003) have provided evidence that Sox5 and Sox6 are expressed not only in the chondrocyte precursors that form the vertebral bodies and the annulus fibrosus but also in the notochord and are critically important for disc development. Sox5 and Sox6 encode two identical transcription factors (L-Sox5 and Sox6); moreover, Sox5 also encodes a short protein (Sox5) that lacks the N-terminal of L-Sox5. L-Sox5 and Sox6 are co-expressed in all cartilages and in a few other tissues. In vitro data have shown that these two transcription factors cooperate with Sox9 in the activation of the collagen II gene (Lefebvre et al. 1998). Moreover, Sox5 and Sox6 have redundant roles in chondrogenesis, and knockout mice for these two transcription factors are characterized by a severe chondrodysplasia (Smits et al. 2001). In a more recent work, Smits and Lefebvre analyzed the spine phenotype of mice lacking Sox5, Sox6, or both through RNA in situ hybridization, cell proliferation, and death assays. First, they showed that Sox5 ^−/− /Sox6 ^−/− mice lack the nucleus pulposus and that the annulus fibrosus has a deficient extracellular matrix. At earlier developmental ages, mutant embryos display an abnormal development of the notochord secondary to massive cell death. Analysis of the possible different genotypes led to the conclusion that Sox5 and Sox6 have redundant functions in notochord development, though Sox6 could be slightly more important.

Taken together, these findings demonstrate that Sox5 and Sox6 are not necessary in early notochord formation but in the survival of notochordal cells. Moreover, these data are consistent with the notion that the nucleus pulposus is derived from the notochord (Choi et al. 2008).

Homozygous Pax-1^−/− mice display abnormalities in the shape of the vertebrae, and the intervertebral discs are replaced by a ventral cartilaginous rodlike structure (Wilm et al. 1998). In contrast to Pax-1 mutants, Pax-9^−/− mutant mice do not exhibit morphological abnormalities of the axial skeleton (Peters et al. 1998). However, double mutants lacking both Pax-1 and Pax-9 have a more severe phenotype in both vertebral bodies and intervertebral discs when ­compared to single Pax-1 null mice (Peters et al. 1999). This study indicates that Pax-1 can completely compensate for the absence of Pax-9 during early development of vertebral bodies and intervertebral discs, whereas Pax-9 has a redundant role in the formation of these structures.