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Whole Mount (whole + mount)
Selected AbstractsMode of attachment and lesions associated with trypanorhynch cestodes in the gastrointestinal tracts of two species of sharks collected from coastal waters of BorneoJOURNAL OF FISH DISEASES, Issue 7 2006J D Borucinska Abstract Lesions associated with two species of tapeworms within the digestive tract of wild-caught specimens of the bull shark, Carcharhinus leucas, and the sicklefin weasel shark, Hemigaleus microstoma, from Malaysian Borneo are described. Portions of the glandular stomach and pyloric gut with parasites were removed and fixed in 10% formalin buffered in sea water. Whole mounts, histological sections of tissues with and without worms in situ, and scanning electron microscopy images of detached worms were examined. Both species of cestodes belonged to the trypanorhynch family Tentaculariidae. Heteronybelinia estigmena was found in large numbers parasitizing the pyloric gut of C. leucas; an unidentified tentaculariid was found in relatively small numbers in both the glandular stomach and pyloric gut of H. microstoma. Both species burrowed their scoleces deeply in the mucosa and attached via hooked tentacles and unciniform microtriches of the scolex. The lesions induced by the parasites were marked in both sharks and ranged from acute necrotizing to chronic granulomatous gastroenteritis. Regenerative hyperplasia and intestinal metaplasia of gastric epithelium were also present. The severity and character of pathology was causally linked to the intensity of infection, the attachment mode of the parasites, and to the anatomophysiological relationships within the gut of the host shark. [source] Quantification and characterization of GABA-ergic amacrine cells in the retina of GAD67-GFP knock-in miceACTA OPHTHALMOLOGICA, Issue 4 2008Christian Albrecht May Abstract. Purpose:, Although the presence of ,-aminobutyrate acid (GABA) in amacrine cells and its co-localization with other neuronal substances is well known, there exists only little information about their quantitative distribution in the mouse eye. The aim of the present study was to characterize GABA-ergic amacrine cells in the retina of the recently introduced glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse. Methods:, Whole mounts of the retina were prepared and the GFP-positive neurons quantified. Immunofluorescence staining was performed with antibodies against GABA, calbindin (CB), calretinin (CR), parvalbumin (PV), choline acetyl transferase (ChAT), tyrosine hydroxylase (TH), vesicular glutamate transporter (VGluT) 1, VGluT2 and VGluT3. Results:, Displaced GABA-ergic amacrine cells in the ganglion cell layer (GCL) showed a density of 1006 ± 170 cells/mm2. In the inner nuclear layer (INL), the density of amacrine cells was 8821 ± 448 cells/mm2 in the central region and 6825 ± 408 cells/mm2 in the peripheral region. GFP-positive amacrine cells co-localized with GABA (99%), CR (INL 18%, GCL 71.3%), CB (INL 6.3%), bNOS (INL 1%, GCL 4%), and ChAT (INL 17%, GCL 92.6%). No co-localization was seen with antibodies against PV, TH, and VGluT 1-3. Conclusions:, This study presents the first quantitative data concerning the co-localization of GABA-ergic neurons in the mouse retina with various neuronal markers. [source] Busulfan-induced central polydactyly, syndactyly and cleft hand or foot: A common mechanism of disruption leads to divergent phenotypesDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2007Takuji Naruse The prevalence of clinical phenotypes that exhibit combinations of central polydactyly, syndactyly, or cleft hand or foot is higher than would be expected for random independent mutations. We have previously demonstrated that maternal ingestion of a chemotherapeutic agent, busulfan, at embryonic day 11 (E11) induces these defects in various combinations in rat embryo limbs. In an effort to determine the mechanism by which busulfan disrupts digital development, we examined cell death by Nile Blue staining and TdT-mediated dUTP nick end labeling (TUNEL) assays; we also carried out whole mount in situ hybridization for fibroblast growth factor-8 (Fgf8), bone morphogenetic protein-4 (Bmp4), and sonic hedgehog (Shh) to examine developmental pathways linked to these defects. In busulfan-treated embryos, diffuse cell death was evident in both ectoderm and mesoderm, peaking at E13. The increased cell death leads to regression of Fgf8 in the apical ectodermal ridge (AER) and Bmp4 and Shh in the underlying mesoderm. The subsequent pattern of interdigital apoptosis and cartilage condensation was variably disrupted. These results suggest that busulfan manifests its teratogenic effects by inducing cell death of both ectoderm and mesoderm, with an associated reduction in tissue and a disruption in the generation of patterning molecules during critical periods of digit specification. [source] Expression of the zebrafish CD133/prominin1 genes in cellular proliferation zones in the embryonic central nervous system and sensory organsDEVELOPMENTAL DYNAMICS, Issue 6 2010Maura McGrail Abstract The CD133/prominin1 gene encodes a pentamembrane glycoprotein cell surface marker that is expressed in stem cells from neuroepithelial, hematopoietic, and various organ tissues. Here we report the analysis of two zebrafish CD133/prominin1 orthologues, prominin1a and prominin1b. The expression patterns of the zebrafish prominin1a and b genes were analyzed during embryogenesis using whole mount in situ hybridization. prominin1a and b show novel complementary and overlapping patterns of expression in proliferating zones in the developing sensory organs and central nervous system. The expression patterns suggest functional conservation of the zebrafish prominin1 genes. Initial analyses of prominin1a and b in neoplastic tissue show increased expression of both genes in a subpopulation of cells in malignant peripheral nerve sheath tumors in tp53 mutants. Based on these analyses, the zebrafish prominin1 genes will be useful markers for examining proliferating cell populations in adult organs, tissues, and tumors. Developmental Dynamics 239:1849,1857, 2010. © 2010 Wiley-Liss, Inc. [source] MicroRNA expression during chick embryo developmentDEVELOPMENTAL DYNAMICS, Issue 11 2006Diana K. Darnell Abstract MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function. Developmental Dynamics 235:3156,3165, 2006. © 2006 Wiley-Liss, Inc. [source] Functional characterization of a neuropeptide F-like receptor from Drosophila melanogasterEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2003Guoping Feng Abstract A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (NPFR76F, also called GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62,66% similarity and 32,34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein G,16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila. [source] Retention of the duplicated cellular retinoic acid-binding protein 1 genes (crabp1a and crabp1b) in the zebrafish genome by subfunctionalization of tissue-specific expressionFEBS JOURNAL, Issue 14 2005Rong-Zong Liu The cellular retinoic acid-binding protein type I (CRABPI) is encoded by a single gene in mammals. We have characterized two crabp1 genes in zebrafish, designated crabp1a and crabp1b. These two crabp1 genes share the same gene structure as the mammalian CRABP1 genes and encode proteins that show the highest amino acid sequence identity to mammalian CRABPIs. The zebrafish crabp1a and crabp1b were assigned to linkage groups 25 and 7, respectively. Both linkage groups show conserved syntenies to a segment of the human chromosome 15 harboring the CRABP1 locus. Phylogenetic analysis suggests that the zebrafish crabp1a and crabp1b are orthologs of the mammalian CRABP1 genes that likely arose from a teleost fish lineage-specific genome duplication. Embryonic whole mount in situ hybridization detected zebrafish crabp1b transcripts in the posterior hindbrain and spinal cord from early stages of embryogenesis. crabp1a mRNA was detected in the forebrain and midbrain at later developmental stages. In adult zebrafish, crabp1a mRNA was localized to the optic tectum, whereas crabp1b mRNA was detected in several tissues by RT-PCR but not by tissue section in situ hybridization. The differential and complementary expression patterns of the zebrafish crabp1a and crabp1b genes imply that subfunctionalization may be the mechanism for the retention of both crabp1 duplicated genes in the zebrafish genome. [source] Patterning of the axial musculature in the developing chick embryo (Gallus)JOURNAL OF ANATOMY, Issue 5 2002P. J. Adds While the development and patterning events of the skeletal, myogenic and connective tissues of the developing limb buds of the chick have been relatively well studied, there is little known about the formation of the epaxial muscles and tendons. The epaxial muscles form the postvertebral muscle groups and develop from the myotome of the somite. The myotome develops from myogenic precursors migrating from the dorsomedial lip of the dermomyotome. These myogenic cells differentiate in a cranial to caudal sequence with the muscle fibres also orientated in a cranial to caudal direction. Here we use immunohistological staining both in whole mount and in transverse sections of chick embryos from various stages of development. Antibodies to the myosin heavy chain or tenascin were used to visualise the development of the epaxial muscles and tendons. The first myotomal muscle fibres are differentiated by Hamburger and Hamilton (H & H) stage 10 in the cranial somites, and differentiation proceeds caudally until at least H & H stage 22,23. Further development of the epaxial muscles does not take place until H & H stage 26,27 with the splitting of the myotome into the individual muscles. We demonstrate how the myotome splits into the individual muscles and how some muscle fibres become reorientated into a more oblique orientation. This delayed development and reorientation of muscle fibres is unique to the epaxial muscles. [source] Non-radioactive in situ detection of mRNA in ES cell-derived cardiomyocytes and in the developing heartMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2002Arnoud C. Fijnvandraat Abstract Non-radioactive in situ hybridisation is an excellent method to visualise mRNA molecules within their topographical context. Recently we have reported a new non-radioactive in situ hybridisation procedure on tissue sections that is essentially based on the whole mount in situ hybridisation procedure. This method is superior in spatial resolution and sensitivity compared to the radioactive in situ hybridisation procedure. Generally, low levels of gene expression, such as found with the developmental onset of gene expression and in differentiating embryonic stem cells, are difficult to detect by in situ hybridisation. Here an application of the protocol is presented which is based on tyramide signal amplification, which enables the detection of very low abundant mRNAs. The significance of this method is two-fold: (1) the molecular phenotype of embryonic stem cell-derived cardiomyocytes can be examined at the cellular level with high sensitivity, and (2) the number of cells that express the gene of interest can be assessed. Microsc. Res. Tech. 58:387,394, 2002. © 2002 Wiley-Liss, Inc. [source] Presumptive pre-sertoli cells express genes involved in cell proliferation and cell signalling during a critical window in early testis differentiationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2007Aron T. Cory In mammals, the pre-Sertoli cell of the male genital ridge is the first cell type to display sex specific differentiation and differential gene expression. The genetic cascade driving the differentiationof pre-Sertoli cells and ultimately testis formationis beginning to be unravelled, but many questions remain. A better understanding of the transcriptome of pre-Sertoli cells immediately after sex determination is essential in order to further understand this differentiationprocess. A mouse model expressing Red Fluorescent Protein (RFP) under the control of a hybrid mouse/pig SRY promoter (HybSRYp-RFP) was used to purify cells from embryonic day 12.0 (e12.0) male genital ridges. To compare the transcriptomes of HybSRYp-RFP cell populations versus age matched whole female genital ridges, RNA was extracted and used to generate molecular probes that were hybridized onto Affymetrix Mouse Genome 430 2.0 micro-arrays. The expression of genes considered markers for pre-Sertoli cells, including Sox9, Mis, Dhh and Fgf9 were identified within the HybSRYp-RFP expressing cell population, while markers for germ cells (Oct4, SSEA-1) and endothelial cells (Ntrk3) were not identified. In contrast, markers for ovarian somatic cell expression, including Fst and Bmp2, were identified as overexpressed within the ovarian cell population. In a general fashion, genes identified as 2.5-fold over expressed in HybSRYp-RFP expressing cells coded notably for cell signalling and extra cellular proteins. The expression of Sox10, Stc2, Fgf18, Fgf13 and Wnt6 were further characterized via whole mount in situ hybridization (WISH) on male and female genital ridges between e11.5 and e14.5. Sox10, Fgf18, Fgf13 and Stc2 gene expression was detected within the male genital ridges while Wnt6 was found diffusely within both the male and female genital ridges. These data represent the earliest comprehensive microarray expression analysis of purified presumptive pre-Sertoli cells available to date. Mol. Reprod. Dev. 74: 1491,1504, 2007. © 2007 Wiley-Liss, Inc. [source] Axin2 expression identifies progenitor cells in the murine prostateTHE PROSTATE, Issue 12 2008Christopher S. Ontiveros Abstract BACKGROUND We previously reported that prostatic stem/progenitor cells are concentrated in the proximal region of prostatic ducts and express stem cell antigen 1 (Sca-1). As Wnt signaling is important for the maintenance of stem cells, we determined whether Sca-1 expressing cells also express Axin2, as Axin2 expression is highly suggestive of active Wnt signaling. METHODS Axin2 promoter reporter mice were used for whole mount and fluorescence activated cell sorting (FACS) analysis to determine its expression in the prostate. Axin2 expressing cells were also examined for the co-expression of Sca-1. We also used a chemical activator of Wnt signaling, BIO, to determine the effects of Wnt signaling on the growth of primary prostate cells in vitro. RESULTS We show that Axin2 expression is present in all lobes and is regulated by androgens with the highest Axin2 expression in the lateral and dorsal prostate. Furthermore, a fraction of Axin2 expressing cells co-express Sca-1, suggesting that some progenitor cells have active Wnt signaling. Lastly, we demonstrate that activation of the Wnt pathway may result in increased growth, consistent with a role for Wnt signaling in maintenance and/or expansion of the progenitor cell population. CONCLUSION Axin2 expressing cells that co-express Sca-1 are present in all prostate lobes suggesting that progenitor cells reside within the Wnt active population. An understanding of the basic biology of signaling pathways mediating growth in the prostate may lead to rational therapies to treat benign prostatic hyperplasia and prostate cancer. Prostate 68:1263,1272, 2008. © 2008 Wiley-Liss, Inc. [source] Correlation of fluorescence and electron microscopy of F-actin-containing sensory cells in the epidermis of Convoluta pulchra (Platyhelminthes: Acoela)ACTA ZOOLOGICA, Issue 1 2002R Pfistermüller Abstract Phalloidin-stained whole mounts of acoel turbellarians show brightly fluorescing club-shaped structures distributed over the epidermis and concentrated especially at the anterior and posterior tips of the body. By correlating electron micrographic images and fluorescence images of Convoluta pulchra, these structures can be seen to be sensory receptors with a central cilium surrounded by a collar of microvilli. The other candidate for showing fluorescence in the epidermis, namely gland necks, can be ruled out since their distribution is too dense to resemble the distribution of the fluorescent structures seen here. The collared sensory receptors were inserted between epidermal cells, and each bore a central cilium surrounded by a collar of 6,18 microvilli and an additional centrally positioned 2,7 microvilli of which 2 or 3 were associated with a modified rootlet called the swallow's nest. Confocal scanning laser microscopy resolved the core of actin filaments within the microvilli of the collar and their rootlet-like connections to the base of the sensory cell. Such receptors could also be identified by fluorescence microscopy in several other species of acoel turbellarians. [source] Transformation of the pectoral girdle in the evolutionary origin of frogs: insights from the primitive anuran DiscoglossusJOURNAL OF ANATOMY, Issue 1 2006Pavla Havelková Abstract Using cleared-and-stained whole mounts and computer-aided three-dimensional reconstructions made from serial histological sections, we studied the development of the pectoral girdle in Discoglossus pictus, an extant member of an ancient frog lineage, represented for example by Eodiscoglossus from the Middle Jurassic to Early Cretaceous periods in Europe. Basic developmental features were compared with those of extinct Temnospondyli, considered to be the most probable anuran ancestors, and with Triadobatrachus, an early Triassic proanuran. In the endochondral girdle, the separate scapula and coracoid of Discoglossus and other anurans (completed by suprascapular and procoracoid cartilages) evolved from the compact scapulocoracoid of temnospondyls by paedomorphosis. In parallel, the dermal ossifications of the girdle were reduced to a small clavicle and cleithrum. The overall reduction in ossification of the anuran pectoral girdle supports the hypothesis of a paedomorphic origin for Anura. The almost simultaneous appearance of dermal and endochondral ossifications may be explained by the accumulation of developmental events during a short, distinct metamorphosis (which did not occur in neotenic temnospondyls living permanently in water). The sternal elements seem to be neomorphs for the most part, which help to cushion the shock of landing in jumping anurans but which also evolved as functional substitutes (insertion area for the pectoralis muscles) of the temnospondyl interclavicle. [source] Development of the pelvis and posterior part of the vertebral column in the AnuraJOURNAL OF ANATOMY, Issue 1 2005Hana Ro, ková Abstract The anuran pelvic girdle is unique among all amphibians in that its acetabular portion is located far posterior to the sacrum, lateral to the postsacral (= caudal) vertebral column, which is reduced to a single rod-like element called the urostyle. This situation in the adult is strikingly different not only from that in ancestral temnospondyls but also in other modern amphibians. Because there is no fossil that would document this evolutionary anatomical modification except for Triadobatrachus, the only data may be inferred from development in modern anurans. We chose seven anuran species (belonging to the genera Discoglossus, Bombina, Pelobates, Bufo, Rana and Xenopus), representing the principal locomotory types (saltation, swimming, crawling and burrowing). Development of the pelvic girdle was studied on cleared and stained whole mounts and partly on serial histological sections. The basic developmental pattern was similar in all species: the pelvis on both sides develops from two centres (puboischiadic and iliac, respectively). The ilium then extends vertically towards the sacral vertebra and later rotates posteriorly so that ultimately the acetabulum is lateral to the tail (= urostyle). Only minor deviations from this pattern were found, mainly associated with differences in water and terrestrial dwelling. [source] The observation of intact hepatic endothelial cells by cryo-electron microscopyJOURNAL OF MICROSCOPY, Issue 2 2003F. Braet Summary Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum-resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo-electron microscopy (cryo-EM) can be used as a good alternative for imaging samples as whole mounts. Cryo-EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 °C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde-fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae-associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation. [source] ,-Aminobutyric acid is present in a spatially discrete subpopulation of hair cells in the crista ampullaris of the toadfish Opsanus tauTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 1 2004Gay R. Holstein Abstract Although ,-aminobutyric acid (GABA) and glutamate are known to be present in the vestibular sensory epithelia of a variety of species, the functional relationship between these two transmitters is not clear. The present study addresses the three-dimensional spatial distribution of GABA and glutamate immunoreactivity in the vestibular labyrinth of the oyster toadfish by using whole end organs labeled by immunofluorescence with monoclonal anti-GABA and/or antiglutamate antibodies and visualized as whole mounts by multiphoton confocal microscopy. We find glutamate-immunoreactive hair cells present throughout the sensory epithelium. In contrast, prominent GABA immunoreactivity is restricted to a small population of hair cells located in the central region of the crista. Double immunofluorescence reveals two distinct staining patterns in GABA-labeled hair cells. Most (,80%) GABA-labeled cells show trace levels of glutamate, appropriate for the metabolic/synthetic role of cytoplasmic glutamate. The remainder of the GABA-stained cells contain substantial levels of both GABA and glutamate, suggesting transmitter colocalization. In the toadfish utricle, glutamatergic hair cells are present throughout the macula. GABA-immunoreactive hair cells follow the arc of the striola, and most GABA-labeled receptor cells coexpress glutamate. The localization of GABA was explored in other species as well. In the pigeon, GABAergic hair cells are present throughout the crista ampullaris. Our findings demonstrate that multiple, neurochemically distinct types of hair cells are present in vestibular sensory epithelia. These observations, together with the excitatory activity generally associated with 8th nerve afferent fibers, strongly suggest that GABA serves an important, specific, and complex role in determining primary afferent response dynamics. J. Comp. Neurol. 471:1,10, 2004. © 2004 Wiley-Liss, Inc. [source] Resident Macrophages are Involved in Intestinal Transplantation-Associated Inflammation and Motoric Dysfunction of the Graft MuscularisAMERICAN JOURNAL OF TRANSPLANTATION, Issue 5 2007N. Schaefer Gut manipulation and ischemia/reperfusion evoke an inflammatory response within the intestinal muscularis that contributes to dysmotility. We hypothesize that resident macrophages play a key role in initiating the inflammatory cascade. Isogenic small bowel transplantation was performed in Lewis rats. The impact of recovery of organs on muscularis inflammation was investigated by comparing cold whole-body perfusion after versus prior to recovery. The role of macrophages was investigated by transplantation of macrophage-depleted gut. Leukocytes were counted using muscularis whole mounts. Mediator expression was determined by real-time RT-PCR. Contractility was assessed in a standard organ bath. Both organ recovery and ischemia/reperfusion induced leukocyte recruitment and a significant upregulation in IL-6, MCP-1, ICAM-1 and iNOS mRNAs. Although organ recovery in cold ischemia prevented early gene expression, peak expression was not changed by modification of the recovery technique. Compared to controls, transplanted animals showed a 65% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted isografts exhibited significant less leukocyte infiltration and only a 19% decrease in contractile activity. In summary, intestinal manipulation during recovery of organs initiates a functionally relevant inflammatory response within the intestinal muscularis that is massively intensified by the ischemia reperfusion injury. Resident muscularis macrophages participate in initiating this inflammatory response. [source] | |||||||||||||