The Morphogenesis of Cranial Sutures in Zebrafish.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

Expression of glutathione peroxidase 1 in the spheno-occipital synchondrosis and its role in ROS-induced apoptosis.

BACKGROUND/OBJECTIVE: Chondrogenesis is an integral part of endochondral bone formation, by which the midline cranial base is developed. Reactive oxygen species (ROS) are required in chondrogenic differentiation and antioxidant enzymes regulate their levels. The aim of this study was to localize the antioxidant enzyme glutathione peroxidase 1 (Gpx1) at the spheno-occipital synchondrosis, as well as its effect on ROS challenge and its expression pattern in the course of differentiation. MATERIALS AND METHODS: Gpx1 was semiquantified in immunohistochemically stained sections of spheno-occipital synchondroses of rats. The effect of Gpx1 on ROS-induced apoptosis was investigated by manipulating the expression of Gpx1 in ATDC5 cells. The temporal pattern of Gpx1 expression was determined during chondrocyte differentiation for 21 days in vitro. RESULTS: Proliferating chondrocytes exhibited the greatest Gpx1 immunoreactivity and hypertrophic ones the lowest (P = 0.02). Cells transfected with Gpx1-siRNA had the highest apoptotic rate, while cells overexpressing Gpx1 the lowest one (P < 0.001). Gpx1 was significantly increased on days 10 (P = 0.02) and 14 (P = 0.01). CONCLUSIONS: Hypertrophic chondrocytes have the lowest Gpx1 activity in the spheno-occipital synchondrosis. Gpx1 is implicated in the ROS-induced apoptosis in chondrocytes. Its expression was not constitutive during chondrogenic differentiation.

Temporal expression of SOX9 and type II collagen in spheno-occipital synchondrosis of mice after mechanical tension stimuli.

OBJECTIVE: To associate the expressions of SOX9 and type II collagen during growth in the synchondrosis with and without tensile stress in order to understand the role of these factors in the growth of cartilage in spheno-occipital synchondrosis. MATERIALS AND METHODS: Sixty 1-day-old male BALB/c mice were randomly divided into experimental and control groups. Each group was subdivided again into five different time points which were 6, 24, 48, 72, and 168 hours. Each subgroup consisted of five mice. Each mouse was sacrificed using an overdose of pentobarbitone sodium. The synchondroses were aseptically removed and incubated in a 24-well plate with or without tensile stress in tissue culture. Tissue sections were stained immunohistochemically to quantitatively analyze the expression of SOX9 and type II collagen. RESULTS: There was a statistically significant increase of 57% (P < .001) in the expression of SOX9 between the experimental and control groups at 24 hours, followed by a significant increase of 44.4% (P < .001) in the expression of type II collagen at 72 hours. CONCLUSIONS: SOX9 may play an important role for early differentiation of chondrocytes and increase the expression of type II collagen, a major component of the extracellular matrix, during the growth of cartilage in the spheno-occipital synchondrosis.

Chondrocyte proliferation of the cranial base cartilage upon in vivo mechanical stresses.

Whereas the growth of the cranial base cartilage is thought to be regulated solely by genes, epiphyseal growth plates are known to respond to mechanical stresses. This disparity has led to our hypothesis that chondrocyte proliferation is accelerated by mechanical stimuli above natural growth. Two-Newton tensile forces with static and cyclic waveforms were delivered in vivo to the premaxillae of actively growing rabbits for 20 min/day over 12 consecutive days. The average number of BrdU-labeled chondrocytes in the proliferating zone treated with cyclic forces was significantly higher than both static forces of matching peak magnitude and sham controls representing natural chondral growth. Cyclic forces also evoked greater area of the proliferating zone than both static forces and sham controls. Thus, chondrocyte proliferation is enhanced by mechanical stresses in vivo, especially those with oscillatory waveform. Analysis of these data suggests that genetically coded chondral growth is up-regulated by mechanical signals.

Postparietal and prehatching ontogeny of the supraoccipital in Alligator mississippiensis (Archosauria, Crocodylia).

The first record of the postparietal bone of Alligator mississippiensis, documented by transverse histological sections, is presented. It is the first evidence of the presence of this bone within Recent reptiles. The postparietal is present in a specimen with a head length of 32.3 mm. The bone is a small dermal plate lying ventrally and posteriorly to the posterior margin of the parietal and dorsally to the trabecular bone, forming a dorsal surface of the supraoccipital portion of the neural endocranium. The trabecular bone develops perichondrally from the dorsal surface of the tectal cartilaginous bridge spanning between the dorsal portions of the otic capsules and occipital pilae. The bridge probably represents the fused tectum synoticum posterior plus tectum posterius. Later in ontogeny, the bridge ossifies endochondrally. The endochondrally ossifying bridge together with its perichondrally ossifying trabecular bone form the future supraoccipital. The trabecular bone is the integral part of the cranial endoskeleton and ontogenetically distinct from the dermal postparietal bone.

Hyaluronan is essential for the expansion of the cranial base growth plates.

Exquisite control of chondrocyte function in the zone of hypertrophy results in expansive growth of cartilaginous growth plates, and is a prerequisite for normal skeletal lengthening. We hypothesize that hyaluronan-mediated hydrostatic pressure causes lacunae expansion in the zone of hypertrophy; an important mechanism in cartilaginous growth plate and associated skeletal expansion. The role of hyaluronan and CD44 in this mechanism was studied using organ culture of the bipolar cranial base synchondroses. Hyaluronan was present in the hypertrophic zones, pericellular to the hypertrophic chondrocytes, while no hyaluronan was detected in the resting, proliferating and maturing zones. This localization of hyaluronan was associated with increased lacunae size, suggesting that chondrocytes deposit and retain pericellular hyaluronan as they mature. In comparison, Toluidine Blue staining was associated with the territorial matrix. Hyaluronidase, the hyaluronan-degrading enzyme, and CD44, the receptor for hyaluronan which also participates in the uptake and degradation of hyaluronan, were co-localized within the zone of ossification. This pattern of expression suggests that cells in the early zone of ossification internalize and degrade hyaluronan through a CD44-mediated mechanism. Treatment of the cultured segments with either Streptomyces hyaluronidase or hyaluronan hexasaccharides inhibited lacunae expansion. These observations demonstrate that hyaluronan-mediated mechanisms play an important role in controlling normal skeletal lengthening.

Regional distribution of presenilin-1 messenger RNA in the embryonic rat brain: comparison with beta-amyloid precursor protein messenger RNA localization.

The messenger RNA expression of presenilin-1, an important gene responsible for early-onset familial Alzheimer's disease, was investigated in the embryonic rat brain with in situ hybridization histochemistry using an oligonucleotide probe specific to the messenger RNA. It was also compared with that of beta-amyloid precursor protein messenger RNA. Presenilin-1 and beta-amyloid precursor protein messenger RNA were abundantly expressed throughout the central nervous system in the embryonic day 13, 17 and 20 rat brain. Presenilin-1 messenger RNA was strongly expressed in both neuroepithelium and differentiating fields. In contrast, beta-amyloid precursor protein messenger RNA was preferentially expressed in differentiating fields, while low expression of beta-amyloid precursor protein messenger RNA was seen in neuroepithelium. Although the expression patterns of these two messenger RNAs were basically similar, there seemed to be a tendency that presenilin-1 messenger RNA was preferentially expressed in immature neurons, while beta-amyloid precursor protein messenger RNA was preferentially expressed in mature neurons, suggesting that presenilin-1 is expressed earlier than beta-amyloid precursor protein and that presenilin-1 is involved in beta-amyloid precursor protein processing. These data raise the possibility that presenilin-1 and beta-amyloid precursor protein co-operatively play pivotal roles in rat neurogenesis.

Occipitocervical segmentation in staged human embryos.

Serial sections of 108 human embryos from stage 11 to stage 23 were investigated, and 33 reconstructions were prepared. The existence of 4 occipital somites was confirmed. The important developmental distinction between axial (central) and lateral components obtains in the occipital as well as in the vertebral region. The lateral occipital components begin to show dense areas as the cervical region is approached. The lateral occipital and vertebral components arise in registration with the initial sclerotomes. In both the occipital and the vertebral region the related nerves and intersegmental arteries traverse the loose areas of the sclerotomes. The axial occipital region is not segmented, whereas the cervical components develop from perinotochordal loose areas. Three complete centra (known as XYZ) develop in the atlanto-axial region, although they are related to only 2 1/2 sclerotomes and only 2 neural arches. The height of the XYZ complex equals that of 3 centra elsewhere, and not 2 1/2, as previously maintained. The experimental findings in the occipitocervical region of the chick embryo show both similarities to, as well as differences from, the data for the human embryo. A scheme showing the early development of the entire vertebral column is included.

The GAGs of the bone: a study on human calva.

The glycosaminoglycans of the human bone are Chondroitin-4 sulfate, Chondroitin-6 sulfate and Keratosulfate. Their quantity presents variations with ageing. The Keratosulfate shows a prevalent localisation in the bone matrix just around the osteocytes.

[Spheno-occipital synchondrosis--a fluorescence and polarization microscopy study in Cercopithecus aethiops monkeys]. / Die Synchondrosis sphenooccipitalis--eine fluoreszenz- und polarisationsmikroskopische Untersuchung am Cercopithecus-aethiops-Affen.

On 17 Cercopithecus aethiops monkeys we investigated with the method of the polychromic sequential dye marking system the histomorphology as well as the dynamics of growth and calcification of the spheno-occipital synchondrosis. The age of the animals ranged from change to teeth to late adolescence. The animals had been divided into four different groups: I: late change of teeth; II: young adult; III: fully grown; IV: late adolescence. In the first group the spheno-occipital synchondrosis showed no ossification in the gap which is filled with cartilage. It showed the characteristic structure with a central zone of equally distributed chondrocytes. Adjacent to this we found a zone of proliferation cell hypotrophy and cell degeneration. With increasing age there is decrease of the density of cells (groups II to IV) and after change to teeth the synchondrosis starts to ossify (group II). Due to the ossification the synchondrosis subdivides into different cartilage regions. We found that the closing of the synchondrosis started in the cranial region and progressed toward the caudal region. During this procedure the synchondrosis never ossified completely. Several cartilage regions persisted uncalcified until late adolescence. Interstitial growth of the synchondrosis was found until the end of the change of the teeth (group I). This growth which was always found in a sagittal direction ceased after bony connections had been formed between both poles of the synchondrosis. The sphenoidal and occipital pole of the synchondrosis showed equal growth potential.

The effect of parathyroid hormone (1-34) on cyclic AMP level, ornithine decarboxylase activity, and glycosaminoglycan synthesis of chondrocytes from mandibular condylar cartilage, nasal septal cartilage, and spheno-occipital synchondrosis in culture.

Previously, we reported methods for isolating chondrocytes from the craniofacial complex and their culture in vitro. The response of these chondrocyte cultures to bovine parathyroid hormone (1-34) (PTH) has now been investigated. PTH stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype in cultured chondrocytes isolated from mandibular condylar cartilage (MCC), nasal septal cartilage (NSC), and spheno-occipital synchondrosis (SOS). These stimulations of GAG synthesis by PTH were dose-dependent. PTH also increased accumulation of cyclic AMP (cAMP) and the activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis. However, PTH did not stimulate DNA synthesis. The increases in the cAMP level, ODC activity, and GAG synthesis after addition of PTH (10(-7) mol/L) were greatest in MCC-chondrocytes and least in NSC-chondrocytes. The difference in the responses to PTH of these three types of chondrocytes may reflect differences of the characteristics of these cells in vivo.

Studies on chondrocytes from mandibular condylar cartilage, nasal septal cartilage, and spheno-occipital synchondrosis in culture. I. Morphology, growth, glycosaminoglycan synthesis, and responsiveness to bovine parathyroid hormone (1-34).

Methods for isolating chondrocytes from the craniofacial complex and culturing them in vitro were established. Chondrocytes which were isolated by collagenase digestion from mandibular condylar cartilage, nasal septal cartilage, and spheno-occipital synchondrosis grew well in vitro. All three types of chondrocytes actively synthesized glycosaminoglycans, a differentiated phenotype of chondrocytes, and responded well to parathyroid hormone. However, some different characteristics were noted among the three types of chondrocytes in culture.