Environmental regulation of notochordal gene expression in nucleus pulposus cells

Cells of the nucleus pulposus (NP) in the intervertebral disc are derived directly from the embryonic notochord. In humans, a shift in NP cell population coincides with the beginning of age‐related changes in the extracellular matrix that can lead to spinal disorders. To begin identifying the bases of these changes, the manner by which relevant environmental factors impact cell function must be understood. This study investigated the roles of biochemical, nutritional, and physical factors in regulating immature NP cells. Specifically, we examined cell morphology, attachment, proliferation, and expression of genes associated with the notochord and immature NP (Sox9, CD24, and type IIA procollagen). Primary cells isolated from rat caudal discs were exposed to different media formulations and physical culture configurations either in 21% (ambient) or 2% (hypoxic) O₂. As expected, cells in alginate beads retained a vacuolated morphology similar to chordocytes, with little change in gene expression. Interestingly, NP tissues not enzymatically digested were more profoundly influenced by oxygen. In monolayer, α‐MEM preserved vacuolated morphology, produced the highest efficiency of attachment, and best maintained gene expression. DMEM and Opti‐MEM cultures resulted in high levels of proliferation, but these appeared to involve small non‐vacuolated cells. Gene expression patterns for cells in DMEM monolayer cultures were consistent with chondrocyte de‐differentiation, with the response being delayed by hypoxia. Overall, results indicate that certain environmental conditions induce cellular changes that compromise the notochordal phenotype in immature NP. These results form the foundation on which the mechanisms of such changes can be elucidated. J. Cell. Physiol. 220: 698–705, 2009. © 2009 Wiley‐Liss, Inc.     

Morpho‐histological and ultra architectural changes during early development of endangered golden mahseer Tor putitora

Ultrastructural and histological changes in the embryonic and larval surface during ontogenesis of the endangered golden mahseer Tor putitora is studied here for the first time. Embryonic development was completed 91–92 h after fertilization at an ambient temperature of 23° ± 1° C (mean ± s.d. ). The gastrula stage was characterized by presence of the Kupffer's vesicle, notochord, ectoderm and endoderm cells. Primordial germ cells were clearly identifiable from c. 55 h post‐fertilization at the organogenesis stage. Mean total length of newly hatched larvae was 7·0 ± 0·5 mm. Scanning electron microscopy of newly hatched larvae demonstrated vitelline arteries, microridged epithelial cells and mucous gland openings over much of the body surface. Eye, oral cavity, pharyngeal arches, heart, intestinal loop, prosencephalon, cephalic vesicle and nasal epithelium were clearly distinguished in 3 day old hatched individuals. In 6 day old individuals, caudal‐fin rays and internal organs were evident. The dorsal fin became prominent at this stage and larvae began swimming at the surface. The reserved yolk material was totally absorbed 8–11 days after hatching and larvae began feeding exogenously. Tor putitora exhibited a longer early developmental period than other cyprinids reared at similar temperatures.     

Morphology,mechanics, and locomotion: the relation between the notochord and swimming motions in sturgeon

Synopsis To examine the relation between morphology and performance, notochordal morphology was correlated with notochordal mechanics and with steady swimming motions in white sturgeon, Acipenser transmontanus. In a still-water tank, motions of four sturgeon varied with changes in swimming speed and axial position along the body. For a 1..34 m sturgeon, slow and fast swimming modes were distinguished, with speeds at the fast mode more than two times those at the slow mode without changes in tailbeat frequency. This increase in speed is correlated with an increase in the body's maximal midline curvature (m^–1), suggesting a role for curvature-related mechanical properties of the notochord. Maximal midline curvature also varied with axial position, and surprisingly was uncorrelated with axial changes in the notochord's cross-sectional shape - as measured by height, width, inner diameter, and lateral thickness of the sheaths. On the other hand, maximal midline curvature was negatively correlated with the axial changes in the notochord's angular stiffness (N m rad^–1) and change in internal pressure (% change from baseline of 58.6 kPa), both of which were measured during in vitro bending tests. In vivo curvature and in vitro angular stiffness were then used to estimate the bending moments (Nm) in the notochord during swimming. In the precaudal notochord, the axial pattern of maximal stiffness moments was congruent with the pattern of maximal notochordal curvature in the precaudal region, but in the caudal notochord maximal angular stiffness was located craniad to maximal curvature. One interpretation of this pattern is that the precaudal notochord resists bending moments generated by the muscles and that the caudal notochord resists bending moments generated by hydrodynamic forces acting on the tail.     

Histological and histochemical characteristics of the bovine notochord.

In 20 bovine embryos and fetuses 6-65 mm long (crown-rump length) and 23 to 60-70 days old, the structure and localization of acid and neutral mucopolysaccharides and glycogen in their notochord were investigated. Also, the localization in the notochord was examined of the activity of alkaline and acid phosphatases, alpha-glycerophosphate-, glucose-6-phosphate-, isocitrate-, glutamate-, lactate- and succinic- dehydrogenases, and nicotinamide-adenine-dinucleotide- and nicotinamide-adenine-dinucleotide-phosphate- diaphorases. It was found that the bovine notochord begins decomposing at the end of embryonal and the beginning of fetal development (45-50 days old) and that in the fetus aged 55-65 days it no longer represents an unbroken cord of notochordal cells. Secretory activity of notochordal cells which produce the notochordal sheath starts very early (in 10 mm-long embryos), and interruptedly increases up to the end of the embryonal developmental period when regression appears at the beginning of the fetal period. These findings agree with findings in the human embryo where, however, they relate to earlier developmental periods.     

The ontogeny of the alimentary tract of larval pandora, Pagellus erythrinus L.

The ontogenesis of the alimentary tract and its associated structures (liver, pancreas, gall bladder) was studied in common pandora Pagellus eythrinus L., a promising species for diversification in Mediterranean aquaculture. Mass production of pandora has been limited so far by high larval and juvenile mortalities, which appear to be related to nutritional deficiencies. The development of the larval digestive system was studied histologically from hatching (0 DAH) until day 50 (50 DAH) in reared specimens, obtained by natural spawning from a broodstock adapted to captivity. At first feeding (3–4 DAH) both the mouth and anus had opened and the digestive tract was differentiated in four portions: buccopharynx, oesophagus, incipient stomach and intestine. The pancreas, liver and gall bladder were also differentiated at this stage. Soon after the commencement of exogenous feeding (5–6 DAH), the anterior intestinal epithelium showed large vacuoles indicating the capacity for absorption of lipids, whereas acidophilic supranuclear inclusions indicating protein absorption were observed in the posterior intestinal epithelium. Both the bile and main pancreatic ducts had opened in the anterior intestine, just after the pyloric sphincter, at this stage. Intestinal coiling was apparent since 4 DAH, while mucosal folding began at 10 DAH. Scattered mucous cells occurred in the oral cavity and the intestine, while they were largely diffused in the oesophagus. Gastric glands and pyloric caeca were firstly observed at 28 DAH and appeared well developed by 41 DAH, indicating the transition from larval to juvenile stage and the acquisition of an adult mode of digestion.     

Wnt signaling maintains the notochord fate for progenitor cells and supports the posterior extension of the notochord

The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific β-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs’ notochordal fate.     

The Effect of Adriamycin Exposure on the Notochord of Mouse Embryos

The notochord has important structural and signaling properties during vertebrate development with key roles in patterning surrounding tissues, including the foregut. The adriamycin mouse model is an established model of foregut anomalies where exposure of embryos in utero to the drug adriamycin leads to malformations including oesophageal atresia and tracheoesophageal fistula. In addition to foregut abnormalities, treatment also causes branching, displacement, and hypertrophy of the notochord. Here, we explore the hypothesis that the notochord may be a primary target of disruption leading to abnormal patterning of the foregut by examining notochord position and structure in early embryos following adriamycin exposure. Treated (n = 46) and control (n = 30) embryos were examined during the crucial period when the notochord normally delaminates away from the foregut endoderm (6–28 somite pairs). Transverse sections were derived from the anterior foregut and analyzed by confocal microscopy following immunodetection of extracellular matrix markers E‐cadherin and Laminin. In adriamycin‐treated embryos across all stages, the notochord was abnormally displaced ventrally with prolonged attachment to the foregut endoderm. While E‐cadherin was normally detected in the foregut endoderm with no expression in the notochord of control embryos, treated embryos up to 24 somites showed ectopic notochordal expression indicating a change in characteristics of the tissue; specifically an increase in intracellular adhesiveness, which may be instrumental in structural changes, affecting mechanical and signaling properties. This is consistent with disruption of the notochord leading to altered signaling to the foregut causing abnormal patterning and congenital foregut malformations.     

Presence of cilia in the chick embryonic notochord

Cilia have been observed in cells of the chick embryonic notochord in different developmental stages. The ciliated notochordal cells were present both at the center and at the periphery of the organ. A well outlined central cavity in the notochord was not observed.     

The notochordal sheath in amphioxus--an ultrastructural and histochemical study

The notochord and notochordal sheath of 10 adult amphioxus were investigated ultrastructurally and histochemically. The notochord in amphioxus consists of parallel notochordal cells (plates) and each plate consists of parallel thicker and thinner fibrils and numerous profiles of smooth endoplasmic reticulum situated just beneath the cell membrane. Histochemical staining shows that the notochordal plates resemble neither the connective tissue notochordal sheath nor the typical muscular structure myotomes. The notochordal sheath has a complex three-layered organization with the outer, middle and inner layer The outer and middle layer are composed of collagen fibers of different thickness and course, that correspond to collagen type I and collagen type III in vertebrates, respectively, and the inner layer is amorphous, resembles basal lamina, and is closely attached to the notochord by hemidesmosome junctions. These results confirm the presence of collagen fibers and absence of elastic fibers in amphioxus.     

Development of the digestive tract in first feeding larvae of Betta splendens Regan, 1910

The aim of this study was to analyse the development of the digestive tract of Siamese fighting fish larvae (Betta splendens Regan, 1910), from hatching to 92 hours post‐hatching (hph) at 28.0°C, in order to determine the most appropriate time to begin exogenous feeding (live feed) and to investigate the digestive tract and its function during larval development. At hatching (29 hours post‐fertilization), the digestive tract was a simple and straight undifferentiated tube lined by a single layer of columnar epithelial cells; the mouth and anus were closed. At 18 hph, the mouth was open. At 32 hph, the tongue was located in the depression of the buccopharyngeal cavity floor and supported by basal cartilage. Goblet cells were present in the epithelium lining the pharynx and oesophagus. At 56 hph, the midgut had four distinct layers: the mucosa with typical villi, the submucosa, the smooth muscle layer, and the serosa. Histological analysis revealed that the larvae retained endogenous yolk reserves until 74 hph. Lipid accumulation was observed in the liver, which coincided with complete yolk absorption. At this time, the digestive tract was fully open and functional. Thus, it can be concluded that live feed should be given to B. splendens larvae from 74 hph onward, when the larvae are able to consume the food provided. This study also provides useful data for the improvement of husbandry techniques and for the formulation of diets specific to ornamental fish larvae.     

Inductive interactions required for mesodermal differentiation in Bufo arenarum gastrulae

 Dorso-marginal epithelium, a distinct crescent-shaped region in the early gastrula of Bufo arenarum, appears after involution as a narrow dorso-median strip of archenteric endoderm close to the notochord. In explant cultures, this layer showed an extreme dorsalizing activity, promoting the formation of notochordal structures from ventro-mesodermal cells. In the presence of ectoderm, this inductive activity was expanded resulting in a wide range of dorso-lateral components such as notochord, muscle, nephric tubules, mesothelium and mesenchyme. The mesodermal origin of these derivatives was confirmed by the use of FDA (fluorescein-dextran-amine)-labelled explants. Extensive mesodermal development in cultures seems to require cell contacts between the inner aspect of the dorso-marginal epithelium and mesodermal cells. When such contacts were prevented, cultures would only differentiate erythrocytes as a result of a purely ectodermal stimulus. Bisection of the dorso-marginal epithelium in whole embryos resulted in the development of a duplicated set of axial structures, clearly showing the role of the epithelium as a dorsal organizer. Received: 30 July 1996 / Accepted: 20 February 1997     

Intermediate filament typing of the human embryonic and fetal notochord

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.     

Role of morphogens in brain growth

During the last century, experiments on the chick embryo established that the ballooning expansion of the early forebrain and midbrain vesicles is dependent on the underlying axial (notochordal) mesoderm. Transient separation of the early midbrain primordium from the notochord causes subsequent collapse of both midbrain and forebrain (telencephalic) vesicles, accompanied by pronounced folding of the neural epithelium. More recent experiments have shown that vesicle collapse is caused by defective Sonic Hedgehog (Shh) signaling from the notochord and floor plate. Separation of the notochord from the brain causes loss of ventral Shh expression, resulting in reduced cell proliferation and increased cell death in the expanding neural epithelium, and culminating in vesicle collapse. These experiments are reviewed here, and set in the context of other studies illustrating the wide range of molecular and cellular processes that cause abnormal brain morphogenesis when perturbed. We also speculate that variation in the regulation of signaling pathways such as Hedgehog may have played a significant part in generating rapid morphogenetic changes during the evolution of the vertebrate brain.     

In vitro development of rabbit pronuclear embryos in rabbit peritoneal fluid

The use of rabbit peritoneal fluid (PF) for the culture of rabbit embryos in vitro was evaluated. Development of zygotes cultured in PF and Earle's balanced salts solution (EBSS) + 10% fetal calf serum (EBSS/FCS) was compared. The effects of increasing the concentration of PF in EBSS and of culturing embryos in fractionated PF were also investigated. In addition, embryonic development in PF was compared to that in vivo. Development to hatching blastocysts was enhanced with PF (73%) compared to EBSS/FCS (3%, p less than 0.001). PF manifested greater mitogenic activity than EBSS/FCS, as indicated by higher cell number in embryos at 48, 72, and 96 h post-mating/hCG (p less than 0.001). PF also promoted blastocyst cell proliferation in a dose-dependent manner (r = 0.98, p less than 0.01); however, embryo growth remained slower than in vivo. Culture in the high (greater than 30,000 Da) molecular mass fraction of PF reduced incidence of hatching (56% vs. 92%, p less than 0.001) and mean cell number in Day 4 blastocysts (151 +/- 4 vs. 243 +/- 5, p less than 0.001). Rates of blastocyst hatching (10%) and cell number (110 +/- 3) were further reduced in the low (less than 30,000 Da) molecular mass fraction. When the high molecular mass fraction was dialyzed, embryos did not develop beyond the early morula stage. This suggests that the interaction or the synergy of high and low molecular mass components of PF is necessary for optimum development of rabbit embryos.     

Notochordal cell disappearance and modes of apoptotic cell death in a rat tail static compression-induced disc degeneration model

Introduction

The intervertebral disc has a complex structure originating developmentally from both the mesenchyme and notochord. Notochordal cells disappear during adolescence, which is also when human discs begin to show degenerative signs. During degeneration later in life, disc cells decline because of apoptosis. Although many animal models have been developed to simulate human disc degeneration, few studies have explored the long-term changes in cell population and phenotype. Our objective was to elucidate the time-dependent notochordal cell disappearance and apoptotic cell death in a rat tail static compression-induced disc degeneration model.

Methods

Twenty-four 12-week-old male Sprague–Dawley rat tails were instrumented with an Ilizarov-type device and loaded statically at 1.3 MPa for up to 56 days. Loaded and distal-unloaded discs were harvested. Changes in cell number and phenotype were assessed with histomorphology and immunofluorescence. Apoptosis involvement was determined with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and immunohistochemistry.

Results

The number of disc nucleus pulposus and annulus fibrosus cells decreased with the loading period; particularly, the decrease was notable at day 7 in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, indicating notochordal origin. Subsequently, the proportion of cells positive for TUNEL and cleaved caspase-3, markers of apoptosis induction, increased from day 7 through day 56. Although the percentage of cells immunopositive for cleaved caspase-8, a marker of apoptosis initiation through the death-receptor pathway, increased only at day 7, the percentage of cells immunopositive for cleaved caspase-9 and p53-regulated apoptosis-inducing protein 1 (p53AIP1), markers of apoptosis initiation through the p53-mediated mitochondrial pathway, increased from day 7 through day 56. The percentage of cells immunopositive for B-cell lymphoma 2 (Bcl-2) and silent mating type information regulation 2 homolog 1 (SIRT1), antiapoptotic proteins, decreased consistently with compression.

Conclusions

This rat tail model mimics notochordal cell disappearance and apoptotic cell death in human disc aging and degeneration. Sustained static compression induces transient activation of apoptosis through the death-receptor pathway and persistent activation of apoptosis through the p53-mediated mitochondrial pathway in disc cells. The increased proapoptotic and decreased antiapoptotic proteins observed at all time points signify static compression-induced disc cell death and degeneration.     

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.     

Reproductive behaviour and early development of the Drysdale hardyhead, Craterocephalus sp. nov. (Pisces: Atherinidae), from the Alligator Rivers System, Northern Territory, Australia

Craterocephalus sp. nov. showed sexual dimorphism in body shape during the breeding season. Pairing occurred during daylight with a single release of eggs amongst submerged vegetation between sunrise and the early afternoon on the same day. The eggs were demersal, adhesive, spherical (0.87–0.96 mm diameter) and had 12 adhesive filaments (0.5–1.5 mm long) at the animal pole. Approximately 24 oil droplets (0.02–0.08 mm diameter) persisted throughout egg and larval development. Hatching occurred 155–160 h after spawning at 25–27° C.
The yolk-sac larvae were 3.85–3.95 mm notochord length at hatching and began feeding at the surface after absorption of the yolk (3–12 h after hatching). All fin rays were developed in 9.4 mm standard length fry, which moved from midwater to feed on the substrate. Aquarium reared fish first spawned at 30 mm s.L. when 165 days of age.
Features of Craterocephalus reproduction, as they relate to a specific survival strategy, are briefly discussed.     

Early ontogeny of Ancherythroculter nigrocauda and effects of delayed first feeding on larvae

Development of embryos and larvae in Ancherythroculter nigrocauda Yih et Woo (1964) and effects of delayed first feeding on larvae were observed after artificial fertilization. The fertilized eggs were incubated at an average temperature of 26.5°C (range: 25.7–27) and the larvae reared at temperatures ranging from 21.8 to 28°C. First cleavage was at 50 min, epiboly began at 7 h 5 min, heartbeat reached 72 per min at 24 h 40 min and hatching occurred at 43 h 15 min after insemination. Mean total length of newly hatched larvae was 4.04 ± 0.03 mm (n = 15). A one‐chambered gas bladder was observed at 70 h 50 min, two chambers occurred at 15 days, and scales appeared approximately 30 days after hatching. Larvae began to feed exogenously at day 4 post‐hatch at an average temperature of 24°C. Food deprivation resulted in a progressive atrophy of skeletal muscle fibres, deterioration of the larval digestive system and cessation of organ differentiation. Larval growth under food deprivation was significantly affected by the time of first exogenous feeding. Starved larvae began to shrink, with negative growth from day 6 post‐hatch. The point of no return (PNR) was reached at day 11 after hatching. Mortality of starved larvae increased sharply from day 12 after hatching.     

The effect of aging on the neural competence of the presumptive ectoderm and the effect of aged ectoderm on the differentiation of the trunk organizer inCynops pyrrhogaster

Summary The effect of aging on the neural competence of the presumptive ectoderm of the early gastrula, and the effect of aged ectoderm on the differentiation of the still uninvaginated dorsal blastoporal lip at the small yolk-plug stage — representing the trunk organizer — were examined by the sandwich method inCynops pyrrhogaster.The presumptive ectoderm to be used as reaction system was taken from 0 to 36 h exogastrulae obtained by operation at the early gastrula stage and combined with trunk organizer. In the 0 to 12 h explants typical trunktail structures were formed. With further aging of the presumptive ectoderm a decrease in frequency of spinal cord, notochord, and muscle and a simultaneous increase in frequency of mesenchyme and mesothelium were observed. In the 30 and 36 h explants neural competence had largely disappeared, the frequency of notochord and muscle become very low and their differentiation very poor, whereas the frequency of mesenchyme and mesothelium reached very high levels.We infer a reciprocal relationship between the induced spinal cord and the differentiation of notochord and muscle, as well as a transformation of notochordal material into mesenchyme and mesothelium under the influence of the aged ectoderm. The mode of action of the trunk organizer in normal development is discussed.     

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.