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In JoVE (1)
Other Publications (14)
- International Journal of Cancer. Journal International Du Cancer
- Clinical Chemistry and Laboratory Medicine : CCLM / FESCC
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Clinical Chemistry and Laboratory Medicine : CCLM / FESCC
- Stroke; a Journal of Cerebral Circulation
- Human Genetics
- CSH Protocols
- Transfusion
- Developmental Biology
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- PLoS Biology
- Genesis (New York, N.Y. : 2000)
- PLoS Biology
- The Journal of Comparative Neurology
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Articles by Sandra Rieger in JoVE
JoVE General
שני הפוטונים axotomy ואת זמן לשגות הדמיה confocal עוברי דג הזברה לחיות
1Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, 2Departments of Neurology and Neurobiology, University of California, Los Angeles
כאן אנו מתארים שיטה הרכבה עוברי דג הזברה הדמיה לטווח ארוך, שני הפוטונים הדמיה רקמות נזק טכניקות זמן לשגות הדמיה confocal.
Other articles by Sandra Rieger on PubMed
Identification and Validation of Novel ERBB2 (HER2, NEU) Targets Including Genes Involved in Angiogenesis
V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2; synonyms HER2, NEU) encodes a transmembrane glycoprotein with tyrosine kinase-specific activity that acts as a major switch in different signal-transduction processes. ERBB2 amplification and overexpression have been found in a number of human cancers, including breast, ovary and kidney carcinoma. Our aim was to detect ERBB2-regulated target genes that contribute to its tumorigenic effect on a genomewide scale. The differential gene expression profile of ERBB2-transfected and wild-type mouse fibroblasts was monitored employing DNA microarrays. Regulated expression of selected genes was verified by RT-PCR and validated by Western blot analysis. Genome wide gene expression profiling identified (i) known targets of ERBB2 signaling, (ii) genes implicated in tumorigenesis but so far not associated with ERBB2 signaling as well as (iii) genes not yet associated with oncogenic transformation, including novel genes without functional annotation. We also found that at least a fraction of coexpressed genes are closely linked on the genome. ERBB2 overexpression suppresses the transcription of antiangiogenic factors (e.g., Sparc, Timp3, Serpinf1) but induces expression of angiogenic factors (e.g., Klf5, Tnfaip2, Sema3c). Profiling of ERBB2-dependent gene regulation revealed a compendium of potential diagnostic markers and putative therapeutic targets. Identification of coexpressed genes that colocalize in the genome may indicate gene regulatory mechanisms that require further study to evaluate functional coregulation. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.)
Simultaneous Analysis of MDR1 C3435T, G2677T/A, and C1236T Genotypes by Multiplexed Mutagenically Separated PCR
P-glycoprotein (PGP) encoded by the multi-drug-resistance 1 (MDR1) gene is a member of the ATP-binding cassette (ABC) transporter family, drug-transporting proteins involved in the bioavailability and pharmacokinetics of various drugs. Several single nucleotide polymorphisms (SNPs) in the MDR1 gene have been identified so far that may influence PGP expression levels and function. Thus, genotyping for MDR1 polymorphisms and determining specific haplotypes may become an important tool in predicting individual susceptibility to developing drug resistance. We developed a new multiplexed allele-specific PCR method based on the principle of mutagenically separated PCR (MS-PCR) for rapid and reliable simultaneous genotyping of the C3435T polymorphism in exon 26 of the MDR1 gene and two additional SNPs (G2677T/A in exon 21 and C1236T in exon 12), which are in linkage disequilibrium with MDR1 C3435T. The accuracy and reliability of this method was confirmed by sequencing the respective regions in the MDR1 gene. This newly developed MDR1 MS-PCR will facilitate fast, accurate and economic analysis of MDR1 genotypes and will provide important information in optimizing individual therapeutic approaches.
Quantum Dots Are Powerful Multipurpose Vital Labeling Agents in Zebrafish Embryos
Recently, inorganic fluorescent contrast agents composed of semiconductor materials have been introduced to biological imaging approaches. These so-called quantum dots provide unique and promising properties unreached by organic fluorophores, but their use as contrast agents within live organisms has been limited, probably due in part to concerns about their in vivo tolerance. Using transparent zebrafish embryos, we challenged quantum dots with a series of intravital imaging problems. We show that quantum dots provide a high fluorescent yield within targeted tissues, possess immense photostability, can be targeted to specific subcellular compartments, remain within targeted cells as lineage tracers, are easily separable from conventional organic fluorescent dyes, and are fixable, allowing them to be used in combination with immunohistochemistry after live recordings. Thus, quantum dots combine the specific advantages of different organic fluorescent contrast agents and promise to become the first fluorophore feasible for long-lasting intravital time-lapse studies. Finally, we show by co-labeling blood vessels of the vasculature and major axon tracts of the nervous system that, for establishing these networks, the same guidance cues might be used in some body parts, whereas in others, both networks appear to develop independently from one another. Thus, the bright fluorescence of quantum dots will help to unravel many open questions in the fields of embryology, cell biology, as well as phenotyping and disease diagnosis.
Reference Materials (RMs) for Analysis of the Human Factor II (prothrombin) Gene G20210A Mutation
The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.
The Thr715Pro Polymorphism of the P-selectin Gene is Not Associated with Ischemic Stroke Risk
A Thr>Pro polymorphism at codon 715 in the coding region of the P-selectin gene has recently been described. Individuals carrying the Pro715 allele were reported to have a reduced risk of myocardial infarction. A possible association of this polymorphism with the risk of ischemic stroke is currently under discussion.
Protein C Promoter Polymorphisms Associate with Sepsis in Children with Systemic Meningococcemia
Meningococcal disease may present as sepsis, meningitis or a combination of both. Protein C (PC) is an important regulator of thrombin activity. Two polymorphisms in the promoter region of PC (C-1654T, A-1641G) have been shown to affect PC levels. In patients with meningococcal sepsis, low PC levels have been correlated with increased severity and poor outcome. We established a multicenter case-control study to determine whether PC promoter polymorphisms are associated with occurrence and outcome of meningococcal disease and sepsis. 288 previously healthy children with meningococcal infection from 97 pediatric hospitals in Germany, Switzerland, Italy, and Austria and 309 healthy controls were included in the study. A strong age-dependant effect was found. Patients younger than 1 year carried significantly more often the CG-CG genotype than healthy controls (28.6% vs. 17.8%, P = 0.04). Carriers of the CG allele showed a 3.43-fold increased odds ratio (OR) to develop sepsis (95% CI: 1.05-11.20; 85.7% vs. 63.6%, P = 0.036). The TA-TA genotype conferred a protective role for the development of sepsis (P = 0.017) with a Haldane OR of 0.09 (95% CI: 0.01-0.94). Systolic blood pressure values were significantly decreased in patients carrying the CG-CG genotype (70 vs. 86 mmHg, P = 0.005), and the need for adrenergic support significantly higher (70% vs. 26%, P = 0.018), resulting in an OR of 6.61 (95% CI: 1.28-34.14). These findings show that in young children PC promoter genotype is associated with susceptibility for meningococcal disease, the development of meningococcal sepsis, lower blood pressure, and need for adrenergic support.
Preparation of Zebrafish Embryos for Transmission Electron Microscopy
INTRODUCTIONThis protocol describes a procedure for the fixation and embedding of zebrafish embryos at organogenesis stages (48-72 hours post-fertilization [hpf]) for transmission electron microscopy (TEM). The lengths of individual steps may be adjusted according to the developmental stage of the specimen.
Neutralization of Human Parvovirus B19 by Plasma and Intravenous Immunoglobulins
Human parvovirus B19 (B19V) is a highly prevalent pathogen, and plasma pools for manufacturing of plasma-derived products have been shown to contain antibodies against B19V (B19V immunoglobulin G [IgG]).
The Zebrafish Cerebellar Rhombic Lip is Spatially Patterned in Producing Granule Cell Populations of Different Functional Compartments
The upper rhombic lip, a prominent germinal zone of the cerebellum, was recently demonstrated to generate different neuronal cell types over time from spatial subdomains. We have characterized the differentiation of the upper rhombic lip derived granule cell population in stable GFP-transgenic zebrafish in the context of zebrafish cerebellar morphogenesis. Time-lapse analysis followed by individual granule cell tracing demonstrates that the zebrafish upper rhombic lip is spatially patterned along its mediolateral axis producing different granule cell populations simultaneously. Time-lapse recordings of parallel fiber projections and retrograde labeling reveal that spatial patterning within the rhombic lip corresponds to granule cells of two different functional compartments of the mature cerebellum: the eminentia granularis and the corpus cerebelli. These cerebellar compartments in teleosts correspond to the mammalian vestibulocerebellar and non-vestibulocerebellar system serving balance and locomotion control, respectively. Given the high conservation of cerebellar development in vertebrates, spatial partitioning of the mammalian granule cell population and their corresponding earlier-produced deep nuclei by patterning within the rhombic lip may also delineate distinct functional compartments of the cerebellum. Thus, our findings offer an explanation for how specific functional cerebellar circuitries are laid down by spatio-temporal patterning of cerebellar germinal zones during early brain development.
Polysialyltransferase Expression is Linked to Neuronal Migration in the Developing and Adult Zebrafish
Modulation of cell-cell adhesion is crucial for regulating neuronal migration and maintenance of structural plasticity in the embryonic and mature brain. Such modulation can be obtained by the enzymatic attachment of polysialic acid (PSA) to the neural cell adhesion molecule (NCAM) by means of the polysialyltransferases STX and PST. Thus, differential expression of STX and PST is likely to be responsible for varying functions of PSA-NCAM during neuronal differentiation, maintenance, plasticity, and regeneration. We have isolated the zebrafish homologues of STX (St8sia2) and PST (St8sia4) and demonstrate that their expression in the embryonic and adult nervous system is often confined to regions of neuronal migration. Moreover, in the adult cerebellum, the complementary expression pattern of both polysialyltransferases suggests a function in regulating cerebellar neuronal plasticity. Enzymatic removal of PSA in the embryonic cerebellum results in impaired neuronal migration, suggesting that PSA-NCAM is a key regulator of motility for cerebellar neuronal progenitors.
Cadherin-2 Controls Directional Chain Migration of Cerebellar Granule Neurons
Long distance migration of differentiating granule cells from the cerebellar upper rhombic lip has been reported in many vertebrates. However, the knowledge about the subcellular dynamics and molecular mechanisms regulating directional neuronal migration in vivo is just beginning to emerge. Here we show by time-lapse imaging in live zebrafish (Danio rerio) embryos that cerebellar granule cells migrate in chain-like structures in a homotypic glia-independent manner. Temporal rescue of zebrafish Cadherin-2 mutants reveals a direct role for this adhesion molecule in mediating chain formation and coherent migratory behavior of granule cells. In addition, Cadherin-2 maintains the orientation of cell polarization in direction of migration, whereas in Cadherin-2 mutant granule cells the site of leading edge formation and centrosome positioning is randomized. Thus, the lack of adhesion leads to impaired directional migration with a mispositioning of Cadherin-2 deficient granule cells as a consequence. Furthermore, these cells fail to differentiate properly into mature granule neurons. In vivo imaging of Cadherin-2 localization revealed the dynamics of this adhesion molecule during cell locomotion. Cadherin-2 concentrates transiently at the front of granule cells during the initiation of individual migratory steps by intramembraneous transport. The presence of Cadherin-2 in the leading edge corresponds to the observed centrosome orientation in direction of migration. Our results indicate that Cadherin-2 plays a key role during zebrafish granule cell migration by continuously coordinating cell-cell contacts and cell polarity through the remodeling of adherens junctions. As Cadherin-containing adherens junctions have been shown to be connected via microtubule fibers with the centrosome, our results offer an explanation for the mechanism of leading edge and centrosome positioning during nucleokinetic migration of many vertebrate neuronal populations.
Time-lapse Imaging of Neural Development: Zebrafish Lead the Way into the Fourth Dimension
Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults.
Hydrogen Peroxide Promotes Injury-induced Peripheral Sensory Axon Regeneration in the Zebrafish Skin
Functional recovery from cutaneous injury requires not only the healing and regeneration of skin cells but also reinnervation of the skin by somatosensory peripheral axon endings. To investigate how sensory axon regeneration and wound healing are coordinated, we amputated the caudal fins of zebrafish larvae and imaged somatosensory axon behavior. Fin amputation strongly promoted the regeneration of nearby sensory axons, an effect that could be mimicked by ablating a few keratinocytes anywhere in the body. Since injury produces the reactive oxygen species hydrogen peroxide (H(2)O(2)) near wounds, we tested whether H(2)O(2) influences cutaneous axon regeneration. Exposure of zebrafish larvae to sublethal levels of exogenous H(2)O(2) promoted growth of severed axons in the absence of keratinocyte injury, and inhibiting H(2)O(2) production blocked the axon growth-promoting effects of fin amputation and keratinocyte ablation. Thus, H(2)O(2) signaling helps coordinate wound healing with peripheral sensory axon reinnervation of the skin.
Coordinate Development of Skin Cells and Cutaneous Sensory Axons in Zebrafish
Peripheral sensory axons innervate the epidermis early in embryogenesis to detect touch stimuli. To characterize the time course of cutaneous innervation and the nature of interactions between sensory axons and skin cells at early developmental stages, we conducted a detailed analysis of cutaneous innervation in the head, trunk, and tail of zebrafish embryos and larvae from 18 to 78 hours postfertilization. This analysis combined live imaging of fish expressing transgenes that highlight sensory neurons and skin cells, transmission electron microscopy (TEM), and serial scanning electron microscopy (sSEM). In zebrafish, the skin initially consists of two epithelial layers, and all of the axons in the first wave of innervation are free endings. Maturation of the epithelium coincides with, but does not depend on, its innervation by peripheral sensory axons. We found that peripheral axons initially arborize between the two epithelial skin layers, but not within the basal lamina, as occurs in other organisms. Strikingly, as development proceeds, axons become tightly enveloped within basal keratinocytes, an arrangement suggesting that keratinocytes may serve structural or functional roles, akin to Schwann cells, in somatosensation mediated by these sensory neurons.
