Mutations in 19 genes resulting in absence of the notochord have been catalogued by the Mouse Genome Informatics (MGI) database (http:​/​/​www.​informatics.​jax.​org). The most well characterized is the T gene, encoding the brachyury protein (Kispert et al. 1994). Brachyury is the prototype T-box transcription factor. It is also considered a marker of primitive mesoderm and notochord. It starts out being expressed in all mesodermal cells. Later, it becomes restricted to the notochord where expression is maintained. Mice with homozygous mutations in T have defects in the formation of mesoderm, and the trunk notochord is not established (Dobrovolskaia-Zavadskaia 1927). Subsequent malformations in the spine and allantois lead to the early demise of the embryo. Mice with a dominant-negative mutation in T (Tc) survive but demonstrate abnormal nucleus pulposus morphology (Stott et al. 1993). Brachyury is also a marker for chordomas, rare malignant tumors along the spine thought to arise from persistent remnants of the notochord (Vujovic et al. 2006). Duplications in the T gene in humans have been associated with susceptibility to chordoma (Yang et al. 2009). For a complete discussion of chordomas, see Chap.​ 18.

While mice with mutations in T fail to establish the notochord, Danforth’s short-tail (Sd) mutation results in failure to maintain the notochord (Paavola et al. 1980). Sd mice arose from a spontaneous mutation in a yet unknown gene. Sd mice have abnormal nucleus pulposus morphology likely due to defects in the formation and maintenance of the notochord early in development. In Sd mice the notochord is discontinuous and becomes increasingly fragmented, eventually disappearing in mice homozygous for the mutant allele. In heterozygous mice, the intervertebral disc forms but the nucleus pulposus is absent and the disc is occupied with fibrous tissue similar to the annulus fibrosus (Semba et al. 2006).

Mutations in the Sickle tail gene (Skt), on the same chromosome as Sd, have nucleus pulposi; however, the nuclei are shifted to the periphery of the disc (Semba et al. 2006). The boundary between the nucleus pulposus and the annulus fibrosus is altered, and the annulus exhibits thin fibrous layers relative to control mice. The sequence of the Skt gene was identified using the gene-trapped ES clone from which it was derived. The protein product is predicted to have a proline-rich region in the N-terminus and a coiled-coil domain in the middle. The coiled-coil domain was similar to those found in a large number of scaffold proteins including keratins. It was suggested that while Sd is required at early stages of notochord development, Skt is required later in development for proper growth, differentiation, and maintenance of the nucleus pulposus. Furthermore, specific polymorphisms in the human SKT gene have been significantly associated with lumbar disc herniation in Japanese and Finnish case-­controlled populations (Karasugi et al. 2009).

A sheath of extracellular matrix containing collagens and glycoproteins, including collagen II and laminin, surrounds the mesodermal cells of the notochord (Gotz et al. 1995). It has been proposed that the notochord sheath functions to contain and direct internal hydrostatic pressure within the notochord. The notochord is the forerunner of the axial skeleton in non-vertebrate chordates (Box 3.2). During early Xenopus development, osmotic inflation of the notochord against the sheath results in lengthening and straightening of the embryo (Adams et al. 1990). Similar mechanics-based mechanisms involving the notochord sheath may be involved in morphogenesis of the nucleus pulposus.

It has been shown that mutations in genes encoding proteins that affect the formation of the notochord sheath also affect the development of the nucleus pulposus (Smits and Lefebvre 2003; Choi and Harfe 2011). Sox5 and Sox6 are Sry-related HMG box transcription factors that cooperate with Sox9 to mediate chondrogenesis (Lefebvre 2002). Inactivation of Sox5 and Sox6 result in dramatic chondrodysplasia with impaired chondrocyte differentiation (Smits et al. 2001). Sox5/6 is also required for the formation of the notochord sheath and subsequent survival of the notochord and development of the nucleus pulposus (Smits and Lefebvre 2003). The sheath contains many matrix proteins that are also found in cartilage including collagen II and proteoglycans containing sulfated glycosaminoglycans like aggrecan and perlecan. The aggrecan and perlecan content in the notochord sheath from Sox5/6 mutant mice was dramatically reduced, whereas collagen II was not affected. It was suggested that Sox5/6 promotes the formation of the notochord sheath by regulating matrix gene expression in the notochord cells. Failure to maintain the sheath matrix resulted in premature death of notochord cells and an aberrant nucleus pulposus.

Shh, a secreted signaling molecule important for several aspects of development, is also required for the formation and maintenance of the notochordal sheath (Choi and Harfe 2011). Shh is secreted by the notochord as well as the nucleus pulposus in embryonic and postnatal life (Dahia et al. 2009; DiPaola et al. 2005). Shh binds to its receptor, Ptc, on nearby cells, thus relieving repression of Smoothened, Smo, a transmembrane protein that is required to transmit the Shh signal (Ingham and McMahon 2001). In mice containing a germ line null allele of Shh, the notochord was formed, but was not maintained (Chiang et al. 1996). The embryos died shortly thereafter preventing analysis of development of the intervertebral disc. To address the role of Shh in the maintenance of the notochord and the subsequent formation of the nucleus pulposus, a conditionally deleted allele of Smo in mice was used (Choi and Harfe 2011). In these experiments the investigators removed Smo from all Shh-expressing cells, including the notochord. The notochord sheath was missing in the Smo-deleted mice. Furthermore, the nucleus pulposus did not expand into the intervertebral disc region, and notochord cells were scattered throughout the vertebral column. Notochord remnants in the vertebral body could be marked by ROSA26 in ShhCreERT2 mice. If Smo was removed after the notochord sheath formed, the nucleus pulposus was not affected indicating the importance of the sheath in this tissue formation.

Collagen II (Col2) is the major collagen in cartilage and an important component of the notochordal sheath (Swiderski and Solursh 1992; Gotz et al. 1995). The notochord is not removed from vertebral bodies, and intervertebral discs do not form normally in mice with a null mutation in Col2a1 (Aszódi et al. 1998). Cartilage proteins including collagens IX and XI, aggrecan, and COMP appear to be expressed in these mice, but collagens I and II are inappropriately expressed in the cartilage of the presumptive vertebrae. As a result, the collagen fibers in cartilage are disorganized. It was proposed that this disorganization would lead to a weakened structure unable to contain the cartilage osmotic pressure. The reduction in internal pressure within the vertebral body could be responsible for the failure of the notochord to be removed from the vertebrae and expand into the intervertebral disc (Adams et al. 1990), as a result, the nucleus pulposus would not form. Mutations in other genes that are expressed in the sclerotome and affect the development of the vertebral body including Nkx3.2 and Pax1/9 also affect the removal of the notochord from the vertebral body and development of the nucleus pulposus, further supporting the requirement of mechanical pressure for removal/expansion of the nucleus pulposus (Peters et al. 1999; Lettice et al. 1999; Tribioli and Lufkin 1999).

The lineage of the cells in the adult nucleus pulposus has been the source of much debate (Risbud et al. 2010; Erwin 2010; Shapiro and Risbud 2010). It has been proposed that the notochord forms the initial central part of the intervertebral disc, but as this compartment matures, these large notochord cells are replaced with smaller cells that more closely resemble chondrocytes, perhaps recruited from the end plate or the inner annulus (Wamsley 1953; Kim et al. 2003). However, more recently, evidence has accumulated to suggest that all of the cells within the nucleus pulposus are indeed derived from the notochord.

Shh is highly expressed in the notochord and later in the nucleus pulposus (Dahia et al. 2009; DiPaola et al. 2005; Choi et al. 2008). Mice that express an inducible Cre under the control of the Shh promoter were used in fate mapping studies of the notochord (Choi et al. 2008). To exclude the possibility that Shh-Cre would mark cells in the notochord and then again in a new nucleus pulposus cell population, a tamoxifen-inducible Cre was employed. Notochord cells were “pulse labeled” by administration of tamoxifen at an early stage of development and then followed over time with ß-galactosidase ­staining through Cre-mediated ­activation of the ROSA26-LacZ locus. When mouse embryos were pulse labeled at E8.0 days of gestation and examined at E13.5, it was clear that all of the cells of the nucleus pulposus were labeled. Furthermore, when old mice (19 months) were examined, all of the cells of the nucleus pulposus were labeled with no labeling observed in the annulus fibrosus. More recently, notochord cells were traced using a Noto-Cre mouse (McCann et al. 2012). Noto is a highly conserved transcription factor with expression limited to the node and early notochord. Again, when cells were marked by activation of the ROSA26-LacZ locus by Noto-Cre, all of the nucleus pulposus cells in the adult, after dramatic changes in cellular morphology, were still labeled although only a subset of adult nucleus cells stained for K8, a putative marker for nucleus pulposus cells. The fate mapping studies strongly suggest the nucleus pulposus is derived completely from the notochord.

Evidence that the nucleus pulposus is derived from the notochord also comes from gene profiling studies. Recent studies used microarray technology to compare the gene expression patterns in human, bovine, canine, and rodent nucleus pulposus, annulus fibrosus, and articular cartilage (Minogue et al. 2010; Lee et al. 2007; Sakai et al. 2009). Brachyury/T, K8, K18, and K19 were highly expressed in the nucleus pulposus. Brachyury/T is known as a marker of the notochord and also a marker of chordoma, notochord tumors, suggesting that the nucleus pulposus could be derived from these embryonic cells. More importantly, gene expression patterns significantly overlapped, and expression of the above nucleus pulposus markers was similar in the large notochord-like cells and the smaller chondrocytic cells within the nucleus pulposus. In contrast to this result, another group showed heterogeneity in the nucleus pulposus with regard to K8 expression (Gilson et al. 2010). Heterogeneity in K8 expression was also observed in the Noto-Cre cell fate mapping studies described above, even though all of the nucleus pulposus cells were clearly derived from Noto-expressing cells (McCann et al. 2012). A likely explanation is that the notochord can differentiate into all of the cell types including K8-expressing and K8-non-expressing cells, within the nucleus pulposus. This idea is supported by studies showing that notochord cells from rabbit can differentiate into cells with varying morphological characteristics similar to those observed in the adult nucleus pulposus (Kim et al. 2009). Taken together, there is now considerable evidence indicating that all of the cell types in the adult nucleus pulposus are derived from the notochord.

Somites are transient structures formed from the presomitic mesoderm (PSM) that define the anterior-posterior segmented pattern of the embryo. They are essentially balls of cells composed of an outer epithelial layer surrounding a mesenchymal core, the somitocoele (Ferrer-Vaquer et al. 2010). The formation of somites is tightly controlled during development both spatially and temporally. Somite pairs bud off from the anterior end of the PSM at time intervals specific to the species (Pourquie 2011; Brand-Saberi et al. 2008). Somite nomenclature is based on the relationship of the somite to the anterior end of the PSM with the closest somite labeled number SI followed by SII, SIII, etc., counting toward the anterior end of the embryo (Christ and Ordahl 1995). Future somites in the presomitic mesoderm are labeled S0 and SI. These somites have not budded off the PSM, but can be seen histologically or with molecular markers and are frequently referred to as somitomeres. It is important to note that the first five somites to bud off are destined to fuse and form the occipital bone (Couly et al. 1993). The remaining somites will progress into the components of the axial skeleton and skeletal muscle. Disruptions to somitogenesis and segmentation of the somites result in birth defects that can severely affect the function of the spine by disrupting the formation and shape of both vertebral bodies and intervertebral disc (Box 3.3; Turnpenny 2008).

Differentiation begins starting with the anterior-most somite. The dorsal epithelium of the somite will form the dermomyotome, which will later differentiate into the dermis of the back and skeletal muscle. The somitocoele and the ventral epithelium of the somite will undergo an epithelial to mesenchymal transition (EMT) to generate sclerotome, which will form all of the connective tissues of the axial skeleton. The division of sclerotome and dermomyotome begins when the somite reaches SIII (Christ and Ordahl 1995) although the sclerotome segment is still plastic until a much later stage (Dockter and Ordahl 2000). Shh secreted from the notochord and ventral floor plate is the primary inductive signal controlling the delamination of the epithelial somite to form the sclerotome (Borycki et al. 1998; Fan and Tessier-Lavigne 1994; Murtaugh et al. 1999; Dockter 2000; Chiang et al. 1996; Marcelle et al. 1999). Pax3 and Pax7 are expressed in the unsegmented PSM but are downregulated in the ventral somite and somitocoele as the sclerotome differentiates. The expression of Pax1 and Pax9, markers of sclerotome, increases (Brand-Saberi and Christ 2000). Shh has been shown to induce Pax1, a marker of sclerotome, when present ectopically (Fan and Tessier-Lavigne 1994; Johnson et al. 1994). A second permissive signal is also required for the formation of sclerotome (Stafford et al. 2011). BMP from the lateral plate mesoderm normally interrupts differentiation of sclerotome and interferes with Shh signaling, allowing the sclerotome to differentiate only from the ventral medial side of the somite. Several extracellular BMP antagonists are expressed regionally that carefully restrict BMP activity (Stafford et al. 2011; Rider and Mulloy 2010). Noggin (Nog) and Gremlin1 (Grem1) cooperate to antagonize BMP signaling, permitting sclerotome differentiation in the presence of Shh (Stafford et al. 2011). Deletion of Nog and Grem1 in mice results in the complete failure of sclerotome differentiation. Dermomyotome was not affected in these mice. Inhibition of BMP alone is not sufficient to specify sclerotome or expand sclerotome differentiation in vivo suggesting antagonism of BMP is a permissive factor for sclerotome differentiation (Rider and Mulloy 2010). Very recently, using notochord deficient Sd mice, it was shown that the floor plate alone is sufficient for the development of the sclerotome (Ando et al. 2011). Shh from the floor plate could replace Shh from the notochord to allow differentiation of ­sclerotome in the absence of notochord.

Resegmentation of the sclerotome was first proposed by Remak in 1855 (Bagnall et al. 1988) after observing that in relationship to the original somites, there was half-segment realignment of the vertebrae. The initial studies followed somite development in chick embryos through lineage tracing using quail/chick chimeras (Bagnall et al. 1988; Goldstein and Kalcheim 1992), vital dye (Bagnall 1992), or viral transduction (Ewan and Everett 1992). The consensus from these studies was that the sclerotome derived from one somite divides into rostral and caudal halves with the rostral half of one segment joining with the caudal half of the segment immediately rostral to it forming the vertebral body. Accordingly, the intervertebral disc would be derived from the sclerotome at the junction of the two half segments (Fig. 3.3). Since those early reports, it has been determined that resegmentation occurs after the sclerotome differentiates from the ventral portion of the somite. Resegmentation results in a one half-segment stagger between the sclerotome and myotome allowing for the alternating pattern between the musculature and the vertebral body in the axial skeleton. Phenotypes indicative of alterations in resegmentation include fusion of vertebrae where joints should be, split vertebrae and ribs, alterations in migration of neural crest through the sclerotome and disorganization of the dorsal root ganglia. Several mouse models demonstrate these types of resegmentation defects including deficiencies in RAB23 (opb mutant) (Sporle and Schughart 1998), Paraxis (Johnson et al. 2001), and Tgfbr2 (Baffi et al. 2006). Ablation of the neural tube after sclerotome formation also results in resegmentation failure suggesting a role for signals from the neural tube in directing resegmentation (Colbjorn Larsen et al. 2006). Much of the molecular mechanisms defining the process of resegmentation remain to be elucidated.

Sclerotome contains multipotent progenitor cells that differentiate into all of the connective tissue cell types of the axial skeleton (Monsoro-Burq 2005). Sclerotome can differentiate into cartilage that will subsequently undergo endochondral ossification to form the bony vertebrae of the spine. It will also differentiate into the annulus fibrosus of the intervertebral disc and the tendons that link the vertebrae to the muscle. Which cell type is specified is determined by the location of cells within the sclerotome and complex interactions of growth factors (Fig. 3.4). How these factors interact with each other to specify cell lineage in the axial skeleton is just beginning to be determined.