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Elwood Linney

Publications and source records attributed to Elwood Linney.

13 recordsLinked to original sources
PubMed2006

Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish.

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.

Agrin↗
PubMed2005

SNPs in the neural cell adhesion molecule 1 gene (NCAM1) may be associated with human neural tube defects.

Neural tube defects (NTDs) are common birth defects, occurring in approximately 1/1,000 births; both genetic and environmental factors are implicated. To date, no major genetic risk factors have been identified. Throughout development, cell adhesion molecules are strongly implicated in cell-cell interactions, and may play a role in the formation and closure of the neural tube. To evaluate the role of neural cell adhesion molecule 1 (NCAM1) in risk of human NTDs, we screened for novel single-nucleotide polymorphisms (SNPs) within the gene. Eleven SNPs across NCAM1 were genotyped using TaqMan. We utilized a family-based approach to evaluate evidence for association and/or linkage disequilibrium. We evaluated American Caucasian simplex lumbosacral myelomeningocele families (n=132 families) using the family based association test (FBAT) and the pedigree disequilibrium test (PDT). Association analysis revealed a significant association between risk for NTDs and intronic SNP rs2298526 using both the FBAT test (P=0.0018) and the PDT (P=0.0025). Using the HBAT version of the FBAT to look for haplotype association, all pairwise comparisons with SNP rs2298526 were also significant. A replication study set, consisting of 72 additional families showed no significant association; however, the overall trend for overtransmission of the less common allele of SNP rs2298526 remained significant in the combined sample set. In addition, we analyzed the expression pattern of the NCAM1 protein in human embryos, and while NCAM1 is not expressed within the neural tube at the time of closure, it is expressed in the surrounding and later in differentiated neurons of the CNS. These results suggest variations in NCAM1 may influence risk for human NTDs.

Gene Expression Regulation, Developmental↗
PubMed2005

Analysis of ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2 in human neural tube defects suggests a possible association with alleles in ALDH1A2.

BACKGROUND: Vitamin A (retinol), in the form of retinoic acid (RA), is essential for normal development of the human embryo. Studies in the mouse and zebrafish have shown that retinol is metabolized in the developing spinal cord and must be maintained in a precise balance along the anteroposterior axis. Both excess and deficiency of RA can affect morphogenesis, including failures of neural tube closure. METHODS: We chose to investigate 5 genes involved in the metabolism or synthesis of RA, ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2, for their role in the development of human neural tube defects, such as spina bifida. RESULTS: An association analysis using both allelic and genotypic single-locus tests revealed a significant association between the risk for spina bifida and 3 polymorphisms in the gene ALDH1A2; however, we found no evidence of a significant multilocus association. CONCLUSIONS: These results may suggest that polymorphisms in ALDH1A2 may influence the risk for lumbosacral myelomeningocele in humans.

Alleles↗
PubMed2005

Expression of cyp26b1 during zebrafish early development.

We have cloned the zebrafish ortholog of the mammalian cyp26b1 gene. The predicted zebrafish cyp26b1 protein shares greater than 73% identity with mammalian homologues. cDNA transfection assays showed that like human cyp26b1, zebrafish cyp26b1 is involved in limiting the activity of retinoic acid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of embryonic RNAs suggested that no maternal cyp26b1 message is detectable. Zygotic cyp26b1 message could be detected at 75% epiboly by RT-PCR and localized to presumptive rhombomere 3 and rhombomere 4 at the early two-somite (2S) stage (10.5 hpf: hour post fertilization) by whole mount in situ hybridization. As development proceeds expression expands anteriorly to include rhombomere 2 at the 10S stage (14hpf). By 14S (16hpf) expression in the hindbrain has also expanded posteriorly and encompasses rhombomere 2 through rhombomere 6. At later stages, 24 through 48 hpf, additional expression was found in the eyes, diencephalon, midbrain-hindbrain boundary, cerebellum, pectoral fin and the pharyngeal arch primordia.

Amino Acid Sequence↗
PubMed2004

Microarray gene expression profiling during the segmentation phase of zebrafish development.

We analyzed 15,512 unique transcripts from wild-type Danio rerio using a long oligonucleotide microarray containing >16,000 65-mers probes. Total RNA was isolated from staged embryos at 2 h intervals over a 24-h period. On average, at any given time point, 27% of the probe set detected corresponding transcripts in embryonic RNA. There were two predominant patterns in the nearly 4000 genes that changed expression in at least one time point during the first 24 hpf. At 12 hpf, we detected 420 up-regulated and 386 down-regulated genes. By 24 hpf, the number of up- and down-regulated genes had increased to 954 and 766, respectively. While the majority of these genes maintained their new level of expression for the duration of the time course, we identified five genes with phasic regulation over the 24-h time course. Two of these genes, germ cell nuclear factor and mesogenin, have been identified as being expressed during gastrulation (5 1/4 to 10 h postfertilization) and subsequently repressed. A cluster containing 36 distinct ribosomal proteins was up-regulated at 12 h, indicating a capability for de novo protein synthesis during and after this stage. Twenty-three muscle-specific genes were up-regulated late during the initial 24 hpf, corresponding to the development and differentiation of the somites.

Animals↗
PubMed2004

Feedback mechanisms regulate retinoic acid production and degradation in the zebrafish embryo.

Retinoic acid (RA) signaling in vertebrate embryos occurs in a distinct physical and temporal pattern. Regulating this spatial distribution is crucial to the development of the embryo, as RA in excess or in inappropriate tissues is teratogenic. In order to understand how RA availability is determined in zebrafish we have investigated the expression of cyp26a1, an enzyme that inactivates RA, and its relationship to raldh2, one of the enzymes that produce RA from retinal. cyp26a1 expression follows three phases: in presumptive anterior neurectoderm and in a circumblastoporal ring during gastrulation, in the tailbud throughout somitogenesis, and in multiple specific tissue types beginning at mid-somitogenesis and continuing through 48 h postfertilization (hpf). This expression was either adjacent or opposite to those tissues expressing raldh2. We then investigated how RA production might regulate these relationships. Endogenous RA produced by raldhs did not play a role in setting cyp26a1 expression in most tissues. However, exogenous RA regulates expression of both enzymes. cyp26a1 is up regulated in the embryo in a time, concentration, and tissue-dependent manner. Conversely, raldh2 expression is reduced with RA treatment. Tests of the raldh2 promoter in cell transfections proved that RA directly represses its activity. These data demonstrate that the feedback mechanisms regulating production and degradation of RA must be considered in any experiments altering levels of RA in the developing vertebrate embryo.

Aldehyde Oxidoreductases↗
PubMed2003

Windows into development: historic, current, and future perspectives on transgenic zebrafish.

The recent explosion of transgenic zebrafish lines in the literature demonstrates the value of this model system for detailed in vivo analysis of gene regulation and morphogenetic movements. The optical clarity and rapid early development of zebrafish provides the ability to follow these events as they occur in live, developing embryos. This article will review the development of transgenic technology in zebrafish as well as the current and future uses of transgenic zebrafish to explore the dynamic environment of the developing vertebrate embryo.

Animals↗
PubMed2002

Genomic structure and embryonic expression of estrogen receptor beta a (ERbetaa) in zebrafish (Danio rerio).

Estrogenic steroid hormones mediate complex actions important in both embryonic and adult life. The hormones signal through ligand-inducible transcription factors known as estrogen receptors (ERs). In this study, we have isolated a zebrafish estrogen receptor with homology to human estrogen receptor beta (ERbeta). This zebrafish ERbeta (ERbetaa) has a conserved genomic structure of eight coding exons with boundaries similar to those of human ERbeta. The coding exon structures of two other zebrafish estrogen receptors (ERalpha and ERbetab) are presented as well. We also analyzed 3.3 kb of the promoter region and identified numerous putative transcription factor binding sites, including SP1 and ER half sites. Zebrafish ERbetaa message RNA is maternally loaded, but quickly degraded after fertilization, as detected by reverse transcriptase polymerase chain reaction. ERbetaa transcripts are detected again between 24 and 48 h post fertilization. These results indicate that ERbeta has been highly conserved during evolution and is likely used during later embryogenesis in zebrafish. Future identification of the expression levels and patterns of this and other estrogen receptors in zebrafish will allow a better understanding of estrogen signaling during embryogenesis.

Amino Acid Sequence↗
PubMed2002

Isolation and functional analysis of the mouse RXRgamma1 gene promoter in anterior pituitary cells.

The retinoid X receptor (RXR) isoform RXRgamma has limited tissue expression, including brain, skeletal muscle, and anterior pituitary gland. Within the anterior pituitary gland, RXRgamma expression is limited primarily to the thyrotropes. In this report, we have isolated approximately 3 kb of 5'-flanking DNA of the mouse RXRgamma1 gene. We have identified the major transcription start site in the thyrotrope-derived TtT-97 cells. Transient transfection studies show that a 1.4-kb promoter fragment has full promoter activity in TtT-97 cells. This promoter has much less activity in thyrotrope-derived alphaTSH cells, pituitary-derived GH3 somatomammotropes, and non-pituitary CV-1 cells. None of these cell lines has detectable RXRgamma1 mRNA. A previous report has identified a non-consensus direct repeat (DR-1) element in the RXRgamma2 gene promoter region that mediates stimulation of promoter activity by 9-cis-retinoic acid (9-cis-RA). Inspection of the RXRgamma1 promoter region revealed a non-consensus DR-1 element at -232 bp from the transcription start site. Interestingly, RXRgamma1 promoter activity was suppressed 50% by 9-cis-RA in the TtT-97 thyrotropes. Further experiments in non-pituitary cells showed that suppression of RXRgamma1 promoter activity was RXR-dependent. Mutagenesis of the DR-1 element abrogated suppression of promoter activity by 9-cis-RA, suggesting that this negative regulation requires both RXR and this specific DR-1 element. In summary, we have isolated the mouse RXRgamma1 gene promoter region and identified the major start site in thyrotropes. Promoter activity is uniquely suppressed by 9-cis-RA through a DR-1 element. Isolation and characterization of the mouse RXRgamma1 promoter region provides a tool for further investigation focusing on thyrotrope-specific gene expression as well as negative regulation of genes by retinoic acid.

5' Untranslated Regions↗
PubMedDate not supplied

Developmental chlorpyrifos effects on hatchling zebrafish swimming behavior.

Chlorpyrifos (CPF), a widely used organophosphate insecticide and potent acetylcholinesterase inhibitor, interferes with neurobehavioral development. Rat models have been key in demonstrating that developmental CPF exposure causes learning deficits and locomotor activity alterations, which persist into adulthood. Complementary nonmammalian models can be useful in determining the neurodevelopmental mechanisms underlying these persisting behavioral effects. Zebrafish (Danio rerio) with their clear chorion and extensive developmental information base provide an excellent model for assessment of molecular processes of toxicant-impacted neurodevelopment. We have developed methods for assessing spatial discrimination learning in adult zebrafish and have documented persisting effects of developmental CPF exposure on swimming activity and learning after low and high doses of CPF (10 and 100 ng/ml) administered to zebrafish embryos on Days 1-5 postfertilization (pf). In the current study, we developed methods for behavioral assessment of CPF exposure on swimming activity in newly hatched zebrafish. An equal area segmented annular grid (concentric circles divided into quadrants through the diameter) was made in a 16-mm diameter cylinder. The test area was placed on a heating device secured to an Olympus SZH10 dissecting scope stage. Zebrafish embryos were exposed to 10 ng/ml CPF, 100 ng/ml CPF, or vehicle control (25 microl/ml DMSO) (n=8-10/treatment group). Each treatment group was kept in a total volume of 25 ml of egg water (60 mg/ml Instant Ocean) including DMSO with or without CPF mixed to above dilutions in an incubator set at 28.5 degrees C. CPF dilutions or vehicle were changed daily with exposure ending on Day 5 pf. Testing of larval zebrafish was performed on Days 6 and 9 pf. The fish were placed in the test cylinder with 1.5 ml of egg H(2)O (28.5 degrees C). After a 2-min acclimation period, the swimming activity of the fish was measured for a 3-min testing session. The 100 ng/ml CPF dose caused significant slowing of swimming activity on Days 6 and 9 pf and had persisting effects of impairing spatial discrimination and decreasing response latency in adulthood. Developmental exposure to 10 ng/ml of CPF did not cause a significant change in locomotor activity during the period soon after hatching. CPF exposure during early development caused clear behavioral impairments detectable during the posthatching period. In a previous study, we found that early developmental CPF exposure caused behavioral alterations in zebrafish, which lasted throughout adulthood. The molecular mechanisms by which early developmental CPF exposure produces these behavioral impairments expressed in adulthood can now be studied in the zebrafish model.

Animals↗
PubMedDate not supplied

Zebrafish as a neurotoxicological model.

At a time when common regulatory pathways are being identified in several different species and genomics is beginning to allow comparisons of genes, how they are arranged on chromosomes and how they are regulated, zebrafish has emerged as a valuable and complementary vertebrate model. Some of the characteristics that prove of value are described and illustrated. Fluorescent transgenic lines of zebrafish embryos are presented for time-line studies with neurotoxicants. While genetic knockout technology has yet to be developed for the model, the anti-sense, morpholino approach allows for knockdown of expression of genes for the 3 day, embryonic period. This can provide for phenocopies of mutant genes for those genes essential to embryonic development or it can provide for a limited inhibition of gene expression that allows subsequent development of the fish. With the zebrafish genomic sequencing effort, microarray technology is now developing for the model system. These resources and technologies allow one to challenge the system with toxicants, and to view the immediate effects of the toxicants with transgenic embryos that fluoresce in part or all of the nervous system. Behavioral and learning protocols have been developed for the organism so that early exposures can be assayed for effects upon adult fish. Microarray technology should allow for one to identify specific genes and pathways affected by a neurotoxicant. In the future, these approaches should provide a working protocol for exploring molecular mechanisms of neurotoxicants. This type of complementary approach should then allow for more efficient examination and testing of mechanisms in mammalian models.

Animals↗
PubMedDate not supplied

Chlorpyrifos exposure of developing zebrafish: effects on survival and long-term effects on response latency and spatial discrimination.

Chlorpyrifos (CPF) is a widely used insecticide, which has been shown to interfere with neurobehavioral development. Rat models have been key in demonstrating that prenatal CPF exposure causes choice accuracy deficits and motor alterations, which persist into adulthood. Complementary nonmammalian models can be useful in determining the molecular mechanisms underlying the persisting behavioral effects of developmental CPF exposure. Zebrafish with their clear chorion and extensive developmental information base provide an excellent model for assessment of molecular processes of toxicant impacted neurodevelopment. To facilitate the use of the zebrafish model and to compare it to the more typical rodent models, the behavioral phenotype of CPF toxicity in zebrafish must be well characterized. Our laboratory has developed methods for assessing spatial discrimination learning in zebrafish, which can differentiate response latency from choice accuracy in a three chambered fish tank. Low and high doses of CPF (10 and 100 ng/ml on days 1-5 postfertilization) both had significant persisting effects on both spatial discrimination and response latency over 18 weeks of testing. The high, but not the low dose, significantly accelerated mortality rates of the fish during the study from 20-38 weeks of age. Developmental exposure to either 10 or 100 ng/ml of CPF caused significant spatial discrimination impairments in zebrafish when they were adults. The impairment caused by 10 ng/ml was seen during early but not later testing, while the impairment caused by 100 ng/ml became more pronounced with continued testing. The higher dose caused a more pervasive impairment. The 10 and 100 ng/ml doses had opposite effects on response latency. The low 10 ng/ml dose significantly slowed response latency, while the high 100 ng/ml dose significant increased response latency. Both of these effects diminished with continued testing. CPF exposure during early development caused clear behavioral impairments, which lasted throughout adulthood in zebrafish. The molecular mechanisms by which early developmental CPF exposure produces these behavioral impairments expressed in adulthood can now be studied in the zebrafish model.

Animals↗