The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation.Introduction: Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates. Materials and Methods: Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization. Results: Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter. Conclusion: Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones.
During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and -9 and the membrane gelatinase MMP-14.
We have generated transgenic mice harboring the deletion of exon 48 in the mouse alpha1(II) procollagen gene (Col2a1). This was the first dominant negative mutation identified in the human alpha1(II) procollagen gene (COL2A1). Patients carrying a single allele with this mutation suffer from a severe skeletal disorder called spondyloepiphyseal dysplasia congenita (SED). Transgenic mice phenotype was neonatally lethal with severe respiratory failure, short bones, and cleft palate. Transgene mRNA was expressed at high levels. Growth plate cartilage of transgenic mice presented morphological abnormalities and reduced number of collagen type II fibrils. Chondrocytes carrying the mutation showed altered expression of several differentiation markers, like fibroblast growth factor receptor 3 (Fgfr3), Indian hedgehog (Ihh), runx2, cyclin-dependent kinase inhibitor P21CIP/WAF (Cdkn1a), and collagen type X (Col10a1), suggesting that a defective extracellular matrix (ECM) depleted of collagen fibrils affects chondrocytes differentiation and that this defect participates in the reduced endochondral bone growth observed in chondrodysplasias caused by mutations in COL2A1.
PurposeTo assess disease-related patterns of in vivo pathology in 11 patients with Corticobasal Syndrome (CBS) compared to 20 healthy controls and 33 mild cognitive impairment (MCI) patients due to Alzheimer’s disease.MethodsWe assessed tau aggregates with [18F]AV1451 PET, amyloid-β depositions with [18F]AV45 PET, and volumetric microstructural changes with MRI. We validated for [18F]AV1451 standardised uptake value ratio (SUVRs) against input functions from arterial metabolites and found that SUVRs and arterial-derived distribution volume ratio (DVRs) provide equally robust measures of [18F]AV1451 binding.ResultsCBS patients showed increases in [18F]AV1451 SUVRs in parietal (P < 0.05) and frontal (P < 0.05) cortices in the affected hemisphere compared to healthy controls and in precentral (P = 0.008) and postcentral (P = 0.034) gyrus in the affected hemisphere compared to MCI patients. Our data were confirmed at the histopathological level in one CBS patient who underwent brain biopsy and showed sparse tau pathology in the parietal cortex co-localizing with increased [18F]AV1451 signal. Cortical and subcortical [18F]AV45 uptake was within normal levels in CBS patients. In parietal and frontal cortices of the most affected hemisphere we found also grey matter loss (P < 0.05), increased mean diffusivity (P < 0.05) and decreased fractional anisotropy (P < 0.05) in CBS patients compared to healthy controls and MCI patients. Grey matter loss and white matter changes in the precentral gyrus of CBS patients were associated with worse motor symptoms.ConclusionsOur findings demonstrate disease-related patterns of in vivo tau and microstructural pathology in the absence of amyloid-β, which distinguish CBS from non-affected individuals and MCI patients.Electronic supplementary materialThe online version of this article (10.1007/s00259-018-4104-2) contains supplementary material, which is available to authorized users.
ExtractThe dipeptidase activity toward L-glutaminyl-L-proline and glycyl-L-proline has been studied in the small intestinal mucosa of human fetuses and newborns between 14 and 36 weeks of fetal age.The small intestine was removed within 15 minutes of death. I t was immediately frozen and used for enzymatic studies within 2 to 30 days. The activity of both enzymes was stable for as long a period as three months at a temperature of -20". Mucosal scrapings were homogenized in 0.01 M Tris-HC1 buffer, p H 7, and then centrifuged at 1500 x g for 10 minutes. The supernatant, which contained more than 95 % of the enzyme activity, was used for assay; it was incubated in 20 m M substrate, at optimum pH, under conditions permitting calculation of initial velocity. Free proline was measured at the end of the incubation period. By the 14th to 16th week of age, levels of activity of both enzymes were comparable with those of the adult ( fig. 1).In order to investigate the distribution of dipeptidase activity, the small bowel Gom fetuses between 22 and 34 weeks of age was examined. Each sample was homogenized separately before studying. Uniformly high levels of dipeptidase activity were found throughout the proximal six-tenths of the small bowel, while lower values were observed in the terminal ileum ( fig. 2). A significant difference in the activity of both enzymes was found between the proximal and the distal third of the small bowel when calculated according to Student's t-test, p <0.02 (table I). The activity ratios were constant throughout the length of the intestine during the entire age span studied.After centrifugation of the homogenate of intestine a t 105,000 x g for one hour, gel filtration on Sephadex G 200 of the supernatant resulted in the partial purification of the enzyme, as indicated by a 4.5-to 6.5-fold increase in specific activity.The enzymes of the crude and the partially purified extracts were studied to determine the influence on enzyme activity of pH, Co++ and M n + + ions, heat treatment, substrate concentrations. Co++ and Mnf + ions clearly activated hydrolysis ofglycyl-L-proline, but did not affect that of L-glutaminyl-L-proline. Heating at 40" in a solution of 0.002 M Mnf + resulted in an increase of activity of glycyl-L-proline dipeptidase and the nearly complete disappearance of that of L-glutaminyl-L-proline dipeptidase (fig. 5) ; however, the activity of both enzymes was not separated by gel filtration, ammonium sulphate precipitation, or DEAE cellulose chromatography. Further studies are necessary to determine if a specific enzyme (s) of the small bowel mucosa, other than glycyl-L-proline dipeptidase (EC.3.4.3.7), causes hydrolysis of L-glutaminyl-L-proline.Glycyl-L-proline dipeptidase activity did not change during the age period studied; however, L-glutaminyl-L-proline dipeptidase activity of 14-to 24-week-old fetuses displayed a p H dependence a n d a kinetic versus substrate concentration not observed in fetuses from 26 to 36 weeks of age (fig.4). These results may suggest that a qualitat...
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