Project description:To further characterize the roles of cortisol signaling via the glucocorticoid receptor (GR) in developing zebrafish, we have used morpholino oligonucleotides to knockdown GR protein translation and measured gene expression in RNA extracted from 24 and 36 hours post fertilization (hpf) embryos. The GR morpholino was characterized previously in Nesan et al., 2012, Endocrinology 181, 35-44) Single-cell zebrafish embryos were microinjected with either active morpholino or mispair control. Embryos were frozen at 24 and 36 hpf and total RNA extracted for microarray analysis. Three independent replicates (different breeding events on separate days) were performed for each treatment per timepoint.
Project description:In triplicate for each condition, 12 WT and acbd6 F0 crispant Danio rerio (zebrafish) embryos were incubated with 20 μM YnMyr for 24 h, either between 48-72 hpf or 96-120 hpf. After labelling, zebrafish were washed twice with fresh egg water, deyolked, flash frozen in liquid nitrogen and stored at -80°C until further analysis.
Project description:To further characterize the roles of cortisol signaling via the glucocorticoid receptor (GR) in developing zebrafish, we have used morpholino oligonucleotides to knockdown GR protein translation and measured gene expression in RNA extracted from 24 and 36 hours post fertilization (hpf) embryos. The GR morpholino was characterized previously in Nesan et al., 2012, Endocrinology 181, 35-44)
Project description:In triplicate for each condition, 12 WT and acbd6 F0 crispant Danio rerio (zebrafish) embryos were incubated with 20 μM YnMyr for 24 h, either between 48-72 hpf or 96-120 hpf. After labelling, zebrafish were washed twice with fresh egg water, deyolked, flash frozen in liquid nitrogen and stored at -80°C until further analysis.
Project description:Increasing frequency and geographical distribution of harmful algal blooms (HABs) presents a growing threat to the public health. Saxitoxin (STX) is a potent neurotoxin naturally produced by dinoflagellates and cyanobacteria during HAB events. Consumption of seafood contaminated with STX is responsible for paralytic shellfish poisoning (PSP). STX inhibits voltage-gated sodium channels, affecting the propagation of action potentials. Humans are among the species most sensitive to PSP, and neurological symptoms of exposure range from tingling of the extremities to severe paralysis. To protect humans against PSP, there is a ban on harvesting of seafood when the STX levels reach 80 μg/100 g of shellfish tissue. However, shellfish with toxin levels below this regulatory limit often are harvested for consumption. Our objective is to understand the potential health effects of exposure to low levels of STX during sensitive windows of development. Zebrafish embryos were exposed to STX (24 or 48 pg) or vehicle (0.3 mM HCl) at 6 hours post fertilization (hpf) via microinjection. There was no overt toxicity, but starting at 36 hpf there was a temporary lack of pigmentation in STX-injected embryos, which resolved by 72 hpf. Using HPLC, we found that STX was retained in embryos up to 72 hpf in a dose dependent manner. We examined transcriptional profiles in embryos at 24, 36 and 48 hpf. There were no differentially expressed genes (DEGs) in STX-injected embryos at 24 hpf, but at 36 and 48 hpf there were_x000B_3547 and 3356 DEGs, respectively, in response to STX. KEGG pathway analysis revealed significant enrichment of genes related to focal adhesion, adherens junction and regulation of actin cytoskeleton, suggesting that cell-cell and cell-extracellular matrix interactions were affected by STX. The genes affected are critical for axonal growth and the development of functional neural networks. We also observed differential expression of axon guidance factors (netrins, semaphorins, and ephrins), which can control axon outgrowth. We are currently using immunohistochemistry to confirm these findings. Overall, these results suggest that STX exposure might affect axon outgrowth by modulating cell adhesion molecules. [NIH P01ES021923 and NSF OCE-1314642].
Project description:This project aimed at identifying developmental stage specific transcript profiles for catecholaminergic neurons in embryos and early larvae of zebrafish (Danio rerio). Catecholaminergic neurons were labeled using transgenic zebrafish strains to drive expression of GFP. At stages 24, 36, 72 and 96 hrs post fertilization, embryos were dissociated and GFP expressing cells sorted by FACS. Isolated RNAs were processed using either polyA selection and libray generation or NanoCAGE. This is the first effort to determine stage specific mRNA profiles of catecholaminergic neurons in zebrafish.
