Histology and histochemistry of the gekkotan notochord and their bearing on the development of notochordal cartilage

The persistence of the notochord into the skeletally mature life stage is characteristic of gekkotans, but is otherwise of rare occurrence among amniotes. The taxonomic diversity of Gekkota affords the opportunity to investigate the structure and development of this phylogenetically ancestral component of the skeleton, and to determine its basic characteristics. The gekkotan notochord spans almost the entire postcranial long axis and is characterized by a moniliform morphology with regularly alternating zones of chordoid and chondroid tissue. Chordoid tissue persists in the region of intervertebral articulations and occupies the cavitations that lie between the centra of the amphicoelous vertebrae. Chondroid tissue is restricted to zones in which the diameter of the notochord is reduced, corresponding to mid‐vertebral locations. In the tail, these zones of chondroid tissue are associated with the autotomic fracture planes. Chondroid tissue first manifests during late embryogenesis, appears to differentiate from pre‐existing chordoid tissue, and has the histological and histochemical characteristics of cartilage. Our observations lend support to the hypothesis that cartilage can be derived directly from notochordal tissue, and suggest that the latter may be an evolutionary and developmental precursor to chordate cartilage. The persistence of chordoid tissue in the intervertebral regions of amphicoelous vertebrae is consistent with a suite of paedomorphic traits exhibited by gekkotans and suggests that the typical hydrostatic nature of notochordal tissue may play a role in mechanically governing patterns of displacement between adjacent amphicoelous vertebrae that lack extensive centrum‐to‐centrum contact. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Notochord-dependent expression of MFH1 and PAX1 cooperates to maintain the proliferation of sclerotome cells during the vertebral column development.

During axial skeleton development, the notochord is essential for the induction of the sclerotome and for the subsequent differentiation of cartilage forming the vertebral bodies and intervertebral discs. These functions are mainly mediated by the diffusible signaling molecule Sonic hedgehog. The products of the paired-box-containing Pax1 and the mesenchyme forkhead-1 (Mfh1) genes are expressed in the developing sclerotome and are essential for the normal development of the vertebral column. Here, we demonstrate that Mfh1 like Pax1 expression is dependent on Sonic hedgehog signals from the notochord, and Mfh1 and Pax1 act synergistically to generate the vertebral column. In Mfh1/Pax1 double mutants, dorsomedial structures of the vertebrae are missing, resulting in extreme spina bifida accompanied by subcutaneous myelomeningocoele, and the vertebral bodies and intervertebral discs are missing. The morphological defects in Mfh1/Pax1 double mutants strongly correlate with the reduction of the mitotic rate of sclerotome cells. Thus, both the Mfh1 and the Pax1 gene products cooperate to mediate Sonic hedgehog-dependent proliferation of sclerotome cells.     

Remodeling of the notochord during development of vertebral fusions in Atlantic salmon (Salmo salar)

Histological characterization of spinal fusions in Atlantic salmon (Salmo salar) has demonstrated shape alterations of vertebral body endplates, a reduced intervertebral space, and replacement of intervertebral cells by ectopic bone. However, the significance of the notochord during the fusion process has not been addressed. We have therefore investigated structural and cellular events in the notochord during the development of vertebral fusions. In order to induce vertebral fusions, Atlantic salmon were exposed to elevated temperatures from fertilization until they attained a size of 15 g. Based on results from radiography, intermediate and terminal stages of the fusion process were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. Examination of structural extracellular matrix proteins such as Perlecan, Aggrecan, Elastin, and Laminin revealed reduced activity and reorganization at early stages in the pathology. Staining for elastic fibers visualized a thinner elastic membrane surrounding the notochord of developing fusions, and immunohistochemistry for Perlecan showed that the notochordal sheath was stretched during fusion. These findings in the outer notochord correlated with the loss of Aggrecan- and Substance-P-positive signals and the further loss of vacuoles from the chordocytes in the central notochord. At more progressed stages of fusion, chordocytes condensed, and the expression of Aggrecan and Substance P reappeared. The hyperdense regions seem to be of importance for the formation of notochordal tissue into bone. Thus, the remodeling of notochord integrity by reduced elasticity, structural alterations, and cellular changes is probably involved in the development of vertebral fusions.     

Vertebral column and associated elements in dipnoans and comparison with other fishes: development and homology.

A vertebral column consisting of a persistent notochord and ossified arcocentra is the primitive condition for Gnathostomata; it still persists in primitive actinopterygians and sarcopterygians. Advanced actinopterygians and sarcopterygians develop numerous types of centra that include, among others, the presence of holocentrum, chordacentrum, and autocentrum. The chordacentrum, a mineralization or calcification of the fibrous sheath of the notochord, is only found in actinopterygians, whereas an autocentrum is a synapomorphy of teleosts above Leptolepis coryphaenoides. The chordacentrum, formed by migration of cartilaginous cells from the arches into the fibrous sheath of the notochord and usually covered by a thin calcification, is a unique feature of chondrichthyans. The actinopterygian chordacentrum and the chondrichthyan chordacentrum are not homologous. The postcaudal cartilaginous centrum is only known in postcaudal vertebrae of living dipnoans. The holocentrum is present in certain fossil dipnoans and actinopterygians, where it has been independently acquired. It is formed by proliferation of cartilage cells around the elastica externa of the notochord. These cells later ossify, forming a compact centrum. A vertebral column formed by a persistent notochord without vertebral centra is the primitive pattern for all vertebrates. The formation of centra, which is not homologous among vertebrate groups, is acquired independently in some lineages of placoderms, most advanced actinopterygians, and some dipnoans and rhipidistians. Several series of structures are associated with the vertebral column such as the supraneurals, interhaemals, radials, and ribs. In living dipnoans median neural spine, "supraneural," and dorsal radial result from growth and distal differentiation of one median cartilage into two or three median bones during ontogeny. The median neural spine articulates with the neural arch and fuses with it in the caudal vertebrae early in ontogeny. Two bones differentiate in the anterior abdominal vertebrae, i.e., the proximal neural spine and the distal "supraneural." Three bones differentiate in front of the dorsal fin, i.e., the proximal neural spine, the middle "supraneural", and the distal radial; the same pattern is observed in front of the anal fin (the proximal haemal spine, the middle interhaemal, and the distal radial). Considering that the three dorsal (and also the three ventral) bones originate from growth of only one cartilage, they cannot be serial homologs of the neural spines, or "supraneural." They are linear homologs of the median neural cartilage in living dipnoans. The development of these elements differs within osteichthyans from sarcopterygians to actinopterygians, in which the neural spine originates as a continuation of the basidorsal arcualia and in which the supraneural and radial originate from independent cartilages that appear at different times during early ontogeny. The ribs of living dipnoans are unique in that they are not articulated with parapophyses, like in primitive fossil dipnoans, but a remnant of the ventral arcuale surrounded by a small arcocentrum remains at its base. A true caudal fin is absent in living dipnoans. The postcaudal cartilages extend to the caudal tip of the body separating dorsal and ventral rays (or the camptotrichia). Actinotrichia are present in young dipnoans. They are also known in extant actinistians and actinopterygians. They probably represent the primitive state for teleostomes. In contrast, the camptotrichia are unique for extant dipnoans (and probably Carboniferous and younger dipnoans). Lepidotrichia apparently developed many times among osteichthyans.     

Embryonic development of the axial column in the little skate,Leucoraja erinacea

The morphological patterns and molecular mechanisms of vertebral column development are well understood in bony fishes (osteichthyans). However, vertebral column morphology in elasmobranch chondrichthyans (e.g., sharks and skates) differs from that of osteichthyans, and its development has not been extensively studied. Here, we characterize vertebral development in an elasmobranch fish, the little skate, Leucoraja erinacea , using microCT, paraffin histology, and whole‐mount skeletal preparations. Vertebral development begins with the condensation of mesenchyme, first around the notochord, and subsequently around the neural tube and caudal artery and vein. Mesenchyme surrounding the notochord differentiates into a continuous sheath of spindle‐shaped cells, which forms the precursor to the mineralized areolar calcification of the centrum. Mesenchyme around the neural tube and caudal artery/vein becomes united by a population of mesenchymal cells that condenses lateral to the sheath of spindle‐shaped cells, with this mesenchymal complex eventually differentiating into the hyaline cartilage of the future neural arches, hemal arches, and outer centrum. The initially continuous layers of areolar tissue and outer hyaline cartilage eventually subdivide into discrete centra and arches, with the notochord constricted in the center of each vertebra by a late‐forming “inner layer” of hyaline cartilage, and by a ring of areolar calcification located medial to the outer vertebral cartilage. The vertebrae of elasmobranchs are distinct among vertebrates, both in terms of their composition (i.e., with centra consisting of up to three tissues layers—an inner cartilage layer, a calcified areolar ring, and an outer layer of hyaline cartilage), and their mode of development (i.e., the subdivision of arch and outer centrum cartilage from an initially continuous layer of hyaline cartilage). Given the evident variation in patterns of vertebral construction, broad taxon sampling, and comparative developmental analyses are required to understand the diversity of mechanisms at work in the developing axial skeleton of vertebrates. J. Morphol. 278:300–320, 2017. © 2017 Wiley Periodicals, Inc.     

MMP‐13 (Matrix MetalloProteinase 13) expression might be an indicator for increased ECM remodeling and early signs of vertebral compression in farmed Atlantic salmon (Salmo salar L.)

Vertebral body compression is a common problem in commercial farming of Atlantic salmon. Although risk factors, such as vaccination and malnutrition, have been identified, the etiology is largely unknown. Histological studies of Atlantic salmon have shown that in a compressed deformity (platyspondyly) the length of the compact bone is reduced while the notochord start to form atypical chrondrogenic structures. In mammals, similar remodeling activities have been linked to inflammatory processes in the tissue. Hence, we wanted to investigate whether the compressed vertebrae in Atlantic salmon showed presence of local (IL‐1β, TNF‐α1), systemic (IgM) and chronic (MMP‐13, MMP‐9) immune responses (measured with quantitative PCR). Unvaccinated groups of Atlantic salmon that would later develop high or low prevalence of vertebral compression during on‐growth in seawater were sampled at seawater transfer, and 3 and 6 weeks after seawater transfer. In addition, compressed and normal vertebrae from the high deformity prevalence group were sampled 44 weeks after transfer to seawater. MMP‐13 was significantly up‐regulated in the group that developed a high prevalence of deformity, and also significantly up‐regulated in compressed vertebrae, 44 weeks after seawater transfer. In compressed vertebrae, MMP‐13 was equally up‐regulated in the notochord, compact bone and trabecular bone. The results of the present study suggest that MMP‐13 may serve as an early indicator for bone remodeling which may lead to vertebral compression, and that there is a relationship between the development of vertebral compression and increased remodeling activities in farmed Atlantic salmon.     

Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model

To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.     

Does the correlation between schmorl's nodes and vertebral morphology extend into the lumbar spine?

Schmorl's nodes are depressions on vertebrae due to herniation of the nucleus pulposus of the intervertebral disc into the vertebral body. This study provides an extension of our previous study which analyzed the shape of the lower thoracic spine and found that vertebral morphology was associated with the presence of Schmorl's nodes. Ninety adult individuals from the late Medieval site of Fishergate House, York, and the Post‐Medieval site of Coach Lane, North Shields, Tyne and Wear, England, were analysed using 2D geometric morphometrics to identify possible relationships between vertebral morphology and Schmorl's nodes at the thoraco‐lumbar junction and in the lumbar spine. A significant correlation was found between vertebral shape and the presence of Schmorl's nodes in the twelfth thoracic vertebrae and the first to third lumbar vertebrae. The findings corroborate previous studies and suggest that vertebral shape may be an important factor in spinal health. It is hypothesized that the pedicle shape of affected vertebrae may not provide adequate structural support for the vertebral bodies, resulting in vertical disc herniation. Am J Phys Anthropol 157:526–534, 2015. © 2015 Wiley Periodicals, Inc.     

The ontogenic development of the vertebrae in some gekkonoid lizards

The ontogeny of amphicoelous vertebrae was studied in Ptyodactylus hasselquistii and Hemidactylus turcicus, and that of procoelous vertebrae, in Sphaerodactylus argus. The embryos were assigned arbitrary stages, drawn to scale, and mostly studied in serial sections. Resegmentation occurs as in all amniotes. A sclerocoel divides each sclerotome into an anterior “presclerotomite” and a denser posterior “postsclerotomite.” Tissue surrounding the intersegmental boundary forms the centrum, which is intersegmental. Tissue around the sclerocoel builds the intervertebral structures, which are midsegmental. In the trunk and neck, postsclerotomites form neural arches, and presclerotomites build zygapophyses. The adult centrum consists of the perichordal primary centrum, plus neural arch bases (= secondary centrum). Between the latter and the arch proper, a neurocentral suture persists until obliterated in maturity. A dorso-ventral central canal persists on either side of the primary centrum, between the latter and the secondary centrum. The notochord becomes true cartilage midvertebrally in all vertebrae, and elastic cartilage intervertebrally in the posterior caudal region. Elsewhere its characteristic tissue persists. Intervertebrally, cervical hypapophyses, caudal chevrons and chevron-bases in the trunk are preformed early in cartilage. Directly ossifying median intercentra are added later in all regions. The first cervical presclerotomite is absent: the hypapophysis (= corpus) of the atlas consists exclusively of postsclerotomitic tissue, there is no proatlas, and the odontoid lacks the apical half-centrum present in other lepidosaurians. In the autotomous caudal region presclerotomites are as prominent as postsclerotomites. Both build neural arches, the two arches of each vertebra remaining distinct and ossifying separately, so that the intersegmental autotomy split persists between them. The last sclerotome is complete, its postsclerotomite forming a half centrum which ossifies. In Sphaerodactylus, while the vertebrae ossify, each intervertebral ring becomes concave anteriorly, convex posteriorly; it remains as a cushion between the condyle and a facet formed by differential growth of the centra. Thus these procoelous centra resemble the amphicoelous centra of Ptyodactylus and Hemidactylus, rather than the procoelus centra of other squamates. The vertebral column of Gekkonoidea closely resembles in its development and microscopical structure that of Sphenodon.     

Collagen II Is Essential for the Removal of the Notochord and the Formation of Intervertebral Discs

Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II–deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice.Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that α1(XI) and α2 (XI) chains form unstable collagen XI molecules, demonstrating that the α3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.     

SOMITE CHONDROGENESIS : A Structural Analysis

Light and electron microscopy are used in this study to compare chondrogenesis in cultured somites with vertebral chondrogenesis These studies have also characterized some of the effects of inducer tissues (notochord and spinal cord), and different nutrient media, on chondrogenesis in cultured somites Somites from stage 17 (54–60 h) chick embryos were cultured, with or without inducer tissues, and were fed nutrient medium containing either horse serum (HS) and embryo extract (EE), or fetal calf serum (FCS) and F12X Amino acid analyses were also utilized to determine the collagen content of vertebral body cartilage in which the fibrils are homogeneously thin (ca. 150 Å) and unbanded. These analyses provide strong evidence that the thin unbanded fibrils in embryonic cartilage matrix are collagen. These thin unbanded collagen fibrils, and prominent 200–800 Å protein polysaccharide granules, constitute the structured matrix components of both developing vertebral cartilage and the cartilage formed in cultured somites Similar matrix components accumulate around the inducer tissues notochord and spinal cord. These matrix components are structurally distinct from those in embryonic fibrous tissue The synthesis of matrix by the inducer tissues is associated with the inductive interaction of these tissues with somitic mesenchyme. Due to the deleterious effects of tissue isolation and culture procedures many cells die in somitic mesenchyme during the first 24 h in culture. In spite of this cell death, chondrogenic areas are recognized after 12 h in induced cultures, and through the first 2 days in all cultures there are larger accumulations of structured matrix than are present in equivalently aged somitic mesenchyme in vivo. Surviving chondrogenic areas develop into nodules of hyaline cartilage in all induced cultures, and in most non-induced cultures fed medium containing FCS and F12X There is more cell death, less matrix accumulation, and less cartilage formed in cultures fed medium containing HS and EE. The inducer tissues, as well as nutrient medium containing FCS and F12X, facilitate cell survival, the synthesis and accumulation of cartilage matrix, and the formation of cartilage nodules in cultured somites.     

A central role for the notochord in vertebral patterning

The vertebrates are defined by their segmented vertebral column, and vertebral periodicity is thought to originate from embryonic segments, the somites. According to the widely accepted 'resegmentation' model, a single vertebra forms from the recombination of the anterior and posterior halves of two adjacent sclerotomes on both sides of the embryo. Although there is supporting evidence for this model in amniotes, it remains uncertain whether it applies to all vertebrates. To explore this, we have investigated vertebral patterning in the zebrafish. Surprisingly, we find that vertebral bodies (centra) arise by secretion of bone matrix from the notochord rather than somites; centra do not form via a cartilage intermediate stage, nor do they contain osteoblasts. Moreover, isolated, cultured notochords secrete bone matrix in vitro, and ablation of notochord cells at segmentally reiterated positions in vivo prevents the formation of centra. Analysis of fss mutant embryos, in which sclerotome segmentation is disrupted, shows that whereas neural arch segmentation is also disrupted, centrum development proceeds normally. These findings suggest that the notochord plays a key, perhaps ancient, role in the segmental patterning of vertebrae.     

Study in situ et in vivo of the acceleration of lumbar vertebrae of a primate exposed to vibration in the Z-axis

In order to study the propagation of vibration along the spine, the authors developed a method allowing in vivo et in situ measurement of vibrational accelerations in the lower lumbar spine of a primate.

The surgical technique used to anchor the miniature accelerometers on the anterior wall of vertebral bodies is described. After recovery, the sitting animal is exposed to a vibration stimulus. Direct recording of data obtained from the lowest four lumbar vertebrae of a healthy live animal provide objective information on the behavior of intervertebral discs and will later permit definition of the role of skeletal muscles in the spinal response to a vibrational stress.     

Age-related x-ray features of the spine in patients with achondroplasia

The paper studies the age-related X-ray features of the spine in patients with achondroplasia. It gives the time course of changes in the shape of vertebrae, the specific features of apophyseal ossification, provides a quantitative account of the shorter caudal lumbar vertebral arch root distance symptom. The time course of changes in the size of the lumbosacral angle was examined. The findings suggest that there are not only considerable static changes in the spine of patients with achondroplasia, but also significant age-related features of vertebral tissue growth and differentiation.     

Expression patterns and function of chromatin protein HMGB2 during mesenchymal stem cell differentiation

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers. Age-related loss of HMGB2 and gene deletion are associated with reduced SZ cellularity and early onset OA. This study addressed HMGB2 expression patterns in MSC and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared with human articular chondrocytes, and its expression declined during chondrogenic differentiation of MSC. Lentiviral HMGB2 transduction of MSC suppressed chondrogenesis as reflected by an inhibition of Col2a1 and Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2(-/-) mice, Col10a1 was more strongly expressed than in wild-type MSC. This is consistent with in vivo results from mouse growth plates showing that Hmgb2 is expressed in proliferating and prehypertrophic zones but not in hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis was also accelerated in Hmgb2(-/-) MSC. The expression of Runx2, which plays a major role in late stage chondrocyte differentiation, was enhanced in Hmgb2(-/-) MSC, and HMGB2 negatively regulated the stimulatory effect of Wnt/β-catenin signaling on the Runx2 proximal promoter. These results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The age-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations.