95 results on '"Cole GJ"'
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2. THE CELLULAR AND HUMORAL RESPONSE TO ANTIGENS IN LAMBS THYMECTOMIZED IN UTERO.
- Author
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Cole, GJ and Morris, Bede
- Published
- 1971
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3. HOMOGRAFT REJECTION AND HYPERSENSITIVITY REACTIONS IN LAMBS THYMECTOMIZED IN UTERO.
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Cole, GJ and Morris, Bede
- Published
- 1971
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4. THE NORMAL LYMPHOCYTE TRANSFER REACTION IN LAMBS THYMECTOMIZED IN UTERO.
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Cole, GJ and Morris, Bede
- Published
- 1971
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5. TECHNIQUES FOR THE LONG-TERM COLLECTION OF LYMPH FROM THE UNANAESTHETIZED FOETAL LAMB IN UTERO.
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Smeaton, TC, Cole, GJ, Simpson-morgan, MW, and Morris, Bede
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- 1969
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6. Embryonic Ethanol but Not Cannabinoid Exposure Affects Zebrafish Cardiac Development via Agrin and Sonic Hedgehog Interaction.
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Zhang C, Ezem N, Mackinnon S, and Cole GJ
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- Animals, Ethanol toxicity, Ethanol metabolism, Hedgehog Proteins metabolism, Agrin metabolism, Edema, Cardiac, Morpholinos pharmacology, Heart, Zebrafish genetics, Cannabinoids metabolism
- Abstract
Recent studies demonstrate the adverse effects of cannabinoids on development, including via pathways shared with ethanol exposure. Our laboratory has shown that both the nervous system and cardiac development are dependent on agrin modulation of sonic hedgehog (shh) and fibroblast growth factor (Fgf) signaling pathways. As both ethanol and cannabinoids impact these signaling molecules, we examined their role on zebrafish heart development. Zebrafish embryos were exposed to a range of ethanol and/or cannabinoid receptor 1 and 2 agonist concentrations in the absence or presence of morpholino oligonucleotides that disrupt agrin or shh expression. In situ hybridization was employed to analyze cardiac marker gene expression. Exposure to cannabinoid receptor agonists disrupted midbrain-hindbrain boundary development, but had no effect on heart development, as assessed by the presence of cardiac edema or the altered expression of cardiac marker genes. In contrast, exposure to 1.5% ethanol induced cardiac edema and the altered expression of cardiac marker genes. Combined exposure to agrin or shh morpholino and 0.5% ethanol disrupted the cmlc2 gene expression pattern, with the restoration of the normal expression following shh mRNA overexpression. These studies provide evidence that signaling pathways critical to heart development are sensitive to ethanol exposure , but not cannabinoid s, during early zebrafish embryogenesis.
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- 2023
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7. Pharmacological activation of the Sonic hedgehog pathway with a Smoothened small molecule agonist ameliorates the severity of alcohol-induced morphological and behavioral birth defects in a zebrafish model of fetal alcohol spectrum disorder.
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Burton DF, Boa-Amponsem OM, Dixon MS, Hopkins MJ, Herbin TA, Toney S, Tarpley M, Rodriguez BV, Fish EW, Parnell SE, Cole GJ, and Williams KP
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- Animals, Embryo, Nonmammalian metabolism, Ethanol toxicity, Female, Hedgehog Proteins metabolism, Humans, Pregnancy, Zebrafish metabolism, Fetal Alcohol Spectrum Disorders drug therapy, Fetal Alcohol Spectrum Disorders metabolism, Prenatal Exposure Delayed Effects
- Abstract
Ethanol exposure during the early stages of embryonic development can lead to a range of morphological and behavioral differences termed fetal alcohol spectrum disorders (FASDs). In a zebrafish model, we have shown that acute ethanol exposure at 8-10 hr postfertilization (hpf), a critical time of development, produces birth defects similar to those clinically characterized in FASD. Dysregulation of the Sonic hedgehog (Shh) pathway has been implicated as a molecular basis for many of the birth defects caused by prenatal alcohol exposure. We observed in zebrafish embryos that shh expression was significantly decreased by ethanol exposure at 8-10 hpf, while smo expression was much less affected. Treatment of zebrafish embryos with SAG or purmorphamine, small molecule Smoothened agonists that activate Shh signaling, ameliorated the severity of ethanol-induced developmental malformations including altered eye size and midline brain development. Furthermore, this rescue effect of Smo activation was dose dependent and occurred primarily when treatment was given after ethanol exposure. Markers of Shh signaling (gli1/2) and eye development (pax6a) were restored in embryos treated with SAG post-ethanol exposure. Since embryonic ethanol exposure has been shown to produce later-life neurobehavioral impairments, juvenile zebrafish were examined in the novel tank diving test. Our results further demonstrated that in zebrafish embryos exposed to ethanol, SAG treatment was able to mitigate long-term neurodevelopmental impairments related to anxiety and risk-taking behavior. Our results indicate that pharmacological activation of the Shh pathway at specific developmental timing markedly diminishes the severity of alcohol-induced birth defects., (© 2022 Wiley Periodicals LLC.)
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- 2022
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8. Highlighting the contributions of Minority Serving Institutions to neuroscience.
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Cole GJ and Marshall SA
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- Hospitals, Minority Groups, Neurosciences
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- 2022
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9. Ethanol and Cannabinoids Regulate Zebrafish GABAergic Neuron Development and Behavior in a Sonic Hedgehog and Fibroblast Growth Factor-Dependent Mechanism.
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Boa-Amponsem O, Zhang C, Burton D, Williams KP, and Cole GJ
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- Animals, Behavior, Animal drug effects, Embryo, Nonmammalian, Gene Expression, Glutamate Decarboxylase drug effects, Glutamate Decarboxylase genetics, Hedgehog Proteins drug effects, In Situ Hybridization, Morpholinos, Neurogenesis genetics, Real-Time Polymerase Chain Reaction, Receptor, Cannabinoid, CB1 agonists, Risk-Taking, Zebrafish, Zebrafish Proteins drug effects, Cannabinoid Receptor Agonists pharmacology, Central Nervous System Depressants pharmacology, Ethanol pharmacology, Fibroblast Growth Factors genetics, GABAergic Neurons drug effects, Hedgehog Proteins genetics, Neurogenesis drug effects, Zebrafish Proteins genetics
- Abstract
Background: Ethanol (EtOH) has diverse effects on nervous system development, which includes development and survival of GABAergic neurons in a sonic hedgehog (Shh) and fibroblast growth factor (Fgf)-dependent mechanism. Cannabinoids also function as inhibitors of Shh signaling, raising the possibility that EtOH and cannabinoids may interact to broadly disrupt neuronal function during brain development., Methods: Zebrafish embryos were exposed to a range of EtOH and/or cannabinoid receptor 1 (CB1R) agonist concentrations at specific developmental stages, in the absence or presence of morpholino oligonucleotides that disrupt shh expression. In situ hybridization was employed to analyze glutamic acid decarboxylase (gad1) gene expression as a marker of GABAergic neuron differentiation, and zebrafish behavior was analyzed using the novel tank diving test as a measure of risk-taking behavior., Results: Combined acute subthreshold EtOH and CB1R agonist exposure results in a marked reduction in gad1 mRNA expression in zebrafish forebrain. Consistent with the EtOH and cannabinoid effects on Shh signaling, fgf8 mRNA overexpression rescues the EtOH- and cannabinoid-induced decrease in gad1 gene expression and also prevents the changes in behavior induced by EtOH and cannabinoids., Conclusions: These studies provide evidence that forebrain GABAergic neuron development and zebrafish risk-taking behavior are sensitive to both EtOH and cannabinoid exposure in a Shh- and Fgf-dependent mechanism, and provide additional evidence that a signaling pathway involving Shh and Fgf crosstalk is a critical target of EtOH and cannabinoids in FASD., (© 2020 by the Research Society on Alcoholism.)
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- 2020
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10. Cannabinoids Exacerbate Alcohol Teratogenesis by a CB1-Hedgehog Interaction.
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Fish EW, Murdaugh LB, Zhang C, Boschen KE, Boa-Amponsem O, Mendoza-Romero HN, Tarpley M, Chdid L, Mukhopadhyay S, Cole GJ, Williams KP, and Parnell SE
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- Animals, Ethanol adverse effects, Ethanol pharmacology, Female, Fetal Alcohol Spectrum Disorders pathology, Mice, Smoothened Receptor metabolism, Cannabinoids toxicity, Fetal Alcohol Spectrum Disorders metabolism, Hedgehog Proteins metabolism, Receptor, Cannabinoid, CB1 metabolism, Signal Transduction drug effects, Teratogenesis drug effects
- Abstract
We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ
9 -THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.- Published
- 2019
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11. Ethanol and cannabinoids interact to alter behavior in a zebrafish fetal alcohol spectrum disorder model.
- Author
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Boa-Amponsem O, Zhang C, Mukhopadhyay S, Ardrey I, and Cole GJ
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- Animals, Cannabinoids pharmacology, Disease Models, Animal, Embryo, Nonmammalian pathology, Ethanol pharmacology, Fetal Alcohol Spectrum Disorders pathology, Fetal Alcohol Spectrum Disorders physiopathology, Cannabinoids adverse effects, Embryo, Nonmammalian embryology, Ethanol adverse effects, Fetal Alcohol Spectrum Disorders metabolism, Gene Expression Regulation, Developmental drug effects, Zebrafish embryology, Zebrafish Proteins biosynthesis
- Abstract
Background: Recent work suggests that endocannabinoids (eCBs) may signal through the sonic hedgehog signaling pathway. We therefore hypothesized that combined ethanol and eCB exposure during defined stages of zebrafish embryogenesis will produce deficits comparable to human fetal alcohol spectrum disorder (FASD)., Methods: Zebrafish embryos were exposed to ethanol or cannabinoid agonists alone or in combination at defined developmental stages and assessed for changes in brain morphology or expression of marker genes such as pax6a. Juvenile fish were then assessed for risk-taking/anxiety-like behavior using the novel tank dive test., Results: Either chronic or acute exposure to high doses of the CB1R agonist ACEA resulted in FASD phenotypes. However, acute subthreshold doses of CB1R agonist alone, or combined with 0.5% ethanol, did not induce morphological phenotypes, but did induce dysmorphogenesis when combined with acute 1% ethanol. Phenotypes were rescued using the CB1R antagonist SR141716A. In addition, JZL195, a dual inhibitor of FAAH and MAGL, two degradative enzymes for eCBs, induced FASD phenotypes in the presence of subthreshold ethanol, confirming the activation of common signaling pathways by ethanol and eCBs. We next analyzed the effects of ethanol and CB1R agonist on juvenile zebrafish behavior and show that ACEA or ethanol alone did not alter behavior, but combined ACEA and ethanol increased risk-taking behavior., Conclusions: These studies demonstrate that pathological and behavioral phenotypes associated with FASD are induced by exposure to CB1R agonists and suggest that combined exposure to lower levels of alcohol and marijuana may be capable of inducing FASD-like morphological and behavioral impairments., (© 2019 Wiley Periodicals, Inc.)
- Published
- 2019
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12. Comparison of molecular marker expression in early zebrafish brain development following chronic ethanol or morpholino treatment.
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Zhang C, Boa-Amponsem O, and Cole GJ
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- Age Factors, Animals, Brain anatomy & histology, Brain embryology, Embryo, Mammalian, Eye drug effects, Eye embryology, Eye metabolism, In Situ Hybridization, Morpholines pharmacology, Tretinoin metabolism, Zebrafish, Zebrafish Proteins genetics, Brain drug effects, Brain metabolism, Central Nervous System Depressants pharmacology, Ethanol pharmacology, Gene Expression Regulation, Developmental drug effects, Zebrafish Proteins metabolism
- Abstract
This study was undertaken to ascertain whether defined markers of early zebrafish brain development are affected by chronic ethanol exposure or morpholino knockdown of agrin, sonic hedgehog, retinoic acid, and fibroblast growth factors, four signaling molecules that are suggested to be ethanol sensitive. Zebrafish embryos were exposed to 2% ethanol from 6 to 24 hpf or injected with agrin, shha, aldh1a3, or fgf8a morpholinos. In situ hybridization was employed to analyze otx2, pax6a, epha4a, krx20, pax2a, fgf8a, wnt1, and eng2b expression during early brain development. Our results showed that pax6a mRNA expression was decreased in eye, forebrain, and hindbrain of both chronic ethanol exposed and select MO treatments. Epha4a expression in rhombomere R1 boundary was decreased in chronic ethanol exposure and aldh1a3 morphants, lost in fgf8a morphants, but largely unaffected in agrin and shha morphants. Ectopic pax6a and epha4a expression in midbrain was only found in fgf8a morphants. These results suggest that while chronic ethanol induces obvious morphological change in brain architecture, many molecular markers of these brain structures are relatively unaffected by ethanol exposure.
- Published
- 2017
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13. Long-term behavioral change as a result of acute ethanol exposure in zebrafish: Evidence for a role for sonic hedgehog but not retinoic acid signaling.
- Author
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Burton DF, Zhang C, Boa-Amponsem O, Mackinnon S, and Cole GJ
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- Animals, Embryo, Nonmammalian drug effects, Embryo, Nonmammalian metabolism, Female, Male, Oligonucleotides, Antisense pharmacology, Pregnancy, Zebrafish, Behavior, Animal drug effects, Ethanol toxicity, Hedgehog Proteins physiology, Morpholinos pharmacology, Tretinoin, Zebrafish Proteins physiology
- Abstract
Background: Developmental exposure to ethanol is recognized to produce long-term neurobehavioral impairment in multiple animal models. However, the molecular mechanisms underlying these deficits remain poorly understood. The present study was undertaken to ascertain whether two well-characterized targets of prenatal alcohol exposure, sonic hedgehog (Shh) and retinoic acid (RA), that induce the hallmark morphological phenotypes of fetal alcohol spectrum disorders (FASD), are involved in the generation of behavioral alterations as a result of alcohol exposure., Methods: Zebrafish embryos were exposed to ethanol (0%, 1%, 3%) at either 8-10 or 24-27h post-fertilization (hpf) and then evaluated during adolescence in the novel tank dive test to assess anxiety and risk-taking behavior. Overt signs of dysmorphogenesis were also scored and behavioral and morphological changes were compared for embryos treated with alcohol alone or in combination with subthreshold doses of shh or alhh1a3 morpholinos (MOs)., Results: Ethanol treated fish displayed altered tank diving behavior that was not exacerbated by combined MO treatment. While treatment of embryos with either shha mRNA or RA prior to ethanol exposure only ameliorated the altered tank diving response in the case of shha mRNA overexpression, dysmorphogenesis was rescued by both treatments., Conclusion: These results suggest that the effects of ethanol exposure on changes in anxiety and risk-taking behavior in adolescent zebrafish is manifested by a blunting of Shh, but not RA, signaling during early development., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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14. Analysis of crosstalk between retinoic acid and sonic hedgehog pathways following ethanol exposure in embryonic zebrafish.
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Zhang C, Anderson A, and Cole GJ
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- Animals, Brain embryology, Eye embryology, Eye Proteins genetics, Homeodomain Proteins genetics, PAX6 Transcription Factor, Paired Box Transcription Factors genetics, Repressor Proteins genetics, Signal Transduction drug effects, Ethanol toxicity, Hedgehog Proteins metabolism, Tretinoin metabolism, Zebrafish embryology, Zebrafish Proteins metabolism
- Abstract
Background: Ethanol is a teratogen affecting numerous regions of the developing nervous system. The present study was undertaken to ascertain whether ethanol independently disrupts distinct signaling pathways or rather disrupts interactive pathways that regulate development of ethanol-sensitive tissues., Methods: Zebrafish embryos were exposed to ethanol in the absence or presence of aldh1a3 or Shh morpholino oligonucleotides (MOs), which disrupt retinoic acid (RA) or sonic hedgehog (Shh) function, respectively. Morphological analysis of ocular or midbrain-hindbrain boundary (MHB) development was conducted, and the ability to rescue ethanol and MO-induced phenotypes was assessed. In situ hybridization was used to analyze Pax6a expression during ocular development., Results: Chronic ethanol exposure, or combined ethanol and MO treatment, results in perturbed MHB formation and microphthalmia. While RA can rescue the MHB phenotype following ethanol combined with either MO, Shh mRNA is unable to rescue the disrupted MHB with combined ethanol and aldh1a3 MO treatment. RA also is unable to rescue microphthalmia induced by ethanol and Shh MO., Conclusion: These studies demonstrate that while reduction of either RA or Shh signaling produces the same disruption of MHB or ocular development, that can be phenocopied using ethanol combined with either MO, RA overexpression can only rescue disrupted MHB, but not microphthalmia, in combined subthreshold Shh MO and ethanol. Our data suggest that MHB development may involve crosstalk between RA and Shh signaling, while ocular development depends on RA and Shh signaling that both are targets of ethanol in fetal alcohol spectrum disorders but do not depend on a mechanism involving crosstalk., (© 2015 Wiley Periodicals, Inc.)
- Published
- 2015
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15. Long-term behavioral impairment following acute embryonic ethanol exposure in zebrafish.
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Bailey JM, Oliveri AN, Zhang C, Frazier JM, Mackinnon S, Cole GJ, and Levin ED
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- Animals, Brain pathology, Female, Habituation, Psychophysiologic drug effects, Learning drug effects, Pregnancy, Reflex, Startle drug effects, Behavior, Animal drug effects, Embryo, Nonmammalian drug effects, Ethanol toxicity, Zebrafish embryology
- Abstract
Background: Developmental exposure to ethanol has long been known to cause persisting neurobehavioral impairment. However, the neural and behavioral mechanisms underlying these deficits and the importance of exposure timing are not well-characterized. Given the importance of timing and sequence in neurodevelopment it would be expected that alcohol intoxication at different developmental periods would result in distinct neurobehavioral consequences., Methods: Zebrafish embryos were exposed to ethanol (0%, 1%, 3%) at either 8-10 or 24-27 h post-fertilization (hpf) then reared to adolescence and evaluated on several behavioral endpoints. Habituation to a repeated environmental stimulus and overall sensorimotor function were assessed using a tap startle test; measurements of anxiety and exploration behavior were made following introduction to a novel tank; and spatial discrimination learning was assessed using aversive control in a three-chambered apparatus. Overt signs of dysmorphogenesis were also scored (i.e. craniofacial malformations, including eye diameter and midbrain-hindbrain boundary morphology)., Results: Ethanol treated fish were more active both at baseline and following a tap stimulus compared to the control fish and were hyperactive when placed in a novel tank. These effects were more prominent following exposure at 24-27 hpf than with the earlier exposure window, for both dose groups. Increases in physical malformation were only present in the 3% ethanol group; all malformed fish were excluded from behavioral testing., Discussion: These results suggest specific domains of behavior are affected following ethanol exposure, with some but not all of the tests revealing significant impairment. The behavioral phenotypes following distinct exposure windows described here can be used to help link cellular and molecular mechanisms of developmental ethanol exposure to functional neurobehavioral effects., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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16. Molecular and morphological changes in zebrafish following transient ethanol exposure during defined developmental stages.
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Zhang C, Frazier JM, Chen H, Liu Y, Lee JA, and Cole GJ
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- Animals, Eye Proteins genetics, Female, Glutamate Decarboxylase genetics, Homeodomain Proteins genetics, PAX6 Transcription Factor, Paired Box Transcription Factors genetics, Pregnancy, Repressor Proteins genetics, Zebrafish embryology, Embryo, Nonmammalian drug effects, Ethanol toxicity, Fetal Alcohol Spectrum Disorders genetics, Fetal Alcohol Spectrum Disorders pathology, Gene Expression Regulation, Developmental drug effects
- Abstract
Alcohol is a teratogen that has diverse effects on brain and craniofacial development, leading to a constellation of developmental disorders referred to as fetal alcohol spectrum disorder (FASD). The molecular basis of ethanol insult remains poorly understood, as does the relationship between molecular and behavioral changes as a consequence of prenatal ethanol exposure. Zebrafish embryos were exposed to a range of ethanol concentrations (0.5-5.0%) during defined developmental stages, and examined for morphological phenotypes characteristic of FASD. Embryos were also analyzed by in situ hybridization for changes in expression of defined cell markers for neural cell types that are sonic hedgehog-dependent. We show that transient binge-like ethanol exposures during defined developmental stages, such as early gastrulation and early neurulation, result in a range of phenotypes and changes in expression of Shh-dependent genes. The severity of fetal alcohol syndrome (FAS) morphological phenotypes, such as microphthalmia, depends on the embryonic stage and concentration of alcohol exposure, as does diminution of retinal Pax6a or forebrain and hindbrain GAD1 gene expression. We also show that changes in eye and brain morphology correlate with changes in Pax6a and GAD1 gene expression. Our results therefore show that transient binge-like ethanol exposures in zebrafish embryos produce the stereotypical morphological phenotypes of FAS, with the severity of phenotypes depending on the developmental stage and alcohol concentration of exposure., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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17. Transgenic overexpression of cdx1b induces metaplastic changes of gene expression in zebrafish esophageal squamous epithelium.
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Hu B, Chen H, Liu X, Zhang C, Cole GJ, Lee JA, and Chen X
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- Animals, Barrett Esophagus metabolism, Barrett Esophagus physiopathology, Cell Proliferation, Epithelium metabolism, Epithelium pathology, Esophagus metabolism, Gene Expression Regulation, Developmental, Homeodomain Proteins metabolism, In Situ Hybridization, Keratin-5 genetics, Keratin-5 metabolism, Larva, Metaplasia genetics, Metaplasia metabolism, Metaplasia pathology, Mucins genetics, Mucins metabolism, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, Promoter Regions, Genetic, Zebrafish Proteins metabolism, Barrett Esophagus genetics, Esophagus pathology, Gene Expression Regulation, Neoplastic, Homeodomain Proteins genetics, Models, Animal, Zebrafish, Zebrafish Proteins genetics
- Abstract
Cdx2 has been suggested to play an important role in Barrett's esophagus or intestinal metaplasia (IM) in the esophagus. To investigate whether transgenic overexpression of cdx1b, the functional equivalent of mammalian Cdx2 in zebrafish, may lead to IM of zebrafish esophageal squamous epithelium, a transgenic zebrafish system was developed by expressing cdx1b gene under the control of zebrafish keratin 5 promoter (krt5p). Gene expression in the esophageal squamous epithelium of wild-type and transgenic zebrafish was analyzed by Affymetrix microarray and confirmed by in situ hybridization. Morphology, mucin expression, cell proliferation, and apoptosis were analyzed by hematoxylin & eosin (HE) staining, Periodic acid Schiff (PAS) Alcian blue staining, proliferating cell nuclear antigen (PCNA) immunohistochemical staining, and TUNEL assay as well. cdx1b was found to be overexpressed in the nuclei of esophageal squamous epithelial cells of the transgenic zebrafish. Ectopic expression of cdx1b disturbed the development of this epithelium in larval zebrafish and induced metaplastic changes in gene expression in the esophageal squamous epithelial cells of adult zebrafish, that is, up-regulation of intestinal differentiation markers and down-regulation of squamous differentiation markers. However, cdx1b failed to induce histological IM, or to modulate cell proliferation and apoptosis in the squamous epithelium of adult transgenic zebrafish.
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- 2013
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18. Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin, Fgf, and sonic hedgehog function.
- Author
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Zhang C, Ojiaku P, and Cole GJ
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- Agrin genetics, Agrin metabolism, Animals, Biomarkers metabolism, Brain embryology, Brain metabolism, Embryo, Nonmammalian drug effects, Embryo, Nonmammalian embryology, Embryo, Nonmammalian metabolism, Fibroblast Growth Factors genetics, Fibroblast Growth Factors metabolism, Gene Expression Regulation, Developmental drug effects, Gene Silencing, Glutamate Decarboxylase genetics, Glutamic Acid metabolism, Hedgehog Proteins genetics, Hedgehog Proteins metabolism, Morpholinos, Neurons drug effects, Neurons metabolism, Prosencephalon drug effects, Prosencephalon embryology, Prosencephalon metabolism, Rhombencephalon drug effects, Rhombencephalon embryology, Rhombencephalon metabolism, Signal Transduction, Brain drug effects, Central Nervous System Depressants toxicity, Embryonic Development drug effects, Ethanol toxicity, Teratogens toxicity, Zebrafish embryology, Zebrafish Proteins metabolism
- Abstract
Background: Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol's effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function., Methods: Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was used to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons., Results: Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8, or Shh mRNA overexpression rescues ethanol-induced decreases in GAD1 and Atonal1a gene expression., Conclusions: These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis., (Copyright © 2012 Wiley Periodicals, Inc.)
- Published
- 2013
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19. Effects of ethanol exposure on nervous system development in zebrafish.
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Cole GJ, Zhang C, Ojiaku P, Bell V, Devkota S, and Mukhopadhyay S
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- Animals, Disease Models, Animal, Female, Fetal Alcohol Spectrum Disorders physiopathology, Motor Neurons cytology, Motor Neurons drug effects, Nervous System cytology, Neural Pathways cytology, Neural Pathways drug effects, Neural Pathways embryology, Neurogenesis drug effects, Pregnancy, Ethanol toxicity, Nervous System drug effects, Nervous System embryology, Zebrafish embryology
- Abstract
Alcohol (ethanol) is a teratogen that adversely affects nervous system development in a wide range of animal species. In humans numerous congenital abnormalities arise as a result of fetal alcohol exposure, leading to a spectrum of disorders referred to as fetal alcohol spectrum disorder (FASD). These abnormalities include craniofacial defects as well as neurological defects that affect a variety of behaviors. These human FASD phenotypes are reproduced in the rodent central nervous system (CNS) following prenatal ethanol exposure. While the study of ethanol effects on zebrafish development has been more limited, several studies have shown that different strains of zebrafish exhibit differential susceptibility to ethanol-induced cyclopia, as well as behavioral deficits. Molecular mechanisms underlying the effects of ethanol on CNS development also appear to be shared between rodent and zebrafish. Thus, zebrafish appear to recapitulate the observed effects of ethanol on human and mouse CNS development, indicating that zebrafish can serve as a complimentary developmental model system to study the molecular basis of FASD. Recent studies examining the effect of ethanol exposure on zebrafish nervous system development are reviewed, with an emphasis on attempts to elucidate possible molecular pathways that may be impacted by developmental ethanol exposure. Recent work from our laboratories supports a role for perturbed extracellular matrix function in the pathology of ethanol exposure during zebrafish CNS development. The use of the zebrafish model to assess the effects of ethanol exposure on adult nervous system function as manifested by changes in zebrafish behavior is also discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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20. Assembly of lamina-specific neuronal connections by slit bound to type IV collagen.
- Author
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Xiao T, Staub W, Robles E, Gosse NJ, Cole GJ, and Baier H
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- Animals, Axons metabolism, Brain metabolism, Nerve Tissue Proteins metabolism, Receptors, Immunologic metabolism, Retinal Ganglion Cells metabolism, Signal Transduction, Zebrafish metabolism, Brain embryology, Collagen Type IV metabolism, Tectum Mesencephali metabolism, Zebrafish embryology, Zebrafish Proteins metabolism
- Abstract
The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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21. Agrin function associated with ocular development is a target of ethanol exposure in embryonic zebrafish.
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Zhang C, Turton QM, Mackinnon S, Sulik KK, and Cole GJ
- Subjects
- Agrin genetics, Agrin metabolism, Animals, Animals, Genetically Modified, Down-Regulation drug effects, Down-Regulation genetics, Embryo, Nonmammalian, Environmental Exposure, Eye metabolism, Gene Expression Regulation, Developmental drug effects, Hedgehog Proteins genetics, Hedgehog Proteins metabolism, Hedgehog Proteins physiology, Microphthalmos chemically induced, Microphthalmos genetics, Microphthalmos pathology, Oligoribonucleotides, Antisense pharmacology, Phenotype, Zebrafish genetics, Zebrafish metabolism, Agrin physiology, Ethanol pharmacology, Eye drug effects, Eye embryology, Zebrafish embryology
- Abstract
Background: Alcohol (ethanol) is a teratogen known to affect the developing eyes, face, and brain. Among the ocular defects in fetal alcohol spectrum disorder (FASD) are microphthalmia and optic nerve hypoplasia. Employing zebrafish as an FASD model provides an excellent system to analyze the molecular basis of prenatal ethanol exposure-induced defects because embryos can be exposed to ethanol at defined developmental stages and affected genetic pathways can be examined. We have previously shown that disruption of agrin function in zebrafish embryos produces microphthalmia and optic nerve hypoplasia., Methods: Zebrafish embryos were exposed to varying concentrations of ethanol in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin function. In situ hybridization was used to analyze ocular gene expression as a consequence of ethanol exposure and agrin knockdown. Morphologic analysis of zebrafish embryos was also conducted., Results: Acute ethanol exposure induces diminished agrin gene expression in zebrafish eyes and, importantly, combined treatment with subthreshold levels of agrin MO and ethanol produces pronounced microphthalmia, markedly reduces agrin gene expression, and perturbs Pax6a and Mbx gene expression. Microphthalmia produced by combined agrin MO and ethanol treatment was rescued by sonic hedgehog (Shh) mRNA overexpression, suggesting that ethanol-mediated disruption of agrin expression results in disrupted Shh function., Conclusions: These studies illustrate the strong potential for using zebrafish as a model to aid in defining the molecular basis for ethanol's teratogenic effects. The results of this work suggest that agrin expression and function may be a target of ethanol exposure during embryogenesis., (Copyright © 2011 Wiley-Liss, Inc.)
- Published
- 2011
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22. Transitin is required for the differentiation of avian QM7 myoblasts into myotubes.
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Jalouli M, Lapierre LR, Guérette D, Blais K, Lee JA, Cole GJ, and Vincent M
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- Animals, Avian Proteins genetics, Cell Differentiation genetics, Cell Line, Fluorescent Antibody Technique, Immunoblotting, Intermediate Filament Proteins genetics, Quail, Reverse Transcriptase Polymerase Chain Reaction, Avian Proteins metabolism, Cell Differentiation physiology, Intermediate Filament Proteins metabolism, Muscle Fibers, Skeletal cytology, Muscle Fibers, Skeletal metabolism, Myoblasts cytology, Myoblasts metabolism
- Abstract
Transitin is a nestin-like intermediate filament protein co-expressed with vimentin in the precursor cells of the myogenic and neurogenic lineages of the avian embryo. To understand its role in myogenesis, stable cell lines expressing transitin-targeted siRNAs were derived from the quail muscle cell line QM7. When cells were cultured in differentiation medium, we found that transitin knockdown prevented myoblast fusion and myotube formation. MyoD mRNA could be detected in transitin siRNA-transfected cells, but upregulation of myogenin and desmin expression was impaired compared to control cells. In addition, transitin siRNA cells maintain high levels of Pax7 expression suggesting that QM7 myoblasts into which transitin expression has been attenuated display a muscle progenitor cell phenotype (Pax7(+)/MyoD(+)/myogenin(-)/desmin(-)). These observations indicate that transitin plays an important role in the initiation of the myogenic program in avian muscle progenitor cells in acting downstream of MyoD and upstream of myogenin during the lineage progression., (© 2010 Wiley-Liss, Inc.)
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- 2010
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23. Age-dependent changes in the structure, composition and biophysical properties of a human basement membrane.
- Author
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Candiello J, Cole GJ, and Halfter W
- Subjects
- Adult, Age Factors, Basement Membrane chemistry, Basement Membrane ultrastructure, Blotting, Western, Female, Fetus, Humans, Immunohistochemistry, Male, Microscopy, Atomic Force, Microscopy, Electron, Transmission, Retina chemistry, Retina ultrastructure, Basement Membrane physiology, Collagen Type IV physiology, Laminin physiology, Retina physiology
- Abstract
Basement membranes (BMs) are considered to be uniform, approximately 100 nm-thin extracellular matrix sheets that serve as a substrate for epithelial cells, endothelial cells and myotubes. To find out whether BMs maintain their ultrastructure, protein composition and biophysical properties throughout life the natural aging history of the human inner limiting membranes (ILM) was investigated. The ILM is a BM at the vitreal surface of the retina that connects the retina with the vitreous. Transmission electron microscopy (TEM) showed that the ILM steadily increases in thickness from 70 nm at fetal stages to several microns at age 90. By the age of 20, the ILM loses its laminated structure to become an amorphous and very irregular extracellular matrix layer. Atomic force microscopy (AFM) showed that the native, hydrated ILMs are on average 4-fold thicker than the dehydrated ILMs as seen by TEM and that their thickness is prominently determined by its water-binding proteoglycans. The morphological changes are accompanied by age-related changes in the biochemical composition, whereby the relative concentrations of collagen IV and agrin increase, and the concentration of laminin decreases with age. Force-indentation measurements by AFM also showed that ILMs become increasingly stiffer with advancing age. The data suggest that BMs from other human tissues may undergo similar age-related changes., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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24. Zebrafish K5 promoter driven GFP expression as a transgenic system for oral research.
- Author
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Hu B, Zhang C, Baawo K, Qin R, Cole GJ, Lee JA, and Chen X
- Subjects
- Animals, Animals, Genetically Modified, Biomedical Research, Gene Expression Regulation, Neoplastic genetics, Green Fluorescent Proteins metabolism, Keratin-15 genetics, Keratin-15 metabolism, Keratin-5 metabolism, Models, Animal, Molecular Sequence Data, Zebrafish anatomy & histology, Gastrointestinal Tract anatomy & histology, Green Fluorescent Proteins genetics, Keratin-5 genetics, Promoter Regions, Genetic genetics, Tongue anatomy & histology, Zebrafish genetics
- Abstract
The zebrafish has become a useful model organism for research on development and diseases. However, there has been no zebrafish model system for studying oral carcinogenesis. In the present study, we first characterized the histology of the upper gastrointestinal tract of zebrafish. We found that zebrafish tongue was covered by a non-keratinized stratified squamous epithelium, which was similar to the oro-esophageal epithelium in humans. In situ hybridization showed that keratin 5, a marker of the basal cell layer of mammalian oral epithelium, was expressed in the squamous epithelium of zebrafish tongue. A highly conserved promoter of zebrafish keratin 5 was cloned to drive transgenic expression of GFP. GFP was found to be expressed in the periderm of embryos. In adult fish, GFP was also abundantly expressed in the tongue and fin. GFP expression in transgenic fish recapitulated endogenous zebrafish keratin 5 gene expression as shown by in situ hybridization. This study indicated a high fidelity of GFP reporter gene expression in the tongue under the control of zebrafish keratin 5 promoter. This zebrafish transgenic model system may be used for future studies on oral development and cancer.
- Published
- 2010
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25. Retina development in zebrafish requires the heparan sulfate proteoglycan agrin.
- Author
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Liu IH, Zhang C, Kim MJ, and Cole GJ
- Subjects
- Acridine Orange, Animals, Animals, Genetically Modified, Cell Death, ELAV Proteins genetics, ELAV-Like Protein 3, Embryo, Nonmammalian drug effects, Enzyme Inhibitors pharmacology, Fibroblast Growth Factor 3 genetics, Fibroblast Growth Factor 3 metabolism, GAP-43 Protein genetics, Gene Expression Regulation, Developmental drug effects, Gene Expression Regulation, Developmental genetics, Green Fluorescent Proteins biosynthesis, Green Fluorescent Proteins genetics, Oligonucleotides, Antisense pharmacology, Pyrroles pharmacology, Retina drug effects, Retina metabolism, Sphingosine analogs & derivatives, Superior Colliculi embryology, Superior Colliculi growth & development, Superior Colliculi metabolism, Transcription Factor Brn-3C genetics, Zebrafish Proteins genetics, Agrin physiology, Gene Expression Regulation, Developmental physiology, Heparan Sulfate Proteoglycans physiology, Retina embryology, Retina growth & development, Zebrafish embryology, Zebrafish growth & development, Zebrafish metabolism
- Abstract
Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.
- Published
- 2008
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26. Olfactomedin-2 mediates development of the anterior central nervous system and head structures in zebrafish.
- Author
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Lee JA, Anholt RR, and Cole GJ
- Subjects
- Animals, Extracellular Matrix Proteins genetics, Eye Proteins genetics, Gene Expression Regulation, Developmental, Glycoproteins genetics, Homeodomain Proteins genetics, PAX6 Transcription Factor, Paired Box Transcription Factors genetics, RNA, Messenger genetics, Repressor Proteins genetics, Body Patterning, Central Nervous System embryology, Extracellular Matrix Proteins physiology, Glycoproteins physiology, Zebrafish embryology
- Abstract
Olfactomedins comprise a diverse family of secreted glycoproteins, which includes noelin, tiarin, pancortin and gliomedin, implicated in development of the nervous system, and the glaucoma-associated protein myocilin. Here we show in zebrafish that olfactomedin-2 (OM2) is a developmentally regulated gene, and that knockdown of protein expression by morpholino antisense oligonucleotides leads to perturbations of nervous system development. Interference with OM2 expression results in impaired development of branchiomotor neurons, specific disruption of the late phase branchiomotor axon guidance, and affects development of the caudal pharyngeal arches, olfactory pits, eyes and optic tectum. Effects of OM2 knockdown on eye development are likely associated with Pax6 signaling in developing eyes, as Pax6.1 and Pax6.2 mRNA expression patterns are altered in the eyes of OM2 morphants. The specific absence of most cartilaginous structures in the pharyngeal arches indicates that the observed craniofacial phenotypes may be due to perturbed differentiation of cranial neural crest cells. Our studies show that this member of the olfactomedin protein family is an important regulator of development of the anterior nervous system.
- Published
- 2008
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27. Generation of transgenic zebrafish expressing green fluorescent protein under control of zebrafish amyloid precursor protein gene regulatory elements.
- Author
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Lee JA and Cole GJ
- Subjects
- Alzheimer Disease etiology, Amyloid beta-Protein Precursor biosynthesis, Amyloid beta-Protein Precursor genetics, Animals, Breeding, Enhancer Elements, Genetic, Female, Gene Expression Profiling veterinary, Green Fluorescent Proteins analysis, Green Fluorescent Proteins biosynthesis, Green Fluorescent Proteins genetics, Male, Models, Animal, Plasmids, Promoter Regions, Genetic, Tissue Distribution, Zebrafish metabolism, Amyloid beta-Protein Precursor physiology, Animals, Genetically Modified, Embryo, Nonmammalian physiology, Gene Expression Regulation, Developmental physiology, Zebrafish genetics
- Abstract
Amyloid precursor protein (APP) encodes a transmembrane protein that is well established as contributing a crucial role to the etiology of Alzheimer's disease. We have generated germline transgenic zebrafish that express green fluorescent protein (GFP) under control of the endogenous zebrafish appb gene. Expression of GFP by the zebrafish appb promoter requires an enhancer element identified in the first intron of the zebrafish appb gene, with this region exhibiting conservation from zebrafish to human. GFP expression in these transgenic zebrafish recapitulates endogenous appb gene expression as shown by in situ hybridization. We show that GFP is expressed in subregions of brain and in spinal cord, as well as being expressed in the developing vasculature of zebrafish embryos. GFP expression is also developmentally regulated, beginning during the first day of development and then increasing in intensity during later development. In 2.5-month-old young adult transgenic zebrafish, GFP expression was abundantly and widely expressed in the brain. The importance of these transgenic zebrafish lines is that it will be possible to assess the effects of environmental factors, natural products, and therapeutic compounds on APP gene expression during nervous system development, using zebrafish as a model system.
- Published
- 2007
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28. Transitin, a nestin-like intermediate filament protein, mediates cortical localization and the lateral transport of Numb in mitotic avian neuroepithelial cells.
- Author
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Wakamatsu Y, Nakamura N, Lee JA, Cole GJ, and Osumi N
- Subjects
- Animals, Cell Differentiation, Cell Membrane metabolism, Chick Embryo, Intermediate Filament Proteins genetics, Membrane Proteins genetics, Nerve Tissue Proteins genetics, Nestin, Protein Binding, Protein Transport, RNA Interference, Gene Expression Regulation, Developmental, Intermediate Filament Proteins metabolism, Membrane Proteins metabolism, Mitosis, Nerve Tissue Proteins metabolism, Neuroepithelial Cells cytology, Neuroepithelial Cells metabolism
- Abstract
Neuroepithelium is an apicobasally polarized tissue that contains neural stem cells and gives rise to neurons and glial cells of the central nervous system. The cleavage orientation of neural stem cells is thought to be important for asymmetric segregation of fate-determinants, such as Numb. Here, we show that an intermediate filament protein, transitin, colocalizes with Numb in the cell cortex of mitotic neuroepithelial cells, and that transitin anchors Numb via a physical interaction. Detailed immunohistological and time-lapse analyses reveal that basal Numb-transitin complexes shift laterally during mitosis, allowing asymmetric segregation of Numb-transitin to one of the daughter cells, even when the cell cleavage plane is perpendicular to the ventricular surface. In addition, RNA interference (RNAi) knockdown of the transitin gene reveals its involvement in neurogenesis. These results indicate that transitin has important roles in determining the intracellular localization of Numb, which regulates neurogenesis in the developing nervous system of avian embryos.
- Published
- 2007
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29. Biomechanical properties of native basement membranes.
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Candiello J, Balasubramani M, Schreiber EM, Cole GJ, Mayer U, Halfter W, and Lin H
- Subjects
- Animals, Basement Membrane embryology, Biomechanical Phenomena, Bruch Membrane physiology, Bruch Membrane ultrastructure, Chick Embryo, Desiccation, Elasticity, Mice, Mice, Mutant Strains, Microscopy, Atomic Force, Basement Membrane physiology
- Abstract
Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.
- Published
- 2007
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30. Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish.
- Author
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Kim MJ, Liu IH, Song Y, Lee JA, Halfter W, Balice-Gordon RJ, Linney E, and Cole GJ
- Subjects
- Agrin analysis, Agrin genetics, Animals, Axons chemistry, Axons physiology, Cell Differentiation, Embryo, Nonmammalian, Embryonic Development genetics, Motor Neurons chemistry, Motor Neurons cytology, Muscle, Skeletal embryology, Muscle, Skeletal innervation, Nervous System chemistry, RNA, Messenger analysis, RNA, Messenger metabolism, Receptors, Cholinergic analysis, Receptors, Cholinergic metabolism, Zebrafish abnormalities, Zebrafish Proteins analysis, Zebrafish Proteins genetics, Agrin physiology, Motor Neurons physiology, Nervous System embryology, Zebrafish embryology, Zebrafish Proteins physiology
- Abstract
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.
- Published
- 2007
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31. Agrin binds alpha-synuclein and modulates alpha-synuclein fibrillation.
- Author
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Liu IH, Uversky VN, Munishkina LA, Fink AL, Halfter W, and Cole GJ
- Subjects
- Agrin metabolism, Alzheimer Disease metabolism, Animals, Antibodies, Monoclonal chemistry, Brain metabolism, Brain pathology, Cell Death, Chickens, Circular Dichroism, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Heparitin Sulfate chemistry, Humans, Immunoblotting, Immunohistochemistry, Lewy Bodies metabolism, Microscopy, Electron, Transmission, Neurodegenerative Diseases pathology, Neurons metabolism, Parkinson Disease metabolism, Prions chemistry, Protein Binding, Protein Conformation, Recombinant Proteins chemistry, Solubility, Substantia Nigra metabolism, Synucleins chemistry, Time Factors, alpha-Synuclein metabolism, Agrin chemistry, alpha-Synuclein chemistry
- Abstract
Recent studies have begun to investigate the role of agrin in brain and suggest that agrin's function likely extends beyond that of a synaptogenic protein. Particularly, it has been shown that agrin is associated with the pathological lesions of Alzheimer's disease (AD) and may contribute to the formation of beta-amyloid (Abeta) plaques in AD. We have extended the analysis of agrin's function in neurodegenerative diseases to investigate its role in Parkinson's disease (PD). Alpha-synuclein is a critical molecular determinant in familial and sporadic PD, with the formation of alpha-synuclein fibrils being enhanced by sulfated macromolecules. In the studies reported here, we show that agrin binds to alpha-synuclein in a heparan sulfate-dependent (HS-dependent) manner, induces conformational changes in this protein characterized by beta-sheet structure, and enhances insolubility of alpha-synuclein. We also show that agrin accelerates the formation of protofibrils by alpha-synuclein and decreases the half-time of fibril formation. The association of agrin with PD lesions was also explored in PD human brain, and these studies shown that agrin colocalizes with alpha-synuclein in neuronal Lewy bodies in the substantia nigra of PD brain. These studies indicate that agrin is capable of accelerating the formation of insoluble protein fibrils in a second common neurodegenerative disease. These findings may indicate shared molecular mechanisms leading to the pathophysiology in these two neurodegenerative disorders.
- Published
- 2005
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32. Embryonic synthesis of the inner limiting membrane and vitreous body.
- Author
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Halfter W, Dong S, Schurer B, Ring C, Cole GJ, and Eller A
- Subjects
- Adult, Animals, Basement Membrane embryology, Basement Membrane metabolism, Blotting, Western, Chick Embryo, Chickens, Extracellular Matrix Proteins genetics, Eye Proteins genetics, Gene Expression Regulation, Developmental physiology, Humans, In Situ Hybridization, Infant, Infant, Newborn, RNA, Messenger metabolism, Retina metabolism, Reverse Transcriptase Polymerase Chain Reaction, Vitreous Body metabolism, Embryonic Development physiology, Extracellular Matrix Proteins biosynthesis, Eye Proteins biosynthesis, Retina embryology, Vitreous Body embryology
- Abstract
Purpose: The inner limiting membrane (ILM) and the vitreous body (VB) are major parts of the extracellular matrix of the eye. The present study was undertaken to investigate the synthesis and turnover of the ILM and VB in chick and human embryonic and postembryonic eye development., Methods: The abundance of ILM and VB proteins was determined by Western blot analysis using samples from chick and human VB of different ages. The mRNA expression of the ILM proteins in lens was determined by in situ hybridization and RT-PCR., Results: Based on the abundance of mRNA expression, the prominent sources of ILM and VB proteins in chick eyes are the lens and ciliary body. In chick, ILM and VB matrix proteins were most abundant in embryonic VB, and their concentration declined precipitously after hatching. Most ILM and VB proteins were no longer detectable in the adult VB. In humans, a similar developmentally regulated expression of ILM and VB proteins in VB was detected: The highest concentrations of ILM and VB proteins were detected in fetal VB, the lowest in the adult VB. The decline in ILM and VB protein synthesis occurred within the first 2 years of life., Conclusions: The abundance of ILM and VB proteins in the embryonic VB, their sharp decline at postembryonic stages, and their very low abundance in the adult VB show that ILM and VB are assembled during embryogenesis and are maintained throughout life with minimum turnover.
- Published
- 2005
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33. Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate/chondroitin sulfate located in two multiple serine-glycine clusters.
- Author
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Winzen U, Cole GJ, and Halfter W
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Blotting, Western, Chickens, Electrophoresis, Polyacrylamide Gel, Glycine chemistry, Glycosylation, Heparitin Sulfate chemistry, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Peptides chemistry, Plasmids metabolism, Protein Structure, Tertiary, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Serine chemistry, Threonine chemistry, Agrin chemistry, Chondroitin Sulfates chemistry, Proteoglycans chemistry
- Abstract
Agrin is a large extracellular matrix protein that plays a key role in the formation and maintenance of the vertebrate neuromuscular junction. The amino acid sequence of agrin encodes a protein with a molecular size of 220 kDa, whereas SDS-PAGE shows a diffuse band around 400 kDa. Further studies showed that agrin is highly glycosylated and belongs to the family of heparan sulfate proteoglycans. By expressing different protein fragments, we localized the glycosaminoglycan (GAG) attachment sites to two locations within the agrin molecule. One site that is located between the seventh and eight follistatin-like domain includes 3 closely spaced serine-glycine (SG) consensus sequences and carries exclusively heparan sulfate side chains. The second site is located further downstream in the centrally located serine-threonine-rich domain and contains a cluster of 4 closely packed SG consensus sequences. This site predominantly carries chondroitin sulfate side chains. Investigating the contribution of individual serines in GAG priming by site-directed mutagenesis showed that each serine of the two SG clusters has the potential to carry GAGs. In accordance with the mixed GAG glycosylation of agrin peptide fragments, it was found that recombinant and in vivo-derived full-length agrin are not exclusively heparan sulfate proteoglycans but also carry chondroitin sulfate side chains.
- Published
- 2003
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34. The heparan sulfate proteoglycan agrin modulates neurite outgrowth mediated by FGF-2.
- Author
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Kim MJ, Cotman SL, Halfter W, and Cole GJ
- Subjects
- Animals, Chick Embryo, Drug Synergism, Fibroblast Growth Factor 2 pharmacology, Genes, fos drug effects, Heparan Sulfate Proteoglycans pharmacology, Humans, Immunoblotting, Mitogen-Activated Protein Kinases metabolism, PC12 Cells, Phosphorylation drug effects, Rats, Retina drug effects, Agrin pharmacology, Neurites drug effects
- Abstract
Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin-binding growth factor basic fibroblast growth factor (FGF-2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF-2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF-2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF-2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c-fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF-2, and augments and sustains FGF-2 mediated increases in c-fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF-2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF-2., (Copyright 2003 Wiley Periodicals, Inc. J Neurobiol 55: 261-277, 2003)
- Published
- 2003
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35. Expression of collagen XVIII and localization of its glycosaminoglycan attachment sites.
- Author
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Dong S, Cole GJ, and Halfter W
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Chickens, Cloning, Molecular, Collagen chemistry, Collagen genetics, Collagen Type XVIII, DNA Primers, DNA, Complementary, Endostatins, Glycosylation, Molecular Sequence Data, Mutagenesis, Site-Directed, Peptide Fragments chemistry, Peptide Fragments genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Collagen metabolism, Glycosaminoglycans metabolism, Peptide Fragments metabolism
- Abstract
Collagen XVIII is the only currently known collagen that carries heparan sulfate glycosaminoglycan side chains. The number and location of the glycosaminoglycan attachment sites in the core protein were determined by eukaryotic expression of full-length chick collagen XVIII and site-directed mutagenesis. Three Ser-Gly consensus sequences carrying glycosaminoglycan side chains were detected in the middle and N-terminal part of the core protein. One of the Ser-Gly consensus sequences carried a heparan sulfate side chain, and the remaining two had mixed chondroitin and heparan sulfate side chains; thus, recombinant collagen XVIII was a hybrid of heparan sulfate and chondroitin proteoglycan. In contrast, collagen XVIII from all chick tissues so far assayed have exclusively heparan sulfate side chains, indicating that the posttranslational modification of proteins expressed in vitro is not entirely identical to the processing that occurs in a living embryo. Incubating the various mutated collagen XVIIIs with retinal basement membranes showed that the heparan sulfate glycosaminoglycan side chains mediate the binding of collagen XVIII to basement membranes.
- Published
- 2003
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36. Association of factor H of the alternative pathway of complement with agrin and complement receptor 3 in the Alzheimer's disease brain.
- Author
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Strohmeyer R, Ramirez M, Cole GJ, Mueller K, and Rogers J
- Subjects
- Aged, Agrin metabolism, Alzheimer Disease metabolism, Alzheimer Disease pathology, Amyloid beta-Peptides analysis, Amyloid beta-Peptides metabolism, Blotting, Western, Brain metabolism, Brain Chemistry, Complement Factor H immunology, Complement Factor H metabolism, Complement Pathway, Alternative, Humans, Immunohistochemistry, Neuroglia immunology, Agrin analysis, Alzheimer Disease immunology, Brain immunology, Complement Factor H analysis, Macrophage-1 Antigen analysis
- Abstract
Factor H, a regulatory protein of the alternative pathway of complement (APC), is present in amyloid-beta (Abeta) plaques in Alzheimer's disease (AD). Abeta plaques also contain significant amounts of heparan sulfate proteoglycans (HSPGs), such as agrin, as well as numerous activated microglia expressing increased levels complement receptor 3 (CR3). Here, we show the colocalization of each of these molecules in the AD brain and the functional capacity for these molecules to bind to one another in vitro. We propose that CR3 receptors expressed by microglia are used for ligand binding to factor H bound to HSPGs and Abeta in plaques in the AD brain.
- Published
- 2002
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37. Molecular polymorphism of the intermediate filament protein transitin.
- Author
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Darenfed H, Ma X, Davis L, Juge N, Savard PE, Cole GJ, and Vincent M
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chick Embryo, Epitope Mapping, Fluorescent Antibody Technique, Indirect, Glial Fibrillary Acidic Protein analysis, Glial Fibrillary Acidic Protein biosynthesis, Glial Fibrillary Acidic Protein immunology, Intermediate Filament Proteins, Mice, Mice, Inbred BALB C, Microscopy, Confocal, Molecular Sequence Data, Nerve Tissue Proteins analysis, Nerve Tissue Proteins biosynthesis, Nerve Tissue Proteins immunology, Nestin, Rats, Recombinant Proteins, Tumor Cells, Cultured, Glial Fibrillary Acidic Protein genetics, Nerve Tissue Proteins genetics, Polymorphism, Genetic
- Abstract
Transitin is an avian intermediate filament protein whose transient expression in the progenitor cells of the muscle and nerve tissues is similar to that of mammalian nestin. Both proteins contain an alpha-helical core domain flanked by a short N-terminal head and a long C-terminal extremity. However, the tail region of transitin is significantly different from that of nestin in that it harbors a unique motif containing more than 50 leucine zipper-like heptad repeats which is not found in any other intermediate filament protein. Despite the absence of introns in this region of the transitin gene, it was reported that different isoforms of the protein were produced by exclusion or inclusion of a number of repeats generated by an unusual splicing mechanism recognizing consensus 5' and 3' splice sites contained within the coding sequence of the heptad repeat domain [Napier et al. (1999) J Mol Neurosci 12:11-22]. Two monoclonal antibodies (mAbs) reacting with repeated epitopes of this motif were used to monitor transitin expression during in vitro myogenesis of the quail myogenic cell line QM7. Confocal microscopy revealed that the subcellular domains decorated with mAbs A2B11 and VAP-5 were mutually exclusive: the intermediate filament network visualized with mAb VAP-5 appeared to abut on a submembranous domain defined by mAb A2B11. When QM7 cells were induced to differentiate by switching to medium containing low serum components, an early effect was the local loss of A2B11 cortical staining at the points of cell-cell contacts. The A2B11 signal also disappeared before that of VAP-5 in newly formed myotubes. Unexpectedly, the mutually exclusive staining pattern of the mAbs could not be explained by alternative splicing since both epitopes mapped to a repeated element preceding the consensus 5' splice sites of the heptad repeat domain. An alternative explanation would be that the central repeat domain of transitin is a polymorphic structure from which different conformations exist depending on the local context. This hypothesis is strengthened by the observation that in cultured neural crest cells, the A2B11 antigen is preferentially expressed by freely migrating crest cells whose intracellular pH and calcium concentrations are different from those of non-migrating cells.
- Published
- 2001
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38. Avian transitin expression mirrors glial cell fate restrictions during neural crest development.
- Author
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Henion PD, Blyss GK, Luo R, An M, Maynard TM, Cole GJ, and Weston JA
- Subjects
- Animals, Antibodies, Monoclonal, Cell Differentiation physiology, Cell Movement physiology, Cells, Cultured, Chick Embryo, Ganglia, Spinal cytology, Gene Expression Regulation, Developmental physiology, Glial Fibrillary Acidic Protein analysis, Glial Fibrillary Acidic Protein immunology, Hybridomas, Intermediate Filament Proteins, Melanocytes cytology, Mice, Nerve Tissue Proteins analysis, Nerve Tissue Proteins immunology, Nestin, Neuroglia chemistry, Neurons chemistry, Neurons cytology, Neurons physiology, Quail, Stem Cells chemistry, Stem Cells cytology, Stem Cells physiology, Glial Fibrillary Acidic Protein genetics, Nerve Tissue Proteins genetics, Neural Crest cytology, Neural Crest embryology, Neuroglia cytology, Neuroglia physiology
- Abstract
During development, trunk neural crest cells give rise to three primary classes of derivatives: glial cells, melanocytes, and neurons. As part of an effort to learn how neural crest diversification is regulated, we have produced monoclonal antibodies (MAbs) that recognize antigens expressed by neural crest cells early in development. One of these, MAb 7B3 (7B3), was found to recognize an avian transitin-like protein by co-immunostaining with a series of transitin-specific monoclonal antibodies and by Western blot analysis. In neural crest cell cultures, we found that 7B3 initially recognizes the majority of neural crest cells as they emerge from the neural tube. Subsequently, 7B3-immunoreactivity (IR) is progressively restricted to a smaller subpopulation of cells. In fully differentiated trunk neural crest cell cultures, 7B3-IR is expressed only by cells that do not express neuronal markers and lack melanin granules. During development in vivo, 7B3-IR is evident in neural crest cells on the medial, but not the lateral migration pathway, suggesting that it is not expressed by melanocyte precursors. Later, the antigen is detected in non-neuronal, presumptive glial cells in dorsal root ganglia (DRG) and sympathetic ganglia, as well as along ventral roots. Cultures of E5 DRG confirm that 7B3-IR is restricted to non-neuronal cells of ganglia, many of which closely associate with neuronal processes. Therefore, of the three major classes of differentiated trunk neural crest derivatives, 7B3 exclusively recognizes glial cells, including both satellite glia and Schwann cells. Since the pattern of 7B3 expression in vitro mirrors the pattern of glial cell fate-restrictions in the trunk neural crest lineage, and is expressed by neural crest-derived glia in vivo, we conclude that 7B3 is an early pan-glial marker for neural crest-derived glial cells and their precursors.
- Published
- 2000
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39. Composition, synthesis, and assembly of the embryonic chick retinal basal lamina.
- Author
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Halfter W, Dong S, Schurer B, Osanger A, Schneider W, Ruegg M, and Cole GJ
- Subjects
- Agrin metabolism, Animals, Blotting, Western, Chick Embryo, Collagen metabolism, Collagenases metabolism, Culture Techniques, Extracellular Matrix, Heparitin Sulfate metabolism, In Situ Hybridization, Laminin metabolism, Membrane Glycoproteins metabolism, Microscopy, Fluorescence, Proteoglycans metabolism, Regeneration, Tenascin metabolism, Time Factors, Vitreous Body embryology, Basement Membrane embryology, Heparan Sulfate Proteoglycans, Nervous System embryology, Retina embryology
- Abstract
To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
40. Localization of transitin mRNA, a nestin-like intermediate filament family member, in chicken radial glia processes.
- Author
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Lee JA and Cole GJ
- Subjects
- Animals, Biological Transport physiology, Cerebellum chemistry, Cerebellum cytology, Chick Embryo, Diencephalon chemistry, Diencephalon cytology, Muscle, Skeletal chemistry, Muscle, Skeletal cytology, Nestin, Spinal Cord chemistry, Spinal Cord cytology, Vimentin genetics, Vimentin metabolism, Glial Fibrillary Acidic Protein genetics, Glial Fibrillary Acidic Protein metabolism, Intermediate Filament Proteins genetics, Intermediate Filament Proteins metabolism, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Neuroglia metabolism, RNA, Messenger analysis
- Abstract
We have examined the gene expression of two radial glia intermediate filament proteins, transitin and vimentin, in the developing chick CNS. Despite global similarities in their mRNA distributions, marked regional differences are observed. Most notably, we show that transitin mRNA is localized along radial glial processes and is localized to radial glia endfeet, whereas vimentin mRNA is not localized in radial glia. Localization of transitin mRNA is best shown in the diencephalic radial glia, as well as cerebellar Bergmann glia. In addition, in the early embryonic optic tectum, telencephalon, and retina, transitin mRNA is highly localized to radial glia endfeet, which is suggestive of its transport in these cells. These in vivo demonstrations of transitin mRNA localization are confirmed by in situ hybridization analysis of cultured chick brain radial glia, which demonstrates the presence of granular staining for transitin mRNA in glial processes. Transitin mRNA distribution in developing muscle also shows a highly regulated expression pattern, especially along the Z-lines of myofibrils. As further support for the transport and localization of transitin mRNA in radial glia and muscle, we have identified a consensus RNA transport signal in transitin mRNA that is absent from vimentin. These data suggest that the local regulation of transitin protein synthesis may contribute to its function as an intermediate filament protein in radial glia., (Copyright 2000 Wiley-Liss, Inc.)
- Published
- 2000
- Full Text
- View/download PDF
41. Agrin binds to beta-amyloid (Abeta), accelerates abeta fibril formation, and is localized to Abeta deposits in Alzheimer's disease brain.
- Author
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Cotman SL, Halfter W, and Cole GJ
- Subjects
- Agrin analysis, Agrin ultrastructure, Amyloid beta-Peptides analysis, Amyloid beta-Peptides ultrastructure, Humans, Microfibrils ultrastructure, Microscopy, Electron, Models, Neurological, Peptide Fragments analysis, Peptide Fragments ultrastructure, Plaque, Amyloid pathology, Protein Binding, Agrin metabolism, Alzheimer Disease pathology, Amyloid beta-Peptides metabolism, Brain pathology, Peptide Fragments metabolism
- Abstract
Agrin is an extracellular matrix heparan sulfate proteoglycan (HSPG) well known for its role in modulation of the neuromuscular junction during development. Although agrin is one of the major HSPGs of the brain, its function there remains elusive. Here we provide evidence suggesting a possible function for agrin in Alzheimer's disease brain. Agrin protein binds the amyloidogenic peptide Abeta (1-40) in its fibrillar state via a mechanism that involves the heparan sulfate glycosaminoglycan chains of agrin. Furthermore, agrin is able to accelerate Abeta fibril formation and protect Abeta (1-40) from proteolysis, in vitro. Supporting a biological significance for these in vitro data, immunocytochemical studies demonstrate agrin's presence within senile plaques and cerebrovascular amyloid deposits, and agrin immunostained capillaries exhibit pathological alterations in AD brain. These data therefore suggest that agrin may be an important factor in the progression of Abeta peptide aggregation and/or its persistence in Alzheimer's disease brain., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
42. Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage.
- Author
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Cheng G, Litchenberg WH, Cole GJ, Mikawa T, Thompson RP, and Gourdie RG
- Subjects
- Adenoviridae genetics, Adenoviridae physiology, Animals, Cell Lineage, Chickens, Embryonic and Fetal Development, Heart Conduction System cytology, Heart Conduction System virology, Humans, Muscles cytology, Neurons cytology, Purkinje Fibers cytology, Purkinje Fibers virology, Retroviridae genetics, Retroviridae physiology, Virus Replication, Heart Conduction System embryology, Purkinje Fibers embryology
- Abstract
The cardiac pacemaking and conduction system sets and maintains the rhythmic pumping action of the heart. Previously, we have shown that peripheral cells of the conduction network in chick (periarterial Purkinje fibers) are selected within a cardiomyogenic lineage and that this recruitment occurs as a result of paracrine cues from coronary arteries. At present, the cellular derivation of other elements of this specialized system (e.g. the nodes and bundles of the central conduction system) are controversial, with some proposing that the evidence supports a neurogenic and others a myogenic origin for these tissues. While such ontological questions remain, it is unlikely that progress can be made on the molecular mechanisms governing patterning and induction of the central conduction system. Here, we have undertaken lineage-tracing strategies based on the distinct properties of replication-incompetent adenoviral and retroviral lacZ-expressing constructs. Using these complementary approaches, it is shown that cells constituting both peripheral and central conduction tissues originate from cardiomyogenic progenitors present in the looped, tubular heart with no detectable contribution by migratory neuroectoderm-derived populations. Moreover, clonal analyses of retrovirally infected cells incorporated within any part of the conduction system suggest that such cells share closer lineage relationships with nearby contractive myocytes than with other, more distal elements of the conduction system. Differentiation birthdating by label dilution using [(3)H]thymidine also demonstrates the occurrence of ongoing myocyte conscription to conductive specialization and provides a time course for this active and localized selection process in different parts of the system. Together, these data suggest that the cardiac conduction system does not develop by outgrowth from a prespecified pool of 'primary' myogenic progenitors. Rather, its assembly and elaboration occur via processes that include progressive and localized recruitment of multipotent cardiomyogenic cells to the developing network of specialized cardiac tissues.
- Published
- 1999
- Full Text
- View/download PDF
43. Identification of extracellular matrix ligands for the heparan sulfate proteoglycan agrin.
- Author
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Cotman SL, Halfter W, and Cole GJ
- Subjects
- Animals, Chick Embryo, Chromatography, Affinity, Immunosorbent Techniques, Ligands, Surface Plasmon Resonance, Agrin metabolism, Extracellular Matrix Proteins metabolism, Fibroblast Growth Factor 2 metabolism, Laminin metabolism, Nerve Tissue Proteins metabolism, Tenascin metabolism, Thrombospondins metabolism
- Abstract
Agrin is a major brain heparan sulfate proteoglycan which is expressed in nearly all basal laminae and in early axonal pathways of the developing central nervous system. To further understand agrin's function during nervous system development, we have examined agrin's ability to interact with several heparin-binding extracellular matrix proteins. Our data show that agrin binds FGF-2 and thrombospondin by a heparan sulfate-dependent mechanism, merosin and laminin by both heparan sulfate-dependent and -independent mechanisms, and tenascin solely via agrin's protein core. Furthermore, agrin's heparan sulfate side chains encode a specificity in interactions with heparin-binding molecules since fibronectin and the cell adhesion molecule L1 do not bind agrin. Surface plasmon resonance studies (BIAcore) reveal a high affinity for agrin's interaction with FGF-2 and merosin (2.5 and 1.8 nM, respectively). Demonstrating a biological significance for these interactions, FGF-2, laminin, and tenascin copurify with immunopurified agrin and immunohistochemistry reveals a partial codistribution of agrin and its ECM ligands in the chick developing visual system. These studies and our previous studies, showing that merosin and NCAM also colocalize with agrin, provide evidence that agrin plays a crucial role in the function of the extracellular matrix and suggest a role for agrin in axon pathway development., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
44. Characterization of the chicken transitin gene reveals a strong relationship to the nestin intermediate filament class.
- Author
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Napier A, Yuan A, and Cole GJ
- Subjects
- Animals, Base Sequence, Cloning, Molecular, Consensus Sequence, Desmin genetics, Evolution, Molecular, Genomic Library, Glial Fibrillary Acidic Protein chemistry, Humans, Intermediate Filament Proteins chemistry, Leucine Zippers, Molecular Sequence Data, Nerve Tissue Proteins chemistry, Nestin, Neurofilament Proteins genetics, Rats, Restriction Mapping, Sequence Alignment, Sequence Homology, Nucleic Acid, Xenopus laevis, Chickens genetics, Glial Fibrillary Acidic Protein genetics, Intermediate Filament Proteins genetics, Nerve Tissue Proteins genetics, Phylogeny
- Abstract
Our laboratory previously reported that transitin is a radial glial intermediate filament protein sharing the basic structural features common to all intermediate filament (IF) proteins. It contains an alpha-helical core domain flanked by a short nonhelical head and a long COOH-terminal tail. The core sequence of transitin shows the greatest similarity to Xenopus tanabin and to rat and human nestin. We also reported that transitin has multiple splice variants derived from the deletion or inclusion of a leucine-zipper heptad repeat domain in the COOH-terminal tail. In the present study, we provide new evidence to support the classification of nestin and transitin in the same group of IF proteins based on the number and position of its introns. In addition, we suggest that the different isoforms of transitin are produced by a splicing mechanism that recognizes consensus 5' and 3' splice sites contained within the coding sequence of the leucine-zipper heptad repeat domain.
- Published
- 1999
- Full Text
- View/download PDF
45. Collagen XVIII is a basement membrane heparan sulfate proteoglycan.
- Author
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Halfter W, Dong S, Schurer B, and Cole GJ
- Subjects
- Amino Acid Sequence, Animals, Basement Membrane metabolism, Cell Adhesion, Chick Embryo, Collagen chemistry, DNA, Complementary, Humans, Immunohistochemistry, Mice, Microscopy, Fluorescence, Molecular Sequence Data, Schwann Cells cytology, Sequence Homology, Amino Acid, Collagen metabolism, Heparan Sulfate Proteoglycans metabolism
- Abstract
The present study shows that collagen XVIII is, next to perlecan and agrin, the third basal lamina heparan sulfate proteoglycan (HSPG) and the first collagen/proteoglycan with heparan sulfate side chains. By using monoclonal antibodies to an unidentified HSPG in chick, 14 cDNA clones were isolated from a chick yolk sac library. All clones had a common nucleotide sequence that was homologous to the mRNA sequences of mouse and human collagen XVIII. The deduced amino acid sequence of the chick fragment shows an 83% overall homology with the human and mouse collagen XVIII. Similar to the human and mouse homologue, the chick collagen XVIII mRNA has a size of 4.5 kilobase pairs. In Western blots, collagen XVIII appeared as a smear with a molecular mass of 300 kDa. After treatment with heparitinase, the protein was reduced in molecular mass by 120 kDa to a protein core of 180 kDa. Collagen XVIII has typical features of a collagen, such as its existence, under non-denaturing conditions, as a non-covalently linked oligomer, and a sensitivity of the core protein to collagenase digestion. It also has characteristics of an HSPG, such as long heparitinase-sensitive carbohydrate chains and a highly negative net charge. Collagen XVIII is abundant in basal laminae of the retina, epidermis, pia, cardiac and striated muscle, kidney, blood vessels, and lung. In situ hybridization showed that the main expression of collagen XVIII HSPG in the chick embryo is in the kidney and the peripheral nervous system. As a substrate, collagen XVIII moderately promoted the adhesion of Schwann cells but had no such activity on peripheral nervous system neurons and axons.
- Published
- 1998
- Full Text
- View/download PDF
46. The developmentally regulated avian protein IFAPa-400 is transitin.
- Author
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Ma X, Charron F, Cole GJ, Savard PE, and Vincent M
- Subjects
- Animals, Blotting, Southern, Cells, Cultured, Chick Embryo, Electrophoresis, Polyacrylamide Gel, Glial Fibrillary Acidic Protein chemistry, Glial Fibrillary Acidic Protein genetics, Intermediate Filament Proteins, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins genetics, Nestin, Precipitin Tests, Retina cytology, Retina embryology, Retina metabolism, Subcellular Fractions drug effects, Gene Expression Regulation, Developmental physiology, Glial Fibrillary Acidic Protein biosynthesis, Nerve Tissue Proteins biosynthesis
- Abstract
Transitin and IFAPa-400 are developmentally regulated high M(r) proteins expressed transiently in early chick embryogenesis. Both are associated with radially oriented fibers in the developing CNS and with various neural and myogenic tissues before their down-regulation at later stages. Previous studies have shown that IFAPa-400 colocalized and copurified with intermediate filament proteins and recent molecular cloning has indicated that transitin is a member of this family of cytoskeletal proteins. Here, we provide evidence that IFAPa-400 and transitin are the same protein. The sequence of a composite cDNA corresponding to more than 700 amino acids of IFAPa-400 carboxy-terminal extremity is identical to that of transitin. Both proteins exhibit identical apparent M(r) and isoelectric point. Immunopurified IFAPa-400 reacts with different antibodies to transitin and vice-versa. The patterns of expression of both proteins show a perfect coincidence at the tissue level. At the subcellular level, most antibodies to IFAPa-400/transitin decorate a typical intermediate filament network. However, monoclonal antibody A2B11, at the origin of transitin identification, exhibits a staining more typical of a cortical component, suggesting that different populations of transitin exist within the cell.
- Published
- 1998
- Full Text
- View/download PDF
47. An alternatively spliced, 5'-truncated MAP1B isoform is expressed in the developing chick nervous system.
- Author
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Burg MA, Lee JA, and Cole GJ
- Subjects
- Animals, Base Sequence, Blotting, Northern, Blotting, Southern, Brain embryology, Chick Embryo, Chondroitin Sulfate Proteoglycans genetics, Cloning, Molecular, DNA, Complementary analysis, Isomerism, Keratan Sulfate genetics, Lumican, Mice, Microtubule-Associated Proteins biosynthesis, Molecular Sequence Data, Polymerase Chain Reaction, Sequence Homology, Alternative Splicing, Brain metabolism, Microtubule-Associated Proteins genetics, Sequence Deletion
- Abstract
Our laboratory has previously characterized a keratan sulfate proteoglycan, named claustrin, and shown by molecular cloning that claustrin and the mouse MAP1B protein share high homology, with claustrin representing a 5'-truncated fragment of MAP1B. In the present study, we examine further the relationship between claustrin and MAP1B, and also describe the isolation of a cDNA encoding the 3'-region of MAP1B, which shares 3'-untranslated sequence, but not coding sequence, with claustrin. We call this partial cDNA 3'-MAP1B-related clone (3'-MRC), since it is homologous to the 3'-region of the mouse MAP1B sequence. We show by Northern analysis that distinct mRNAs are recognized by the claustrin and 3'-MRC cDNAs, and by RT-PCR that mRNAs encoding these distinct MAP1B-related molecules are present in embryonic chick brain and cardiac and smooth muscle. Our data also suggest a higher level of expression of claustrin mRNA in astrocyte cultures, when compared to 3'-MRC. Our data therefore provide new evidence that alternatively spliced variants of MAP1B are expressed in brain, and that at least one of these variants encodes the claustrin proteoglycan.
- Published
- 1997
- Full Text
- View/download PDF
48. Immunocytochemical localization of a novel radial glial intermediate filament protein.
- Author
-
Cole GJ and Lee JA
- Subjects
- Animals, Astrocytes metabolism, Astrocytes ultrastructure, Cells, Cultured, Central Nervous System cytology, Central Nervous System embryology, Central Nervous System metabolism, Chick Embryo, Glial Fibrillary Acidic Protein metabolism, Immunohistochemistry, Intermediate Filament Proteins, Nerve Tissue Proteins metabolism, Nestin, Neuroglia ultrastructure, Vimentin metabolism, Glial Fibrillary Acidic Protein physiology, Nerve Tissue Proteins physiology, Neuroglia physiology
- Abstract
We have examined by immunocytochemistry the subcellular localization of a chick radial glial protein, named transitin, that by molecular cloning has been shown to be a novel member of the intermediate filament protein superfamily. In astrocytes cultured from E10 chick brain, transitin is localized to the intermediate filament network in accordance with its structural properties. Using confocal microscopy we examined the expression of transitin, vimentin and glial fibrillary acidic protein (GFAP) in cultured astrocytes, and show that transitin co-distributes with these other glial intermediate filament proteins. The expression of transitin, vimentin and GFAP was also compared in embryonic chick spinal cord and brain radial glia, with these studies showing that these intermediate filament proteins display distinct expression patterns during CNS development. Of particular note is the absence of vimentin and GFAP in spinal cord midline radial glia that express transitin protein, and a transient expression of transitin in brain midline radial glia that continue to express vimentin. Our studies presented here therefore indicate that transitin, a novel radial glial intermediate filament protein, may have functions that are unrelated to GFAP or vimentin during CNS development, since transitin is localized to the processes of midline radial glia and is transiently expressed during chick CNS development.
- Published
- 1997
- Full Text
- View/download PDF
49. Distribution and substrate properties of agrin, a heparan sulfate proteoglycan of developing axonal pathways.
- Author
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Halfter W, Schurer B, Yip J, Yip L, Tsen G, Lee JA, and Cole GJ
- Subjects
- Animals, Blotting, Western, Cerebellum cytology, Cerebellum ultrastructure, Chick Embryo, Electrophoresis, Polyacrylamide Gel, Ganglia, Spinal cytology, Ganglia, Spinal physiology, In Situ Hybridization, Microscopy, Fluorescence, Neural Pathways metabolism, Neurites drug effects, Neurites ultrastructure, RNA, Messenger biosynthesis, Retina cytology, Retina metabolism, Spinal Cord cytology, Agrin metabolism, Axons metabolism, Heparitin Sulfate metabolism
- Abstract
The distribution and substrate properties of agrin, an extracellular matrix heparan sulfate proteoglycan (HSPG), was investigated in the developing chick nervous system by immunocytochemistry, Western blotting, and in neurite outgrowth assays. By comparing the distribution of agrin with that of laminin-1, merosin (laminin-2), neurofilament, and neural cell adhesion molecule (NCAM), it was found that throughout development, agrin is a constituent of all basal laminae. From embryonic day (E) 4 onwards, agrin is also abundant in axonal pathways of the central nervous system, such as the optic nerve, the tectobulbar pathway, the white matter of the spinal cord, and the marginal and the molecular layers of the forebrain and the cerebellum. The abundance of agrin in brain decreases from E13 onwards. In the peripheral nervous system, agrin is present throughout development as a constituent of the Schwann cell basal laminae. Western blots confirmed the immunocytochemical data, showing maximum expression of agrin occurs during the early to medium stages of brain development. Western blots also showed that in mouse and human brain, agrin exists as an HSPG. Purified agrin did not support neurite outgrowth, rather it inhibited retinal neurite extension on mixed agrin/merosin substrates. Despite the fact that agrin, when used as a substrate inhibited neurite outgrowth, its temporal and spatial overlap with growing axons suggests that agrin has a supportive role in the development of axonal pathways, possibly as a binding component for growth factors and cell adhesion proteins.
- Published
- 1997
50. Molecular cloning of a new intermediate filament protein expressed by radial glia and demonstration of alternative splicing in a novel heptad repeat region located in the carboxy-terminal tail domain.
- Author
-
Yuan Y, Lee JA, Napier A, and Cole GJ
- Subjects
- Amino Acid Sequence, Animals, Astrocytes metabolism, Cells, Cultured, Chickens, Intermediate Filament Proteins, Intermediate Filaments metabolism, Molecular Sequence Data, Nestin, Rats, Rats, Sprague-Dawley, Alternative Splicing, Cloning, Molecular, Glial Fibrillary Acidic Protein genetics, Glial Fibrillary Acidic Protein metabolism, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Neuroglia metabolism, Repetitive Sequences, Nucleic Acid
- Abstract
In the present study we describe the molecular cloning of transitin, formerly named EAP-300. We show that transitin is an intermediate filament protein with a core domain most closely resembling nestin and tanabin. Transitin also contains a novel heptad amino acid repeat domain, comprising multiple leucine zipper repeats, located in its tail region. Based on these structural motifs we propose that a novel intermediate filament protein that is transiently expressed by radial glia during CNS development has been identified. We also show the existence of splice variants of transitin with splicing occurring in the novel heptad repeat domain to give rise to transitin isoforms that lack this heptad repeat. By in situ hybridization analysis we show that transitin mRNA is expressed by midline radial glial structures, by several axon commissures, and by Bergmann glia of the developing cerebelium. Based on the structural properties of the transitin protein, and expression of its mRNA, we suggest that transitin is a new member of the intermediate filament gene superfamily that is transiently expressed by radial glia.
- Published
- 1997
- Full Text
- View/download PDF
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