Unknown,Transcriptomics,Genomics,Proteomics

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Gene Expression analysis of transgenic RAS heatshock zebrafish embryos at 30 hpf

ABSTRACT: Heterogeneous Tg(hsp70-hRASG12V) embryos were obtained by mating the male homozygous transgenic fish to wildtype females. They were raised to 24 hour-post fertilization (hpf) stage and received heatshock at 37°C in waterbath for one hour, and kept in 28.5°C till 30hpf for RNA extraction. Wildtype embryos receiving the same heatshock treatment were used as controls. Each microarray sample was prepared by pooling 50 embryos and biological duplication was used. The zebrafish has been established as a powerful vertebrate model organism for large-scale genetic screens and chemical screens. Here, we seek to establish that zebrafish embryos can be utilized as an in vivo system to dissect crucial pathways for oncogenesis. A microarray analysis was performed by comparing transcription profile of heterogeneous Tg(hsp70-hRASG12V) embryos with heatshock to wildtype embryos with heatshock. Both groups of embryos received heatshock at 37°C for one hour at 24 hour-post fertilization (hpf) stage, and kept in 28.5°C till 30hpf for RNA extraction. Each microarray sample was prepared by pooling 50 embryos and biological triplicate was used. Ingenuity Pathway Analysis (IPA) was performed using the upregulated lists. “Cancer “ was the top diseases and disorders, with “Developmental disorder” and “Organismal Injury and Abnormalities” as the second and third diseases and disorders, validating that transient heatshock induction of oncogenic RAS in embryos activates major pathways in oncogenesis.

ORGANISM(S): Danio rerio

SUBMITTER: Anthony DiBiase

PROVIDER: E-GEOD-44364 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Parallels between phylogeny and ontogeny have been discussed since almost two centuries and a number of theories have been proposed to explain such patterns. Especially elusive is the so-called phylotypic stage, a phase during development where species within a phylum are particularly similar to each other. While this has formerly been interpreted as a recapitulation of phylogeny, it is nowadays thought to reflect an ontogenetic progression phase, where strong constraints on developmental regulation and gene interactions exist. Several studies have shown that genes expressed during this stage evolve slower, but it has so far not been possible to derive an unequivocal molecular signature associated with this stage. We use here a combination of phylostratigraphy and stage specific gene expression data to generate a cumulative index that reflects the evolutionary age of the transcriptome at given ontogenetic stages. Using zebrafish ontogeny and adult development as a model, we find that the phylotypic stage does indeed express the oldest transcriptome set and that younger sets are expressed during early and late development, thus faithfully mirroring the hourglass model of morphological divergence. Reproductively active animals show the youngest transcriptome, with major differences between males and females. Intriguingly, aging animals express also increasingly older genes. Comparisons with similar datasets from flies and nematodes show that this pattern occurs across phyla. Our results suggest that an old transcriptome marks the phylotypic phase and that phylogenetic differences at other ontogenetic stages correlate with the expression of newly evolved genes. Zebrafish (Danio rerio) were kept in 12L flow-through tanks at 26.5 °C (around 60 animals per tank). For accurate staging, fertilized eggs were collected within 15 min intervalls and incubated in Petri dishes at 28.5°C with water changes every 2-6 hours. After hatching, larvae were transferred to 1 liter tanks and kept at 28.5 °C. We took 72 samples in at least two biological replicates (147 experiments in total) that correspond to 61 stages across zebrafish ontogeny (50 stages before the sex could be clearly recognized plus 11 adult stages of males and females each). Staging was done according to post-fertilization time. Embryos were additionally staged under the dissecting microscope to check for the consistency of post-fertilization timing and morphological development at standard temperature (28.5 °C). Only healthy animals that showed the expected morphological features for a given post-fertilization time were sampled. Each sample contained around 50 individuals until the 1 day and 3 hour embryo stage, 15 individuals until the 10 day larval stage, 10 individuals until the 18 day larval stage, 5 individuals until the 45 days juvenile stage, while in later juvenile and adult stages we sampled males and females separately and each sample contained 2 individuals. All samples were snap frozen in liquid nitrogen and stored at -80 °C until RNA extraction. To avoid severe biases due to the excess of unfertilized eggs we squeezed eggs from adult females before freezing them in liquid nitrogen.

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Gene Expression analysis of transgenic RAS heatshock zebrafish embryos at 30 hpf

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