Development of the axial skeleton and intervertebral disc.

Development of the axial skeleton is a complex, stepwise process that relies on intricate signaling and coordinated cellular differentiation. Disruptions to this process can result in a myriad of skeletal malformations that range in severity. The notochord and the sclerotome are embryonic tissues that give rise to the major components of the intervertebral discs and the vertebral bodies of the spinal column. Through a number of mouse models and characterization of congenital abnormalities in human patients, various growth factors, transcription factors, and other signaling proteins have been demonstrated to have critical roles in the development of the axial skeleton. Balance between opposing growth factors as well as other environmental cues allows for cell fate specification and divergence of tissue types during development. Furthermore, characterization of progenitor cells for specific cell lineages has furthered the understanding of specific spatiotemporal cues that cells need in order to initiate and complete development of distinct tissues. Identifying specific marker genes that can distinguish between the various embryonic and mature cell types is also of importance. Clinically, understanding developmental clues can aid in the generation of therapeutics for musculoskeletal disease through the process of developmental engineering. Studies into potential stem cell therapies are based on knowledge of the normal processes that occur in the embryo, which can then be applied to stepwise tissue engineering strategies.

A histocytological and radiological overview of the natural history of intervertebral disk: from embryonic formation to age-related degeneration.

PURPOSE: To elucidate the natural history of intervertebral disk (IVD) and characterize its embryonic beginnings and age-related degeneration. METHODS: Coronal sections of embryonic (E13.5-neonatal) and postnatal (4-60-week-old) Sprague-Dawley rat IVD were stained by a series of histological stainings (hematoxylin and eosin, Alcian blue, Picrosirius red, Masson, Periodic acid-Schiff). Growth kinetics within embryonic IVD were evaluated by immunohistochemical staining of Ki67 and proliferating cell nuclear antigen. Postnatal maturation and degeneration of IVD were visualized on radiology by X-ray, CT, and MR imaging. RESULTS: During the formation of rat IVD, inner annulus fibrosus (AF) and cartilaginous endplate (CEP) shared similar cell density, extracellular matrix, and potential of growth kinetics; notochord provided increased and enlarged cytoplasmic vacuoles to generate nucleus pulposus (NP), part of which was retained within CEP. Postnatally, vacuolated notochord cells were reduced by devacuolation, while chondrocytic NP cells increased; cartilaginous layers of CEP were narrowed by vertebrae growth and secondary ossification; fibrotic portion of AF decreased as cartilaginous matrix accumulated and infiltrated outward. In aged and degenerated IVD, large longitudinal fissures were detected near the boundaries between inner and outer AF, whereas both reduced cellularity and accumulated cell clusters were evident within the dehydrated NP; only part of these histocytological changes could be reported on radiology. CONCLUSIONS: By showing that the natural history of IVD is orchestrated by a dynamic histocytological regulation, our study may facilitate better understanding of the developmental defects, cellular heterogeneity, age-related degenerative mechanisms, and biological regeneration of IVD. These slides can be retrieved under Electronic Supplementary Material.

The embryonic and evolutionary boundaries between notochord and cartilage: a new look at nucleus pulposus-specific markers.

The adult nucleus pulposus (NP) and articular cartilage are similar in terms of their histocytological components and biomechanical functionalities, requiring a deep understanding of NP-specific markers to better evaluate stem-cell-based NP regeneration. Here, we seek to distinguish NP cells from articular chondrocytes (ACs), focusing on differences in their embryonic formation and evolutionary origin. Embryonically, NP cells are conservatively derived from the axial notochord, whereas ACs originate in a diversified manner from paraxial mesoderm and neural crest cells. Evolutionarily, although the origins of vertebrate NP and AC cells can be traced to similar structures within protostomia-like bilaterian ancestors, the distant phylogenetic relationship between the two groups of animals and the differences in the bodily origins of the tissues suggest that the tissues may in fact have undergone parallel evolution within the protostomia and deuterostomia. The numbers of supposedly NP-specific markers are increasing gradually as microarray studies proceed, but no final consensus has been attained on the specificity and physiology of "exclusive" NP markers because of innate variations among species; intrinsic expression of genes that destabilize the circadian clock; and cooperation by, and crosstalk among, different genes in terms of physiology-related phenotypes. We highlight the embryonic and evolutionary boundaries between NP and AC cells, to aid in recognition of the challenges associated with evaluation of the role played by nucleopulpogenic differentiation during stem-cell-based intervertebral disc regeneration.

Expression of Carbonic Anhydrase III, a Nucleus Pulposus Phenotypic Marker, is Hypoxia-responsive and Confers Protection from Oxidative Stress-induced Cell Death.

The integrity of the avascular nucleus pulposus (NP) phenotype plays a crucial role in the maintenance of intervertebral disc health. While advances have been made to define the molecular phenotype of healthy NP cells, the functional relevance of several of these markers remains unknown. In this study, we test the hypothesis that expression of Carbonic Anhydrase III (CAIII), a marker of the notochordal NP, is hypoxia-responsive and functions as a potent antioxidant without a significant contribution to pH homeostasis. NP, but not annulus fibrosus or end-plate cells, robustly expressed CAIII protein in skeletally mature animals. Although CAIII expression was hypoxia-inducible, we did not observe binding of HIF-1α to select hypoxia-responsive-elements on Car3 promoter using genomic chromatin-immunoprecipitation. Similarly, analysis of discs from NP-specific HIF-1α null mice suggested that CAIII expression was independent of HIF-1α. Noteworthy, silencing CAIII in NP cells had no effect on extracellular acidification rate, CO2 oxidation rate, or intracellular pH, but rather sensitized cells to oxidative stress-induced death mediated through caspase-3. Our data clearly suggests that CAIII serves as an important antioxidant critical in protecting NP cells against oxidative stress-induced injury.

Whole Transcriptome Analysis of Notochord-Derived Cells during Embryonic Formation of the Nucleus Pulposus.

Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.