alcian-blue and alizarin

Topics: Alcian Blue; Animals; Anthraquinones; Bone and Bones; Cartilage; Ethanol; Formaldehyde; Glycerol; Hydroxides; Morphogenesis; Potassium Compounds; Staining and Labeling; Tissue Fixation

The axolotl is a great model for studying cartilage, bone and joint regeneration, fracture healing, and evolution. Stainings such as Alcian Blue/Alizarin Red have become workhorses in skeletal analyses, but additional methods complement the detection of different skeletal matrices. Here we describe protocols for studying skeletal biology in axolotls, particularly Alcian Blue/Alizarin Red staining, microcomputed tomography (μCT) scan and live staining of calcified tissue. In addition, we describe a method for decalcification of skeletal elements to ease sectioning.

Topics: Alcian Blue; Ambystoma mexicanum; Animals; Biology; Staining and Labeling; X-Ray Microtomography

Craniofacial morphogenesis is underpinned by orchestrated growth and form-shaping activity of skeletal and soft tissues in the head and face. Disruptions during development can lead to dysmorphology of the skull, jaw, and the pharyngeal structures. Developmental disorders can be investigated in animal models to elucidate the molecular and cellular consequences of the morphogenetic defects. A first step in determining the disruption in the development of the head and face is to analyze the phenotypic features of the skeletal tissues. Examination of the anatomy of bones and cartilage over time and space will identify structural defects of head structures and guide follow-up analysis of the molecular and cellular attributes associated with the defects. Here we describe a protocol to simultaneously visualize the cartilage and bone elements by Alcian blue and Alizarin red staining, respectively, of wholemount specimens in mouse models.

Topics: Alcian Blue; Animals; Anthraquinones; Cartilage; Mice; Skull; Staining and Labeling

The present study was carried out to study the larval skeletal development in Labeo calbasu by using a modified double skeletal staining technique with Alizarin red and Alcian blue. The larval samples were obtained after induced breeding of wild L. calbasu germplasm from the River Ganga. Samples from 2 to 20 dph (day post hatching) were preserved in 4% neutral phosphate buffered formalin solution. Alizarin red and Alcian blue were used to stain the bony and cartilage parts of the skeleton, respectively. The size of the specimens ranged from 6.6 ± 0.16 to 15.6 ± 1.15 mm. The development of skeleton was observed at very early stages. A straight notochord throughout its length and origin of caudal fin rays were seen on 2 dph. The ventral spines, unbranched caudal fin rays and hypurals at ventral side of notochord were clearly visible from 4 dph. Most of the head skeletal elements and vertebral column with vertebral centrum and neural spines started appearing at 4 dph. The dorsal and caudal fins with branched rays and the opercular and jaw bones started ossifying between 10 and 20 dph. The present study gives an idea about the skeletal development process as well as detects the skeletal abnormalities in Indian major carp, L. calbasu.

Topics: Alcian Blue; Animals; Cyprinidae; Formaldehyde; Osteogenesis; Phosphates; Staining and Labeling

Understanding proteolytic remodeling of extracellular matrix involves the generation of global or conditional knockout mice by homologous recombination in embryonic stem cells or their manipulation through new advanced technologies such as CRISPR-Cas9. These models provide opportunities to understand the roles of ADAMTS genes in skeletogenesis. Whole-mount skeletal preparations are necessary for assessment of the skeletal phenotype. They allow for facile visualization of skeletal patterning, size and shape of skeletal elements, and skeletal structure. This protocol describes the staining of the murine skeleton using Alcian blue to identify cartilage and alizarin red to identify bone.

Topics: Alcian Blue; Animals; Animals, Newborn; Anthraquinones; Body Patterning; Bone and Bones; CRISPR-Cas Systems; Embryonic Development; Mice

Caffeine is a natural methylxanthine widespread throughout the food industry. Many research studies have shown that caffeine readily crosses the placenta causing teratogenic and embryotoxic effects. The objective of this study was to assess the influence of caffeine, administered at 10°C, on the development of a rat's bone tissue, with particular reference to elemental bone composition using an X-ray microprobe.. The research was conducted on white rats of the Wistar strain. The fertilized females were divided into two groups: an Experimental Group (Group E) and a Control Group (Group C). The females in Group E were given caffeine orally (at 10°C) in 30 mg/day doses from the 8(th) - 21(st) day of pregnancy. The females in Group C were given water at the same temperature. The foetuses were used to assess the growth and mineralization of the skeleton. Qualitative analysis of the morphology and mineralization of bones was conducted using the alcian-alizarin method. For calcium and potassium analysis, an X-ray microprobe was used.. By staining the skeleton using the alcian-alizarin method, changes in 47 Group E foetuses were observed. The frequency of the development variants in the Group E rats was statistically higher, compared with Group C.. On the basis of these results, it can be concluded that caffeine in high doses disturbs the development of bone tissue. An additional factor which enhances the adverse effects of this substance on bone tissue is the temperature of the administered solution (10(o)C). In the Experimental Group, a significant decrease in the calcium level, as well as an increase in the potassium level, was observed. The X-ray microprobe can be a perfect complement to the methods which enable determination of the mineralization of osseous tissue.

Topics: Alcian Blue; Animals; Anthraquinones; Bone and Bones; Bone Development; Caffeine; Calcium; Female; Microscopy, Electron, Scanning; Potassium; Rats; Rats, Wistar; Temperature

Jaw formation involves an intricate series of molecular events, whereby a chondrogenic scaffold precedes osteogenesis. The mechanisms coupling timing of cartilage maturation to onset of bone differentiation are poorly understood, particularly for neural crest-derived bones of the head. Here we present a novel zebrafish gene/protein-trap Citrine-fusion line that reveals transient expression of the zinc-finger protein Znf385C in maturing chondrocytes of the jaw. Functional analysis shows that loss of Znf385C disrupts a distinct peak of p21(cip1/waf1) expression in the chondrocytes, as well as causes premature ossification of the zebrafish jaw. We find that Znf385C is expressed as two splice variants which act differentially to activate p21(cip1/waf1) and/or interact with p53 in subcellular compartments. Taken together, the results suggest that Znf385C acts as a developmental switch for p53 function that modulates cell cycle arrest of chondrocytes and regulates timing of jaw cartilage maturation and ossification.

Topics: Alcian Blue; Alternative Splicing; Amino Acid Sequence; Animals; Anthraquinones; Binding Sites; Blotting, Western; Chondrocytes; Chromatin Immunoprecipitation; Cloning, Molecular; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Gene Expression Profiling; Gene Expression Regulation, Developmental; In Situ Hybridization; Jaw; Microscopy, Fluorescence; Models, Biological; Molecular Sequence Data; Osteogenesis; Time Factors; Tumor Suppressor Protein p53; Zebrafish; Zebrafish Proteins

Most gnathostomata craniofacial structures derive from pharyngeal arches (PAs), which are colonized by cranial neural crest cells (CNCCs). The anteroposterior and dorsoventral identities of CNCCs are defined by the combinatorial expression of Hox and Dlx genes. The mechanisms associating characteristic Hox/Dlx expression patterns with the topology and morphology of PAs derivatives are only partially known; a better knowledge of these processes might lead to new concepts on the origin of taxon-specific craniofacial morphologies and of certain craniofacial malformations. Here we show that ectopic expression of Hoxa2 in Hox-negative CNCCs results in distinct phenotypes in different CNCC subpopulations. Namely, while ectopic Hoxa2 expression is sufficient for the morphological and molecular transformation of the first PA (PA1) CNCC derivatives into the second PA (PA2)-like structures, this same genetic alteration does not provoke the transformation of derivatives of other CNCC subpopulations, but severely impairs their development. Ectopic Hoxa2 expression results in the transformation of the proximal Meckel's cartilage and of the malleus, two ventral PA1 CNCCs derivatives, into a supernumerary styloid process (SP), a PA2-derived mammalian-specific skeletal structure. These results, together with experiments to inactivate and ectopically activate the Edn1-Dlx5/6 pathway, indicate a dorsoventral PA2 (hyomandibular/ceratohyal) boundary passing through the middle of the SP. The present findings suggest context-dependent function of Hoxa2 in CNCC regional specification and morphogenesis, and provide novel insights into the evolution of taxa-specific patterning of PA-derived structures.

Topics: Alcian Blue; Animals; Anthraquinones; Branchial Region; DNA Primers; Gene Expression Profiling; Gene Expression Regulation, Developmental; Homeodomain Proteins; In Situ Hybridization; Mice; Mice, Mutant Strains; Morphogenesis; Neural Crest; Real-Time Polymerase Chain Reaction

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies.

Topics: Adipocytes; Alcian Blue; Alkaline Phosphatase; Animals; Anthraquinones; Azo Compounds; Cell Differentiation; Chondrocytes; Coloring Agents; Fibroblasts; Flow Cytometry; Genes, Reporter; Luminescent Proteins; Mesenchymal Stem Cells; Mice; Mice, Inbred C3H; Musculoskeletal System; Optical Imaging; Osteocytes; Polymerase Chain Reaction; Tissue Engineering; Transfection

Historically, some fetuses for regulatory developmental toxicity studies have been stained with alizarin red S and cleared with glycerol to visualize the ossified portion of their skeletons. Interest in examining cartilage arose owing to its inclusion in some regulatory guidelines. Methods for double staining rat skeletons have been published previously. The method described here for staining mouse skeletons is fully automated and uses alizarin red S to stain bone and Alcian blue to stain cartilage. Pregnant mice (Crl:CD1) were euthanized on gestation day 18 to obtain fetal specimens. Day 0 post-partum mouse pups also were stained. Our method was developed using the Shandon Pathcentre , which is a fully enclosed automated staining system that allows staining to be carried out at 30° C with a final clearing at 35° C. Our method uses the same solutions as for fetal rat processing, but with reduced time periods for the smaller size of mice vs. rat specimens. Staining, maceration and clearing of the specimens requires approximately 2 days. The time required of laboratory personnel, however, is minimal, because all solutions are changed automatically and the specimens do not require examination or removal from the processor until processing is complete. After processing, the specimens are suitable for immediate assessment of bone and cartilage. A mouse developmental toxicity study using 20 animals/group and approximately 10 fetuses/animal could be processed in only three runs using one machine.

Topics: Alcian Blue; Animals; Animals, Newborn; Anthraquinones; Bone and Bones; Cartilage, Articular; Cells, Cultured; Coloring Agents; Diagnosis, Differential; Mice; Staining and Labeling

The emergence of craniofacial skeletal elements, and of the jaw in particular, was a crucial step in the evolution of higher vertebrates. Most facial bones and cartilage are generated during embryonic development by cranial neural crest cells, while an osteochondrogenic fate is suppressed in more posterior neural crest cells. Key players in this process are Hox genes, which suppress osteochondrogenesis in posterior neural crest derivatives. How this specific pattern of osteochondrogenic competence is achieved remains to be elucidated. Here we demonstrate that Hox gene expression and osteochondrogenesis are controlled by epigenetic mechanisms. Ezh2, which is a component of polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 in histone 3 (H3K27me3), thereby functioning as transcriptional repressor of target genes. Conditional inactivation of Ezh2 does not interfere with localization of neural crest cells to their target structures, neural development, cell cycle progression or cell survival. However, loss of Ezh2 results in massive derepression of Hox genes in neural crest cells that are usually devoid of Hox gene expression. Accordingly, craniofacial bone and cartilage formation is fully prevented in Ezh2 conditional knockout mice. Our data indicate that craniofacial skeleton formation in higher vertebrates is crucially dependent on epigenetic regulation that keeps in check inhibitors of an osteochondrogenic differentiation program.

Topics: Alcian Blue; Animals; Anthraquinones; Cartilage; Chondrogenesis; Chromatin Immunoprecipitation; DNA Methylation; Enhancer of Zeste Homolog 2 Protein; Epigenesis, Genetic; Facial Bones; Flow Cytometry; Galactosides; Gene Expression Regulation, Developmental; Histones; Immunohistochemistry; Indoles; Mice; Mice, Transgenic; Microarray Analysis; Neural Crest; Osteogenesis; Polycomb Repressive Complex 2; Real-Time Polymerase Chain Reaction

Integrative taxonomy, in which multiple disciplines are combined to address questions related to biological species diversity, is a valuable tool for identifying pelagic marine fish larvae and recognizing the existence of new fish species. Here we combine data from DNA barcoding, comparative morphology, and analysis of color patterns to identify an unusual fish larva from the Florida Straits and demonstrate that it is the pelagic larval phase of a previously undescribed species of Liopropoma sea bass from deep reefs off Curaçao, southern Caribbean. The larva is unique among larvae of the teleost family Serranidae, Tribe Liopropomini, in having seven elongate dorsal-fin spines. Adults of the new species are similar to the golden bass, Liopropoma aberrans, with which they have been confused, but they are distinct genetically and morphologically. The new species differs from all other western Atlantic liopropomins in having IX, 11 dorsal-fin rays and in having a unique color pattern-most notably the predominance of yellow pigment on the dorsal portion of the trunk, a pale to white body ventrally, and yellow spots scattered across both the dorsal and ventral portions of the trunk. Exploration of deep reefs to 300 m using a manned submersible off Curaçao is resulting in the discovery of numerous new fish species, improving our genetic databases, and greatly enhancing our understanding of deep-reef fish diversity in the southern Caribbean. Oh the mother and child reunion is only a moment away. Paul Simon.

Topics: Alcian Blue; Animal Distribution; Animal Fins; Animals; Anthraquinones; Bass; Classification; Cluster Analysis; Coral Reefs; DNA Barcoding, Taxonomic; Florida; Histological Techniques; Larva; Netherlands Antilles; Photomicrography; Phylogeny; Skin Pigmentation; Species Specificity

The vertebrate T-box transcription factor gene Tbx18 performs a vital role in development of multiple organ systems. Tbx18 insufficiency manifests as recessive phenotypes in the upper urinary system, cardiac venous pole, inner ear, and axial skeleton; homozygous null mutant animals die perinatally. Here, we report a new regulatory mutation of Tbx18, a reciprocal translocation breaking 78kbp downstream of the gene. 12Gso homozygotes present urinary and vertebral defects very similar to those associated with Tbx18-null mutations, but 12Gso is clearly not a global null allele since homozygotes survive into adulthood. We show that 12Gso down-regulates Tbx18 expression in a manner that is both spatially- and temporally-specific; combined with other data, the mutation points particularly to the presence of an essential urogenital enhancer located near the translocation breakpoint site. In support of this hypothesis, we identify a distal enhancer element, ECR1, which is active in developing urogenital and other tissues; we propose that disruption of this element leads to premature loss of Tbx18 function in 12Gso mutant mice. These data reveal a long-range regulatory architecture extending far downstream of Tbx18, identify a novel and likely essential urogenital enhancer, and introduce a new tool for dissecting postnatal phenotypes associated with dysregulation of Tbx18.

Topics: Alcian Blue; Animals; Anthraquinones; Base Sequence; Chromosome Mapping; DNA Primers; Enhancer Elements, Genetic; Gene Expression Regulation; Histological Techniques; Immunohistochemistry; Mice; Mice, Transgenic; Molecular Sequence Data; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; T-Box Domain Proteins; Translocation, Genetic; Urogenital System

In eukaryotic cells, newly synthesized secretory proteins require COPII (coat protein complex II) to exit the endoplasmic reticulum (ER). COPII contains five core components: SAR1, SEC23, SEC24, SEC13, and SEC31. SEC23 is a GTPase-activating protein that activates the SAR1 GTPase and also plays a role in cargo recognition. Missense mutations in the human COPII paralogues SEC23A and SEC23B result in craniolenticulosutural dysplasia and congenital dyserythropoietic anemia type II, respectively. We now report that mice completely deficient for SEC23B are born with no apparent anemia phenotype, but die shortly after birth, with degeneration of professional secretory tissues. In SEC23B-deficient embryonic pancreas, defects occur in exocrine and endocrine tissues shortly after differentiation. Pancreatic acini are completely devoid of zymogen granules, and the ER is severely distended. Similar ultrastructural alterations are also observed in salivary glands, but not in liver. Accumulation of proteins in the ER lumen activates the proapoptotic pathway of the unfolded protein response, suggesting a central role for apoptosis in the degeneration of these tissues in SEC23B-deficient embryos. Although maintenance of the secretory pathway should be required by all cells, our findings reveal a surprising tissue-specific dependence on SEC23B for the ER exit of highly abundant cargo, with high levels of SEC23B expression observed in professional secretory tissues. The disparate phenotypes in mouse and human could result from residual SEC23B function associated with the hypomorphic mutations observed in humans, or alternatively, might be explained by a species-specific shift in function between the closely related SEC23 paralogues.

Topics: Alcian Blue; Animals; Anthraquinones; Apoptosis; Endoplasmic Reticulum; Fluorescent Antibody Technique; Humans; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Immunoelectron; Mutation; Pancreas; Real-Time Polymerase Chain Reaction; Secretory Pathway; Species Specificity; Vesicular Transport Proteins

Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene cause defective ribosome assembly and are associated with exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. However, the mechanism underlying these phenotypes remains unclear. Here we show that knockdown of the zebrafish sbds ortholog fully recapitulates the spectrum of developmental abnormalities observed in the human syndrome, and further implicate impaired proliferation of ptf1a-expressing pancreatic progenitor cells as the basis for the observed pancreatic phenotype. It is thought that diseases of ribosome assembly share a p53-dependent mechanism. However, loss of p53 did not rescue the developmental defects associated with loss of zebrafish sbds. To clarify the molecular mechanisms underlying the observed organogenesis defects, we performed transcriptional profiling to identify candidate downstream mediators of the sbds phenotype. Among transcripts displaying differential expression, functional group analysis revealed marked enrichment of genes related to ribosome biogenesis, rRNA processing and translational initiation. Among these, ribosomal protein L3 (rpl3) and pescadillo (pes) were selected for additional analysis. Similar to knockdown of sbds, knockdown or mutation of either rpl3 or pes resulted in impaired expansion of pancreatic progenitor cells. The pancreatic phenotypes observed in rpl3- and pes-deficient embryos were also independent of p53. Together, these data suggest novel p53-independent roles for ribosomal biogenesis genes in zebrafish pancreas development.

Topics: Alcian Blue; Animals; Anthraquinones; Bone Marrow Diseases; Disease Models, Animal; Exocrine Pancreatic Insufficiency; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Knockdown Techniques; In Situ Hybridization; Lipomatosis; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Pancreas; Ribosomal Protein L3; Ribosomal Proteins; Ribosomes; Shwachman-Diamond Syndrome; Tumor Suppressor Protein p53; Zebrafish; Zebrafish Proteins

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.

Topics: Alcian Blue; Animals; Anthraquinones; Bone and Bones; Bone Development; Cartilage; Chick Embryo; Decalcification Technique; Hydrogen-Ion Concentration; Staining and Labeling

Zebrafish craniofacial, skeletal, and tooth development closely resembles that of higher vertebrates. Our goal is to identify viable adult zebrafish mutants that can be used as models for human mineralized craniofacial, dental, and skeletal system disorders. We used a large-scale forward-genetic chemical N-ethyl-nitroso-urea mutagenesis screen to identify 17 early lethal homozygous recessive mutants with defects in craniofacial cartilage elements, and 7 adult homozygous recessive mutants with mineralized tissue phenotypes including craniofacial shape defects, fused sutures, dysmorphic or missing skeletal elements, scoliosis, and neural arch defects. One mutant displayed both an early lethal homozygous phenotype and an adult heterozygous phenotype. These results extend the utility of the zebrafish model beyond the embryo to study human bone and cartilage disorders.

Topics: Alcian Blue; Animals; Anthraquinones; Cartilage; Craniofacial Abnormalities; Disease Models, Animal; Ethylnitrosourea; Genes, Recessive; Mutagenesis; Phenotype; Zebrafish

Mesenchymal stem cells (MSCs) are multipotent cells that have the potential to differentiate into various mesenchymal lineages in vitro and in vivo. Due to their availability from tissues such as bone marrow, synovium, fat, and muscle, and their highly proliferative capacity, MSCs have evoked interest as a potential cell source for repair and regeneration of various types of tissues. Characterization by the expression of a panel of surface markers and the ability of MSCs to undergo multilineage differentiation is the benchmark for identifying this stem cell population. In this chapter, the protocols for the differentiation of MSC to chondrogenic, osteogenic, and adipogenic lineages and histological and immunostaining protocols for confirming trilineage differentiation of the MSC cells are described.

Topics: Adipogenesis; Alcian Blue; Animals; Anthraquinones; Azo Compounds; Bone Marrow Cells; Cell Culture Techniques; Cell Separation; Chondrogenesis; Collagen; Humans; Immunohistochemistry; Mesenchymal Stem Cells; Minerals; Osteogenesis; Phenazines; Protein Biosynthesis; Proteoglycans; Staining and Labeling

The COL2A1 gene encodes the α1(II) chain of the homotrimeric type II collagen, the most abundant protein in cartilage. In humans, COL2A1 mutations create many clinical phenotypes collectively termed type II collagenopathies; however, the genetic basis of the phenotypic diversity is not well elucidated. Therefore, animal models corresponding to multiple type II collagenopathies are required. In this study we identified a novel Col2a1 missense mutation--c.44406A>C (p.D1469A)--produced by large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis in a mouse line. This mutation was located in the C-propeptide coding region of Col2a1 and in the positions corresponding to a human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). The phenotype was inherited as a semidominant trait. The heterozygotes were mildly but significantly smaller than wild-type mice. The homozygotes exhibited lethal skeletal dysplasias, including extremely short limbs, severe spondylar dysplasia, severe pelvic hypoplasia, and brachydactyly. As expected, these skeletal defects in the homozygotes were similar to those in PLSD-T patients. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by abnormally expanded rough endoplasmic reticulum (ER) and upregulation of ER stress-related genes, such as Grp94 and Chop, in chondrocytes. These findings suggested that the accumulation of mutant type II collagen in the ER and subsequent induction of ER stress are involved, at least in part in the PLSD-T-like phenotypes of the mutants. This mutant should serve as a good model for studying PLSD-T pathogenesis and the mechanisms that create the great diversity of type II collagenopathies.

Topics: Alcian Blue; Animals; Anthraquinones; Bone and Bones; Chromosome Mapping; Collagen Type II; Disease Models, Animal; DNA Primers; Ethylnitrosourea; Genotype; Immunohistochemistry; Mice; Mice, Mutant Strains; Microscopy, Electron, Transmission; Mutagenesis; Mutation, Missense; Osteochondrodysplasias; Reverse Transcriptase Polymerase Chain Reaction

MeCP2 plays a critical role in interpreting epigenetic signatures that command chromatin conformation and regulation of gene transcription. In spite of MeCP2's ubiquitous expression, its functions have always been considered in the context of brain physiology. In this study, we demonstrate that alterations of the normal pattern of expression of MeCP2 in cardiac and skeletal tissues are detrimental for normal development. Overexpression of MeCP2 in the mouse heart leads to embryonic lethality with cardiac septum hypertrophy and dysregulated expression of MeCP2 in skeletal tissue produces severe malformations. We further show that MeCP2's expression in the heart is developmentally regulated; further suggesting that it plays a key role in regulating transcriptional programs in non-neural tissues.

Topics: Alcian Blue; Animals; Anthraquinones; Bone and Bones; Bromodeoxyuridine; Crosses, Genetic; DNA Primers; Gene Expression Regulation, Developmental; Heart; In Situ Hybridization; In Situ Nick-End Labeling; Methyl-CpG-Binding Protein 2; Mice; Mice, Transgenic; Microscopy, Fluorescence; Myocardium; Osteogenesis; Reverse Transcriptase Polymerase Chain Reaction; Transgenes

The aim of the present study was to evaluate the biocompatibility and bone mineralization potential of 45S5 Bioglass-derived glass-ceramic scaffolds using a chick embryo shell-less (ex ovo) culture system. Chick embryos were divided into two groups: control (C) and experimental (E). Scaffolds were placed on the chorioallantoic membrane (CAM) in embryos of group E at 10 days of total incubation. The 45S5 Bioglass-derived glass-ceramic scaffolds proved to be biocompatible in terms of the absence of inflammatory response at the implant site (CAM). Moreover, no alterations in the other end-points assessed, i.e. survival, stage of embryonic development and body weight, were detected. However, body length was greater in group E embryos than in group C embryos (p0.05). A marked reduction (93%) in Ca content in the scaffolds was evidenced by energy-dispersive X-ray analysis at 5 days post-implantation. Calcium release from the scaffold implanted on the CAM might have been responsible for the restoration of the bone-like phenotype in chick embryonic skeleton of group E as detected by Alcian blue-Alizarin red double staining, as well as by histological and microchemical analyses. Conversely, the control embryos exhibited a chondrogenic phenotype.

Topics: Alcian Blue; Animals; Anthraquinones; Biocompatible Materials; Bone Substitutes; Calcium; Ceramics; Chick Embryo; Inflammation; Ions; Materials Testing; Phenotype; Tissue Engineering; X-Rays

Perfluorooctane sulfonate (PFOS) is found ubiquitously in the environment, and is known to cause developmental toxicity, including cleft plate (CP). The aim of the present study was to elucidate the mechanism of CP associated with in utero exposure to PFOS in mice. We first examined whether the concentration of PFOS in fetal serum was related to susceptibility to CP. We compared palatogenesis following the administration of various concentrations of PFOS to dams. We conducted histological examination on gestational day (GD) 15 and 18, and alizarin red/alcian blue staining of fetal heads on GD18. Finally, we cultured palatal shelves (PSs) of GD14 fetuses, which had not yet made contact with each other, for 48h, to examine whether the shelves maintained the ability to fuse. The incidence of CP increased from 7.3% with a fetal serum concentration of PFOS of 110.7+/-13.4microg/ml (13mg/kg) to 78.3% with 138.6+/-0.9microg/ml (20mg/kg). PFOS at 50mg/kg on GD11-15 caused CP at a rate of 6.1%, meanwhile PFOS at 20mg/kg on GD1-17 caused a CP rate of 89.3%. Failure of palatal shelf elevation was observed with 20mg/kg PFOS. PFOS at 20mg/kg on GD1-17 and 50mg/kg on GD11-15 inhibited mandibular growth to the same extent, even though the rate of CP was different. Explants exposed to PFOS 20mg/kg and Tween 20 showed 94% (34/36) and 100% (31/31) fusion, respectively. We demonstrated that increasing the oral dose of PFOS from 13 to 20mg/kg resulted in a significant increase in CP even though there was only a small increase in serum concentration of PFOS. PFOS prevented elevation of the PSs above the tongue because their growth/fusion potential was maintained. Mandibular hypoplasia did not seem to play a critical role in the pathogenesis of CP.

Topics: Alcian Blue; Alkanesulfonic Acids; Amniotic Fluid; Animals; Anthraquinones; Body Weight; Cleft Palate; Female; Fetal Blood; Fluorocarbons; Indicators and Reagents; Mice; Mice, Inbred ICR; Organ Culture Techniques; Organ Size; Pregnancy

It is generally assumed that the characteristic deregionalized body plan of species with a snake-like morphology evolved through a corresponding homogenization of Hox gene expression domains along the primary axis. Here, we examine the expression of Hox genes in snake embryos and show that a collinear pattern of Hox expression is retained within the paraxial mesoderm of the trunk. Genes expressed at the anterior and most posterior, regionalized, parts of the skeleton correspond to the expected anatomical boundaries. Unexpectedly however, also the dorsal (thoracic), homogenous rib-bearing region of trunk, is regionalized by unconventional gradual anterior limits of Hox expression that are not obviously reflected in the skeletal anatomy. In the lateral plate mesoderm we also detect regionalized Hox expression yet the forelimb marker Tbx5 is not restricted to a rudimentary forelimb domain but is expressed throughout the entire flank region. Analysis of several Hox genes in a caecilian amphibian, which convergently evolved a deregionalized body plan, reveals a similar global collinear pattern of Hox expression. The differential expression of posterior, vertebra-modifying or even rib-suppressing Hox genes within the dorsal region is inconsistent with the homogeneity in vertebral identity. Our results suggest that the evolution of a deregionalized, snake-like body involved not only alterations in Hox gene cis-regulation but also a different downstream interpretation of the Hox code.

Topics: Alcian Blue; Amphibians; Animals; Anthraquinones; Body Patterning; Bone and Bones; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Homeodomain Proteins; Lizards; Mesoderm; Mice; Snakes; Somites; T-Box Domain Proteins

Topics: Alcian Blue; Animals; Animals, Newborn; Anthraquinones; Bone and Bones; Cartilage; Chiroptera; Female; Pregnancy; Staining and Labeling

Topics: Alcian Blue; Animals; Animals, Newborn; Anthraquinones; Bone and Bones; Cartilage; Female; Mice; Pregnancy; Staining and Labeling

Mesenchymal cell aggregation is critical for cartilage formation in the vertebrate limb. The extracellular matrix (ECM) plays a critical role in governing cell behavior and cell phenotype in this tissue, and the hyalectin versican is highly expressed in the ECM of precartilage mesenchymal cells and developing synovial joints. Although several in vitro studies have been conducted in an attempt to address versican's role during limb mesenchymal condensation, factors such as differences in cell density in culture, variations between chondrogenic cell lines, and the inability to prolong the viability of limb explants have led to conflicting data, mandating an in vivo analysis. By using a morpholino directed strategy in ovo, we performed knock-down of versican expression in the presumptive ulnar region of the developing chick wing at time points critical to skeletogenesis. These data indicate that in ovo misexpression of versican compromised mesenchymal condensation with resulting ulnar cartilages reduced in length distally by an average of 53% relative to contralateral control limbs. In select versican morphants the olecranon process was also reduced in size proximally and failed to cup the humerus, likely impairing joint morphogenesis. This study represents the first report assessing the role of versican in the developing chick limb in ovo, further demonstrating the importance of versican proteoglycan expression during chondrogenesis and extending previous findings to suggest a role for versican during synovial joint development.

Topics: Alcian Blue; Animals; Anthraquinones; Apoptosis; Chick Embryo; Chondrogenesis; Embryonic Development; Immunohistochemistry; In Situ Nick-End Labeling; Limb Buds; Versicans

Biglycan (Bgn) and decorin (Dcn) are highly expressed in numerous tissues in the craniofacial complex. However, their expression and function in the cranial sutures are unknown. In order to study this, we first examined the expression of biglycan and decorin in the posterior frontal suture (PFS), which predictably fuses between 21 and 45 days post-natal and in the non-fusing sagittal (S) suture from wild-type (Wt) mice. Our data showed that Bgn and Dcn were expressed in both cranial sutures. We then characterized the cranial suture phenotype in Bgn deficient, Dcn deficient, Bgn/Dcn double deficient, and Wt mice. At embryonic day 18.5, alizarin red/alcian blue staining showed that the Bgn/Dcn double deficient mice had hypomineralization of the frontal and parietal craniofacial bones. Histological analysis of adult mice (45-60 days post-natal) showed that the Bgn or Dcn deficient mice had no cranial suture abnormalities and immunohistochemistry staining showed increased production of Dcn in the PFS from Bgn deficient mice. To test possible compensation of Dcn in the Bgn deficient sutures, we examined the Bgn/Dcn double deficient mice and found that they had impaired fusion of the PFS. Semi-quantitative RT-PCR analysis of RNA from 35 day-old mice revealed increased expression of Bmp-4 and Dlx-5 in the PFS compared to their non-fusing S suture in Wt tissues and decreased expression of Dlx-5 in both PF and S sutures in the Bgn/Dcn double deficient mice compared to the Wt mice. Failure of PFS fusion and hypomineralization of the calvaria in the Bgn/Dcn double deficient mice demonstrates that these extracellular matrix proteoglycans could have a role in controlling the formation and growth of the cranial vault.

Topics: Alcian Blue; Animals; Anthraquinones; Biglycan; Cranial Sutures; Decorin; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Developmental; Male; Mice; Mice, Knockout; Phenotype; Pregnancy; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staining and Labeling

The potential of in utero exposure to fluconazole to initiate teratogenesis was analyzed in ICR (CD-1) mice. Developmental phase specificity was determined by treating mice with single oral doses of 700 mg/kg on gestational day 8, 9, 10, 11, or 12. Control animals received vehicle on gestational days 8-12. Gestational day 10 was identified as the phase of maximal sensitivity for induction of cleft palate, the predominant teratogenic effect induced by fluconazole, with 50% of fetuses exposed on this developmental phase being affected. After treatments on gestational day 8, 9, 11, or 12, cleft palate occurred with lower frequencies: 12, 21, 28.7, and 2.7%, respectively. Examination of skeletal morphology revealed anomalies of the middle ear apparatus in 15% of the fetuses that were exposed on gestational day 8. Dysmorphic tympanic ring and absence of the incus were the more common ear anomalies recorded. Reduced humeral length was noted in 22% of fetuses that were exposed on gestational day 10. Dose-response relationship was investigated by treating animals with 0 (vehicle), 87.5, 175, or 350 mg/kg on gestational day 10, coincident with the phase of peak teratogenic sensitivity. Besides showing that fluconazole operates under a strict dose-response mechanism, the study identified 175 mg/kg as the lowest observed adverse effect level for cleft palate induction, with 7.6% of the exposed fetuses being affected.

Topics: Abnormalities, Drug-Induced; Alcian Blue; Animals; Anthraquinones; Antifungal Agents; Body Weight; Cartilage; Cleft Palate; Dose-Response Relationship, Drug; Ear, Middle; Female; Fluconazole; Forelimb; Mice; Mice, Inbred ICR; Pregnancy; Pregnancy, Animal; Teratogens; Teratology; Time Factors

Topics: Acid Phosphatase; Alcian Blue; Alkaline Phosphatase; Animals; Anthraquinones; Biomarkers; Bone and Bones; Bone Development; Bromodeoxyuridine; Cartilage; Chondrocytes; Dissection; Fluoresceins; Histological Techniques; Isoenzymes; Mutagenesis; Mutation; Osteoblasts; Osteoclasts; Phenotype; Radiography; Staining and Labeling; Tartrate-Resistant Acid Phosphatase; Zebrafish

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.

Topics: Alcian Blue; Amino Acid Sequence; Animals; Annexin A5; Anthraquinones; Apoptosis; Blotting, Western; Cartilage; Cell Differentiation; Cell Line; Cell Membrane; Cell Nucleus; Cells, Cultured; Chondrocytes; Chromosomes; Collagen; Coloring Agents; Cystatins; DNA, Complementary; Genetic Vectors; Immunoblotting; Immunohistochemistry; In Situ Hybridization, Fluorescence; Membrane Potentials; Mice; Mice, Transgenic; Microscopy, Fluorescence; Molecular Sequence Data; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Time Factors; Transfection

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.

Topics: Alcian Blue; Animals; Anthraquinones; Bone Development; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Coloring Agents; DNA, Complementary; Fluoresceins; Fluorescent Dyes; Indicators and Reagents; Mutation; Notochord; Time Factors; Transforming Growth Factor beta; Zebrafish

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 microns in length can be clearly visualized. The method is suitable for both ontogenic studies during early stages of skeletal development in most marine fishes (e.g., Sparus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.

Topics: Alcian Blue; Animals; Anthraquinones; Bone and Bones; Calcification, Physiologic; Cartilage; Fishes; Histocytochemistry; Larva; Staining and Labeling; Time Factors; Tissue Preservation

In this work we study the skeletal teratogenic response in rats exposed to NaVP at different embryonic stages. Crl:CD female rats were treated subcutaneously with 400 mg/Kg b.w. NaVP at presomitic stage (group II) or nearly at 2, 6, 10, 14, 18 or 22 somites (groups III-VIII). The females on group I were treated with saline and served as controls. No treatment-related effects were observed at the level of resorptions, live fetuses and fetal or placental weight. The skeletal examination showed characteristic patterns of malformations strictly related to the period of treatment. In particular, groups II and III showed a significant increase of alterations of cervical vertebrae (mainly 1st to 3rd segment) and a decrease of the frequency of extra lumbar ribs in comparison to control. Group IV showed severe abnormalities localized at the 4th to 7th cervical segment and at the level of the 1st and 2nd thoracic segments, including duplications of thoracic segments 1, 2 or 3. The fetuses of group V were characterized by several alterations of the thoracic segments distributed without a clear specificity. In group VI, the thoracic region was also affected with some specificity at the level of the segments 4th to 9th; in group VII, last thoracic and lumbar segments were affected (mainly duplications) and in group VIII only lumbo-sacral abnormalities were recorded. These results confirm the specific effect of NaVP at the level of the axial skeleton and suggest a possible interaction with the expression of genes identifying the vertebral segments.

Topics: Alcian Blue; Animals; Anthraquinones; Bone and Bones; Coloring Agents; Embryo, Mammalian; Rats; Rats, Inbred Strains; Spine; Teratogens; Valproic Acid