Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of dwarf and normal Coregonus whitefishes at different life history stages

ABSTRACT: The gene expression analysis for this study focuses on the divergence at different life history stages of dwarf and normal whitefish. Eight pairwise (dwarf vs. normal) comparisons for both the embryonic and the juvenile fish stage were performed resulting in two sets of eight microarrays per stage. Pools containing the total RNA of five embryos were used for the embryo experiments. This pooling approach integrates patterns of gene expression over a larger number of individuals but would nevertheless reveal differences between group means as tested for in an analysis of variance. Juvenile fish extractions yielded large quantities of total RNA and were used individually. The same representation of experimental groups was used in both the embryo and juvenile fish experiments. In order to reduce artefacts samples that are similar with respect to developmental features (segment count) were used in the embryos. The sampling of juvenile fish is less difficult as they have finished their morphogenesis. Their ontogeny is also slower and probably reduced to relatively constant growth processes. Juvenile fishes were chosen to represent a similar body mass range

ORGANISM(S): Coregonus clupeaformis

SUBMITTER: arne nolte

PROVIDER: E-MEXP-1973 | 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.

Project description:The MTF-1 transcription factor is considered to be a master regulator of zinc homoestasis. We previously characterized a constitutively nuclear, dominant-negative zebrafish MTF-1/eGFP fusion protein (dnMTF-1). In this study, in vitro transcribed dnMTF-1 mRNA was microinjected into zebrafish embryos (2-cell stage), with controls consisting of zebrafish embryos (2-cell stage) microinjected with in vitro transcribed enhanced green fluorescent protein (eGFP) mRNA. Transcriptomic profiling was performed using an Agilent 4 x 44K array on 4 replicate pools of thirty 28- and 36-hpf embryos for each treatment. The role of MTF-1 signaling in zebrafish embryos was examined by injection of in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (2-cell stage), with controls consisting of zebrafish embryos (2-cell stage) microinjected with in vitro transcribed enhanced green fluorescent protein (eGFP) mRNA. Transcriptomic profiling was performed using an Agilent 4 x 44K array on 4 replicate pools of thirty 28- and 36-hpf embryos for each treatment. A Narishige IM-300 microinjector was used to microinject 1-2 cell embryos with ~100 pg of dnMTF-1 IVT mRNA (~2.1 nL microinjection volume with a concentration 50 ng/M-BM-5L IVT mRNA), or ~100 pg of enhanced green fluorescent protein (eGFP) IVT mRNA. Immediately after microinjection, embryos from each treatment were divided into 4 replicates of 30 pooled embryos and held in 25 mL of 0.3X DanieauM-bM-^@M-^Ys at 28.5M-BM-0C under a 14 hour light/10 hour dark cycle. eGFP positive embryos (dnMTF-1 and eGFP) were collected at 28 and 36 hpf and flash frozen in liquid nitrogen for future RNA isolation and microarray hybridization. Total RNA was prepared using the RNA STAT60 protocol (Tel-Test, Inc., TX). RNA was quantified using a Nanodrop 2000C spectrophotometer and assessed for quality using an Agilent 2100 Bioanalyzer Lab-on-chip system. Only samples with RNA integrity number (RIN) between 9.8-10 were used for microarray analysis. For each RNA sample, a single microarray was hybridized with 750 ng Cy3 labeled cDNA using AgilentM-bM-^@M-^Ys standard conditions for single-color microarrays. The samples were hybridized to a the Agilent 4x44K feature zebrafish microarray using the Agilent In situ Hybridization Kit Plus by the Genome Technology Core at the Whitehead Institute for Biomedical Research (Cambridge, MA). Post-hybridization, microarray slides were washed as per the Agilent In situ Hybridization Kit Plus and scanned with an Agilent DNA Microarray Scanner. Extraction of raw microarray results was performed using AgilentM-bM-^@M-^Ys Feature Extraction software with background detrending (spatial and multiplicative). The data were normalized using a non-linear scaling method based on rank invariant probes. Cy3 signal saturated probes and probes not above background in all replicated were then removed prior to statistical analysis. Statistical tests were performed using the MeV microarray analysis suite. All data were log transformed and median centered for each probe. A two-factor ANOVA was performed for time (28 vs. 36 hpf), treatment (dnMTF-1 vs. eGPF), and their interaction with p-value based on 1000 permutations of the data and alpha of 0.01. For post-hoc analyses to find biologically relevant results, the probes significant for treatment and time-treatment interaction were secondarily examined using Rank Product (RP) analysis. Rank product analysis was performed to provide lists of probes up- and down-regulated between GFP control and MTF-1 for each time point. For each test, a two-class unpaired RP analysis was performed using 1000 permutation of the data with a false discovery rate (FDR) not exceeding 5%.

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Transcription profiling of dwarf and normal Coregonus whitefishes at different life history stages

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