ß1 Integrin regulates convergent extension in mouse notogenesis, ensures notochord integrity and the morphogenesis of vertebrae and intervertebral discs.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/planar cell polarity (PCP) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which the ß1 integrin gene (Itgb1) is ablated at E8.0 in the notochord only or in the notochord and tail bud. These Itgb1 conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-ß1 integrin blocking antibodies, to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of ß1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

NFAT5/TonEBP controls early acquisition of notochord phenotypic markers, collagen composition, and sonic hedgehog signaling during mouse intervertebral disc embryogenesis.

High osmolarity, bound water, and hydrostatic pressure contribute to notochord mechanics and its morphogenesis into the nucleus pulposus (NP) compartment of the intervertebral disc. Indeed, the osmoadaptive transcription factor, nuclear factor of activated T-cells 5 (NFAT5 aka TonEBP), is robustly expressed by resident cells of the notochord and NP. Nevertheless, the molecular mechanisms that drive notochord osmoregulation and the functions of NFAT5 in disc embryogenesis remain largely unexplored. In this study, we show that deletion of NFAT5 in mice results in delayed vertebral column development and a reduced NP aspect ratio in the caudal spine. This phenotype is associated with lower levels of the T-box transcription factor, Brachyury, delayed expression of notochord phenotypic markers, and decreased collagen II deposition in the perinotochordal sheath and condensing mesenchyme. In addition, NFAT5 mutants showed a stage-dependent dysregulation of sonic hedgehog (Shh) signaling with non-classical expression of Gli1. Generation of mice with notochord-specific deletion of IFT88 (ShhcreERT2;Ift88f/f) supported this mode of Gli1 regulation. Using isolated primary NP cells and bioinformatics approaches, we further show that Ptch1 and Smo expression is controlled by NFAT5 in a cell autonomous manner. Altogether, our results demonstrate that NFAT5 contributes to notochord and disc embryogenesis through its regulation of hallmark notochord phenotypic markers, extracellular matrix, and Shh signaling.

The role of the notochord in amniote vertebral column segmentation.

The vertebral column is segmented, comprising an alternating series of vertebrae and intervertebral discs along the head-tail axis. The vertebrae and outer portion (annulus fibrosus) of the disc are derived from the sclerotome part of the somites, whereas the inner nucleus pulposus of the disc is derived from the notochord. Here we investigate the role of the notochord in vertebral patterning through a series of microsurgical experiments in chick embryos. Ablation of the notochord causes loss of segmentation of vertebral bodies and discs. However, the notochord cannot segment in the absence of the surrounding sclerotome. To test whether the notochord dictates sclerotome segmentation, we grafted an ectopic notochord. We find that the intrinsic segmentation of the sclerotome is dominant over any segmental information the notochord may possess, and no evidence that the chick notochord is intrinsically segmented. We propose that the segmental pattern of vertebral bodies and discs in chick is dictated by the sclerotome, which first signals to the notochord to ensure that the nucleus pulposus develops in register with the somite-derived annulus fibrosus. Later, the notochord is required for maintenance of sclerotome segmentation as the mature vertebral bodies and intervertebral discs form. These results highlight differences in vertebral development between amniotes and teleosts including zebrafish, where the notochord dictates the segmental pattern. The relative importance of the sclerotome and notochord in vertebral patterning has changed significantly during evolution.

Early stages of development of the alar fascia (human specimens at 6-12 weeks of development).

In recent years, there has been much discussion concerning the cervical fasciae. The aim of this study is to confirm and to describe the development of the alar fascia as well as its relationship with nearby structures. Histological preparations of 25 human embryos (6-8 weeks of development) and 25 human fetuses (9-12 weeks of development) were studied bilaterally using a conventional optical microscope. Our study confirms the existence of the alar fascia and permits three stages to be established during its development. The initial stage (1st), corresponding to the 6th week of development (Carnegie stages 18-19), is characterized by the beginning of the alar fascia primordium in the retroesophageal space at the level of C7-T1. In the formation stage (2nd), corresponding to the 7th and 8th weeks of development (Carnegie stages 20-23), the alar fascia primordium grows upwards and reaches the level of C2-C3. In the maturation stage (3rd), beginning in the 9th week of development, the visceral, alar and prevertebral fasciae can be identified. The alar fascia divides the retrovisceral space (retropharyngeal and retroesophageal) into two spaces: one anterior (between the alar fascia and the visceral fascia and extending from C1 to T1, named retropharyngeal or retroesophageal space according to the level) and the other posterior (between the alar fascia and the prevertebral fascia, named danger space). We suggest that this latter space be named the retroalar space. This study suggests that alar fascia development is related to mechanical factors and that the alar fascia permits the sliding of the pharynx and the oesophagus during swallowing.

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.

Early development of the vertebral column.

The segmental organization of the vertebrate body is most obviously visible in the vertebral column, which consists of a series of vertebral bones and interconnecting joints and ligaments. During embryogenesis, the vertebral column derives from the somites, which are the primary segments of the embryonic paraxial mesoderm. Anatomical, cellular and molecular aspects of vertebral column development have been of interest to developmental biologists for more than 150 years. This review briefly summarizes the present knowledge on early steps of vertebral column development in amniotes, starting from sclerotome formation and leading to the establishment of the anatomical bauplan of the spine composed of vertebral bodies, vertebral arches, intervertebral discs and ribs, and their specific axial identities along the body axis.

Morphogenesis of aligned collagen fibers in the annulus fibrosus: Mammals versus avians.

The mammalian intervertebral disc (IVD) consists of a gel-like, disordered nucleus pulposus (NP) surrounded by a highly ordered collagen structure, the annulus fibrosus (AF). While this concentric array of lamellae has been amply studied, its physical origin is poorly understood. The notochord is a rod-like organ located in the mid-line of the growing embryo and plays an essential role in IVD development. The aim of this study was to elucidate the effect of notochord development on the collagen fiber arrangement evolution in the AF. To that end, we studied IVD development in mouse embryos and compared these observations to those from chicken embryos, which do not form the typical laminar structure around the NP. In mouse, cross-aligned collagen arrangement of the AF forms from the sclerotome upon bulging of the notochord to become NP. By contrast, the notochord in the chicken embryo swells substantially without the physical restrictions of the future vertebrae and thus do not bulge. From these observations, we conclude that physical and geometrical constrictions are essential for the formation of the highly structured AF.

Surgical anatomy, radiological features, and molecular biology of the lumbar intervertebral discs.

The intervertebral disc (IVD) is a joint unique in structure and functions. Lying between adjacent vertebrae, it provides both the primary support and the elasticity required for the spine to move stably. Various aspects of the IVD have long been studied by researchers seeking a better understanding of its dynamics, aging, and subsequent disorders. In this article, we review the surgical anatomy, imaging modalities, and molecular biology of the lumbar IVD. Clin. Anat. 30:251-266, 2017. © 2017 Wiley Periodicals, Inc.

Disordered vertebral and rib morphology in pudgy mice. Structural relationships to human scoliosis.

Normal and abnormal vertebral development have been studied over the past 200 years at increasing levels of resolution as techniques for biological investigation have improved. Disordered development of the axial skeleton from the early embryonic period on leads to structurally malformed vertebrae and intervertebral discs and ribs causing the severe deformities of scoliosis, kyphosis, and kyphoscoliosis. Developmental malformation of the axial skeleton therefore has led to considerable biological and clinical interest. This work will detail our studies on the structural deformities of the vertebral column and adjacent ribs in the pudgy mouse [1] caused by mutations in the delta-like 3 (Dll3) gene of the Notch family [2]. While gene abnormalities in the pudgy mouse have been outlined, there has been no in-depth assessment of the histopathology of the pudgy vertebral and rib abnormalities that this study will provide. In addition, although congenital scoliosis has been recognized as a clinical problem since the mid-nineteenth century (1800s) [3] and accurately defined by radiography since the early twentieth century (1900s) [4-6], there have been few detailed histopathologic studies of human cases. We will also relate our histopathologic findings in the pudgy mouse to the histopathology of human vertebral and rib malformations in clinical cases of congenital scoliosis, one of which we defined in detail previously [7].

The notochord: structure and functions.

The notochord is an embryonic midline structure common to all members of the phylum Chordata, providing both mechanical and signaling cues to the developing embryo. In vertebrates, the notochord arises from the dorsal organizer and it is critical for proper vertebrate development. This evolutionary conserved structure located at the developing midline defines the primitive axis of embryos and represents the structural element essential for locomotion. Besides its primary structural function, the notochord is also a source of developmental signals that patterns surrounding tissues. Among the signals secreted by the notochord, Hedgehog proteins play key roles during embryogenesis. The Hedgehog signaling pathway is a central regulator of embryonic development, controlling the patterning and proliferation of a wide variety of organs. In this review, we summarize the current knowledge on notochord structure and functions, with a particular emphasis on the key developmental events that take place in vertebrates. Moreover, we discuss some genetic studies highlighting the phenotypic consequences of impaired notochord development, which enabled to understand the molecular basis of different human congenital defects and diseases.

Soluble factors from the notochordal-rich intervertebral disc inhibit endothelial cell invasion and vessel formation in the presence and absence of pro-inflammatory cytokines.

BACKGROUND: Chronic low back pain can be associated with the pathological ingrowth of blood vessels and nerves into intervertebral discs (IVDs). The notochord patterns the IVD during development and is a source of anti-angiogenic soluble factors such as Noggin and Chondroitin sulfate (CS). These factors may form the basis for a new minimally invasive strategy to target angiogenesis in the IVD. OBJECTIVE: To examine the anti-angiogenic potential of soluble factors from notochordal cells (NCs) and candidates Noggin and CS under healthy culture conditions and in the presence of pro-inflammatory mediators. DESIGN: NC conditioned media (NCCM) was generated from porcine NC-rich nucleus pulposus tissue. To assess the effects of NCCM, CS and Noggin on angiogenesis, cell invasion and tubular formation assays were performed using human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor alpha (TNFα [10 ng/ml]). vascular endothelial growth factor (VEGF)-A, MMP-7, interleukin-6 (IL-6) and IL-8 mRNA levels were assessed using qRT-PCR. RESULTS: NCCM (10 & 100%), CS (10 and 100 µg) and Noggin (10 and 100 ng) significantly decreased cell invasion of HUVECs with and without TNFα. NCCM 10% and Noggin 10 ng inhibited tubular formation with and without TNFα and CS 100 µg inhibited tubules in Basal conditions whereas CS 10 µg inhibited tubules with TNFα. NCCM significantly decreased VEGF-A, MMP-7 and IL-6 mRNA levels in HUVECs with and without TNFα. CS and Noggin had no effects on gene expression. CONCLUSIONS: We provide the first evidence that soluble factors from NCs can inhibit angiogenesis by suppressing VEGF signaling. Notochordal-derived ligands are a promising minimally invasive strategy targeting neurovascular ingrowth and pain in the degenerated IVD.

Notochord to Nucleus Pulposus Transition.

A tissue that commonly deteriorates in older vertebrates is the intervertebral disc, which is located between the vertebrae. Age-related changes in the intervertebral discs are thought to cause most cases of back pain. Back pain affects more than half of people over the age of 65, and the treatment of back pain costs 50-100 billion dollars per year in the USA. The normal intervertebral disc is composed of three distinct regions: a thick outer ring of fibrous cartilage called the annulus fibrosus, a gel-like material that is surrounded by the annulus fibrosus called the nucleus pulposus, and superior and inferior cartilaginous end plates. The nucleus pulposus has been shown to be critical for disc health and function. Damage to this structure often leads to disc disease. Recent reports have demonstrated that the embryonic notochord, a rod-like structure present in the midline of vertebrate embryos, gives rise to all cell types found in adult nuclei pulposi. The mechanism responsible for the transformation of the notochord into nuclei pulposi is unknown. In this review, we discuss potential molecular and physical mechanisms that may be responsible for the notochord to nuclei pulposi transition.

Metameric pattern of intervertebral disc/vertebral body is generated independently of Mesp2/Ripply-mediated rostro-caudal patterning of somites in the mouse embryo.

The vertebrae are derived from the sclerotome of somites. Formation of the vertebral body involves a process called resegmentation, by which the caudal half of a sclerotome is combined with the rostral half of the next sclerotome. To elucidate the relationship between resegmentation and rostro-caudal patterning of somite, we used the Uncx4.1-LacZ transgene to characterize the resegmentation process. Our observations suggested that in the thoracic and lumbar vertebrae, the Uncx4.1-expressing caudal sclerotome gave rise to the intervertebral disc (IVD) and rostral portion of the vertebral body (VB). In the cervical vertebrae, the Uncx4.1-expressing caudal sclerotome appeared to contribute to the IVD and both caudal and rostral ends of the VB. This finding suggests that the rostro-caudal gene expression boundary does not necessarily coincide with the resegmentation boundary. This conclusion was supported by analyses of Mesp2 KO and Ripply1/2 double KO embryos lacking rostral and caudal properties, respectively. Resegmentation was not observed in Mesp2 KO embryos, but both the IVD and whole VB were formed from the caudalized sclerotome. Expression analysis of IVD marker genes including Pax1 in the wild-type, Mesp2 KO, and Ripply1/2 DKO embryos also supported the idea that a metameric pattern of IVD/VB is generated independently of Mesp2/Ripply-mediated rostro-caudal patterning of somite. However, in the lumbar region, IVD differentiation appeared to be stimulated by the caudal property and suppressed by the rostral property. Therefore, we propose that rostro-caudal patterning of somites is not a prerequisite for metameric patterning of the IVD and VB, but instead required to stimulate IVD differentiation in the caudal half of the sclerotome.

Impaired intervertebral disc development and premature disc degeneration in mice with notochord-specific deletion of CCN2.

OBJECTIVE: Currently, our ability to treat intervertebral disc (IVD) degeneration is hampered by an incomplete understanding of disc development and aging. The specific function of matricellular proteins, including CCN2, during these processes remains an enigma. The aim of this study was to determine the tissue-specific localization of CCN proteins and to characterize their role in IVD tissues during embryonic development and age-related degeneration by using a mouse model of notochord-specific CCN2 deletion. METHODS: Expression of CCN proteins was assessed in IVD tissues from wild-type mice beginning on embryonic day 15.5 to 17 months of age. Given the enrichment of CCN2 in notochord-derived tissues, we generated notochord-specific CCN2-null mice to assess the impact on the IVD structure and extracellular matrix composition. Using a combination of histologic evaluation and magnetic resonance imaging (MRI), IVD health was assessed. RESULTS: Loss of the CCN2 gene in notochord-derived cells disrupted the formation of IVDs in embryonic and newborn mice, resulting in decreased levels of aggrecan and type II collagen and concomitantly increased levels of type I collagen within the nucleus pulposus. CCN2-knockout mice also had altered expression of CCN1 (Cyr61) and CCN3 (Nov). Mirroring its role during early development, notochord-specific CCN2 deletion accelerated age-associated degeneration of IVDs. CONCLUSION: Using a notochord-specific gene targeting strategy, this study demonstrates that CCN2 expression by nucleus pulposus cells is essential to the regulation of IVD development and age-associated tissue maintenance. The ability of CCN2 to regulate the composition of the intervertebral disc suggests that it may represent an intriguing clinical target for the treatment of disc degeneration.

An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration.

Cell-based regenerative medicine therapies have been proposed for repairing the degenerated intervertebral disc (a major cause of back pain). However, for this approach to be successful, it is essential to characterise the phenotype of its native cells to guarantee that implanted cells differentiate and maintain the correct phenotype to ensure appropriate cell and tissue function. While recent studies have increased our knowledge of the human nucleus pulposus (NP) cell phenotype, their ontogeny is still unclear. The expression of notochordal markers by a subpopulation of adult NP cells suggests that, contrary to previous reports, notochord-derived cells are retained in the adult NP, possibly coexisting with a second population of cells originating from the annulus fibrosus or endplate. It is not known, however, how these two cell populations interact and their specific role(s) in disc homeostasis and disease. In particular, notochordal cells are proposed to display both anabolic and protective roles; therefore, they may be the ideal cells to repair the degenerate disc. Thus, understanding the ontogeny of the adult NP cells is paramount, as it will inform the medical and scientific communities as to the ideal phenotype to implant into the degenerate disc and the specific pathways involved in stem cell differentiation towards such a phenotype.

Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs.

The vertebrae notochord is a transient rod-like structure that produces secreted factors that are responsible for patterning surrounding tissues. During later mouse embryogenesis, the notochord gives rise to the middle part of the intervertebral disc, called the nucleus pulposus. Currently, very little is known about the molecular mechanisms responsible for forming the intervertebral discs. Here we demonstrate that hedgehog signaling is required for formation of the intervertebral discs. Removal of hedgehog signaling in the notochord and nearby floorplate resulted in the formation of an aberrant notochord sheath that normally surrounds this structure. In the absence of the notochord sheath, small nuclei pulposi were formed, with most notochord cells dispersed throughout the vertebral bodies during embryogenesis. Our data suggest that the formation of the notochord sheath requires hedgehog signaling and that the sheath is essential for maintaining the rod-like structure of the notochord during early embryonic development. As notochord cells form nuclei pulposi, we propose that the notochord sheath functions as a "wrapper" around the notochord to constrain these cells along the vertebral column.

Comparative immunolocalisation of fibrillin-1 and perlecan in the human foetal, and HS-deficient hspg2 exon 3 null mutant mouse intervertebral disc.

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ³â»/Δ³â» exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ³â»/Δ³â» exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell-matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-ß a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.

The ovine newborn and human foetal intervertebral disc contain perlecan and aggrecan variably substituted with native 7D4 CS sulphation motif: spatiotemporal immunolocalisation and co-distribution with Notch-1 in the human foetal disc.

Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 µg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 µg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 µg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P < 0.001). Cell populations on the margins of the AF and cartilaginous vertebral rudiments in newborn ovine and human foetal IVD strongly expressed 7-D-4 CS epitope and perlecan, This was co-distributed with Notch-1 expression in human foetal IVDs consistent with the 7-D-4 CS sulphation motif representing a marker of tissue development expressed by disc progenitor cell populations.

Comparative immunolocalisation of perlecan, heparan sulphate, fibroblast growth factor-18, and fibroblast growth factor receptor-3 and their prospective roles in chondrogenic and osteogenic development of the human foetal spine.

PURPOSE: A comparative immunolocalisation study of perlecan, HS, FGF-18 and FGFR-3 in the 12-20-week gestational age human foetal spine was undertaken to identify spatiotemporal associations between these components to provide insights into prospective roles in spinal development. METHODS: Comparative immunolocalisations of matrix and cell associated components in Histochoice-fixed paraffin-embedded human foetal spinal tissues. RESULTS: The 12-14-week-old human foetal spine was a predominantly cartilaginous structure with the discs displaying a relative paucity of proteoglycan compared to the adjacent cartilaginous vertebral rudiments, notochordal remnants were also observed. HS and perlecan had a widespread distribution throughout the spine at 12 weeks, however, FGF-18 was only localised to the outer AF margins and hypertrophic cell condensations in the vertebral bodies. This contrasted with HS distributions at 14-20 weeks, which were prominent in the developing intervertebral disc (IVD). Ossification centres were also evident centrally within the vertebral rudiments surrounded by small columns of hypertrophic chondrocytes which expressed FGFR-3 and FGF-18 and upregulated levels of perlecan. FGF-18 also had a prominent localisation pattern in the developing IVD and the cartilaginous endplate while FGFR-3 was expressed throughout the disc interspace. This suggested roles for perlecan, FGF-18 and FGFR-3 in chondrogenic and osteogenic events which drive discal development and ossification of the vertebral bodies. CONCLUSIONS: The above data supported a role for FGF-18 in discal development and in the terminal osteogenic differentiation of chondroprogenitor cell populations, which promote vertebral ossification during spinal development.

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene.

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5' of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue.