Danio rerio
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ABSTRACT:
PROVIDER: PRJNA668620 | ENA |
REPOSITORIES: ENA
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Project description:To gain a better understanding of the factors necessary for successful CNS regeneration, a temporal analysis of the changes in gene expression in the eye caused by optic nerve injury was conducted. Dual color oligonucleotide microarrays were used to compare total RNA harvested from the eyes of sham-operated and optic nerve-injured fish at 3, 24, and 168 hours following surgery. Optic nerve injured fish are compared to sham-operated fish in order to eliminate gene expression due to non-neuronal damage and inflammatory response. Statistical analyses identified 131 genes with a 2.0-fold or greater difference in expression. Wild type zebrafish were obtained from a local pet store. Optic nerve injury was conducted using a severing model accomplished as follows. Zebrafish were anesthetized in 0.2% MS-222 dissolved in tank water. The muscles surrounding the eye were cut and the eye angled rostrally to expose the nerve. The optic nerve was then severed using microsissors without damaging the ophthalmic artery. In sham operated fish the muscles surrounding the eye were severed but the nerve was not damaged. RNA was extracted from the eye at three time points following surgery 3 hours, 24 hours, and 168 hours. RNA was pooled from multiple fish to achieve 10 ug total RNA. Samples were collected in triplicate per time point. Gene expression was analyzed on a dual color oligonucleotide array where the optic nerve injured fish were compared to sham-operated fish. Four samples of RNA were also collected from control fish and compared to each other on the microarray to confirm that processing did not create expression differences.
2010-05-16 | E-GEOD-17854 | biostudies-arrayexpress
Project description:It is well-established that neurons in the adult mammalian central nervous system are terminally differentiated and, if injured, will be unable to regenerate their connections. In contrast to mammals, zebrafish and other teleosts display a robust neuroregenerative response. Following optic nerve crush (ONX), retinal ganglion cells (RGC) regrow their axons to synapse with topographically correct targets in the optic tectum, such that vision is restored in ~21 days. What accounts for these differences between teleostean and mammalian responses to neural injury is not fully understood. A time course analysis of global gene expression patterns in the zebrafish eye after optic nerve crush can help to elucidate cellular and molecular mechanisms that contribute to a successful neuroregeneration. We used microarrays to detail the global gene expression patterns underlying a successful regeneration or the optic nerve following injury. Experiment Overall Design: Microarray analysis was performed on total RNA extracted from whole eye following optic nerve crush (ONX) or sham surgery at defined intervals (4, 12, & 21 days).
2010-02-21 | E-GEOD-19298 | biostudies-arrayexpress
Project description:RNA-sequencing of Adult Zebrafish Inner Ear Hair Cells
| PRJNA395227 | ENA
Project description:Zebrafish (Danio Rerio) have the capacity for successful adult optic nerve regeneration, unlike mammals. Optic nerve regeneration is frequently studied using optic nerve crush (ONC). For ONC, animals were deeply anesthetized in 0.033% tricaine methane-sulfonate (MS-222). The right optic nerve was exposed by gently removing the connective tissue on the dorsal half of the eye and rotating the eye ventrally out of the orbit with a pair of number 5 forceps. A nerve crush was then performed using number 5 forceps to crush the nerve ~0.5 to 1 mm from the optic nerve head for 5 seconds. Success of crush was assessed by identifying the generation of a translucent stripe in the nerve that completely separated two areas of white myelination with no bleeding. Fish were then revived in fresh aquatic system water in individual tanks. After 1 hour the tanks were returned to the fish system and animals were maintained normally with daily feeding until 3 days post injury.
Female and male (6 month to 1 year old) Zebrafish optic nerves (right side/OD) were crushed and collected three days after. The associated retinas and tecta were also collected under the same conditions. Contralateral, uninjured optic nerves, retinas and tecta were collected as controls. For tissue collection, animals were euthanized by overdose of MS-222 (>400mg/L) followed by dissection. The tissue was collected from the optic nerve head to the optic chiasm. The tissues were immediately frozen on dry ice. Optic nerve samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 31 to obtain sufficient protein concentrations for analysis (pooled optic nerves served as one sample). Retina and tectum samples were pooled using the same categories (female crush, female control, male crush, male control) at n = 10-12 (pooled tissue served as one sample).
Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic peptide standards (Regen III) were added to the samples during the extraction to measure extraction efficiency. Each standard was spiked into samples for a total concentration of 48uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 50ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. All samples were labelled using 2 sets of 14 tags from a 18plex TMT (Tandem Mass Tag) kit (A52045: Thermo Fisher Scientific, Waltham, MA) for quantification. After combination and drying of all peptide samples, the samples were fractionated into 9 fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (84868: Thermo Fisher Scientific, Waltham, MA). After fractionation and drying of all peptide samples, each fractionated TMT sample was spiked with two additional peptide standards (Regen II) containing isobaric labels. The final concentration of the post extraction spiked peptides was 54uM per plex. These standards (Regen II) were spiked directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards serve as a normalization method that may be used to compare protein abundance data across multiple datasets. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The Danio rerio proteome was downloaded from UniProt and used as the target database for protein identification. Max missed cleavage site was set to 2 and minimum peptide length to 6 . Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, carbamidomethylation and TMTpro. The Normalization was set to total peptide amount and confidence to low. Two additional local databases were created for the pre- and post- extraction peptides and ran separately from experimental protein identification.
2024-06-25 | MSV000095140 | MassIVE
Project description:Whole transcriptomic sequencing of zebrafish rx3 mutants during optic vesicle morphogenesis
| PRJNA229539 | ENA
Project description:WDR12 is a ribosome biogenesis factor known to be expressed in Optic tectum Neuroepithelial cells, we define the transcriptome profile of the optic tectum neuroepithelial cells, by cell-sorting followed by RNA-seq (Illumina technology).
2019-04-20 | GSE80497 | GEO
Project description:Mycobacterium marinum chronic infection of adult zebrafish
| PRJNA112215 | ENA