Unknown,Transcriptomics,Genomics,Proteomics

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Stage-specific transcriptional changes during zygotic embryogenesis in maritime pine (Pinus pinaster Ait.)

ABSTRACT: The significant morphological differences observed during embryo development in angiosperms and gymnosperms are expected to be the result of a differential control of gene expression. We used a loblolly pine (Pinus taeda) cDNA microarray to analyze global transcriptional changes along zygotic embryogenesis in maritime pine (Pinus pinaster). A time-course analysis of the data obtained from the five embryo developmental groups used in this study led to the identification of 4,645 genes whose expression varied along P. pinaster embryogenesis. These transcripts were clustered into six distinct expression profiles. The grouping of these profiles in early, mid-embryogenesis and embryo maturation, according to the developmental period where most of the sequences were up-regulated, evidenced that characteristic transcriptional changes are associated to each developmental period. The application of a cut-off value of 1.95-fold change led to the identification of 1,838 differentially expressed transcripts that were categorized by biological process. Metabolism, interaction with the environment, oxidation-reduction and transport were some of the most represented categories. During early embryogenesis genes putatively involved in phytohormone-mediated signaling were identified, whereas in middle stages the overrepresented genes could be associated with cotyledon formation and induction of somatic embryogenesis. Genes associated with the synthesis of storage products were up-regulated in the latest stages of pine embryo development. It was also during this developmental period that the largest number of sequences putatively encoding transcription factors was identified. Pine immature zygotic embryos were pooled into five different developmental groups according to the collection date (Day0; Day5; Day11; Day15 and Day25). A common reference design was used. Total RNA was extracted from each sample. Three extractions were prepared per sample (biological replicates). A defined ammount from each RNA sample was pooled to use as reference. Messenger RNA was amplified for both test samples and reference. These were hybridized together in two separate experiments (technical replicates). In total, 30 slides were analyzed.

ORGANISM(S): Pinus pinaster

SUBMITTER: Marta Simões

PROVIDER: E-GEOD-32551 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: The early stages of D. melanogaster embryogenesis involve cell migration and pattern formation, and lead to the formation of three germ layers (the ectoderm, mesoderm and endoderm). These developmental events are controlled by differential gene activity. In the current study we used a suppressive subtractive hybridization (SSH) procedure to identify a group of genes potentially involved in D. melanogaster early embryogenesis and to study the temporal activity of developmentally regulated genes at five different intervals covering 12 stages of embryogenesis. Results: Macroarrays were constructed to confirm induction of expression and determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes that significantly increased their expression levels at least at one developmental interval compared with the reference interval. 53.4% of them have a phenotype and/or molecular function reported in the literature, whereas 46.6% are essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 30 of the transcripts and of these, 19 (63%) proved to have restricted expression patterns. 11 of the uncharacterized genes that exhibited temporal and spatially restricted patterns of expression in developing embryos encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in S2R+ cells and analyze the subcellular distribution of recombinant proteins. Conclusions: Our data provides a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis, including novel information on the temporal and spatial expression patterns of several previously uncharacterized genes. In particular, we recovered a significant number of novel genes encoding putative secreted and transmembrane proteins, suggesting new components of signalling pathways that might be incorporated within the existing regulatory networks controlling Drosophila embryogenesis; they are also good candidates for more functional targeted analyses. Keywords: Drosophila melanogaster, gastrulation, macroarray, developmental time course We generated a subtracted stage specific cDNA library representing genes differentially expressed between Drosophila melanogaster synciyial blastoderm and gastrula stages. Clones were spotted onto nylon membranes and hybridized against cDNA probes representing mRNA from embryos at five different developmental intervals, in order to obtain time course expression data covering the first 12 stages of Drosophila development. We designed macroarrays containing either 302 non-redundant cDNAs, including protein-coding sequences, ncRNA, transposons, introns and intergenic regions (membranes A, B and C) and the complete subtracted library (579 cDNAs, membrane D). Each clone was spotted in duplicate. The following controls were spotted onto membranes: (1) a fragment of the vector pBluescript II obtained by amplification with the T7 and SP6 universal primers; (2) several spots of DMSO 50% (solvent used); (3) PCR amplified fragment of genes serendipity Î±, twist, tinman, fog and snail as positive controls, and (4) actin, tubulin, and RP49 as housekeeping, and (5) four dilutions of a PCR amplified fragment from a B. subtilis dap cDNA (ATCC; number 87486), which hybridizes to an in vitro synthesized poly(A)-RNA that was added to the embryo RNA samples (dilution 1/200) prior to the labelling process and used as spike mRNA (Kane et al., 2000).

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Stage-specific transcriptional changes during zygotic embryogenesis in maritime pine (Pinus pinaster Ait.)

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