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Comparative analysis of gene expression profiles between four kinds of blastomere at 8-cell stage embryos.

ABSTRACT: The establishment of body axes and specification of early embryonic cells depend on maternally supplied transcripts and/or proteins, several of which are localized at specific regions of fertilized eggs and early embryos. The ascidian is known to exhibit a mosaic mode of development, and this mode is largely dependent on localized maternal factors. Using blastomere isolation, microarray and whole-mount in situ hybridization, the present study of Ciona intestinalis demonstrates that maternal transcripts of a total of 17 genes are localized at the posterior-most region of fertilized eggs and early embryos. Ten of them are newly identified in the present study, while the remaining seven genes have already been characterized in the previous studies. In addition, maternal transcripts of two genes, in addition to 14 genes encoded by the mitochondrial genome, showed a mitochondria-like distribution. Despite the present comprehensive approach, we could not identify maternal transcripts that are clearly localized to the animal-pole side, the vegetal-pole side, the anterior-side or other specific regions of the early embryo. Therefore, we concluded that the posterior-most localization and mitochondria-like distribution appear to be major specialized patterns of maternal transcripts in the early Ciona embryos.

ORGANISM(S): Ciona intestinalis

PROVIDER: GSE15637 | GEO | 2009/04/17

SECONDARY ACCESSION(S): PRJNA115637

REPOSITORIES: GEO

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Project description:Postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs with include at least 2 determinants (Macho1 and PEM1) involved in muscle differentiation, unequal cleavage and posterior patterning of the embryo. Two main classes have been defined - type I (localized in oocytes) and type II (localizing in embryos). Based on the fact that Vasa RNA - the only type II recognized in Ciona - segregates into B8.12 (germ) and sister B8.11 cells, while many others (including Macho1 and PEM1) segregate only into B8.11 (somatic) cells a first attempts at re-classifying these RNAs has been made. We have analyzed the associations of postplasmic/PEM RNAs with cortical structures in 2 ascidians species Ciona intestinalis and Phallusia mammillata using: - very high resolution fluorescent in situ hybridization localization in eggs and embryos and in their cortical fragments, - comparisons of RNAs extracted from whole oocytes and from isolated cortices using microarray analysis â screening of the Ciona intestinalis genome for the presence in 3â UTR regions of repeats containing the tri-nucleotide CAC motif (xCACx) followed by localization of xCACx rich RNAs using in situ hybridization. Based on our combined approaches we observe that: - 1) the vast majority of Ciona intestinalis postplasmic/PEM RNAs (including Ci-Vasa) are cortically localized in the eggs (type I) - 2) postplasmic/PEM RNAs are much more likely to contain xCACx motifs in their 3âUTR than other transcripts. Using this criterion we have found 2 new postplasmic/PEM RNAs: MnK and PSD - 3) some postplasmic/PEM RNAs are anchored to cER while others are located into granules. We therefore come to the conclusion that there are at least 2 distinct categories of postplasmic/PEM RNAs based on their cortical anchorage and cell fate: I) cER sub-domain associated postplasmic/PEM RNAs (Macho1 like) which segregate in the somatic B8.11 cells. II) postplasmic/PEM RNAs associated with (putative germ) granules (Vasa like) which in addition to B8.11 cells segregate into B8.12 germ cells. Two kinds of sample, Dye Swap design with 2 arrays

Project description:Microarray analysis of zygotic gene expression in 2-to-3 hour wild-type (wt) and smg mutant embryos. Expression is relative to mature, stage 14 oocytes, which contain the full maternal pool of mRNA. Strictly maternal genes that are not transcribed at the MZT contribute approximately 80% of transcripts in early embryos, and are not shown. Class I zygotic genes showed high levels of expression in 2-to-3 hour embryos. 142 of the 166 Class I genes were not expressed in smg mutants. The remaining zygotically expressed genes were also present in oocytes. These genes were divided into two classes, based on analysis of 4-to-6 hour old unfertilized eggs (4-6h unf), which are transcriptionally inactive. Class-II genes produce maternal transcripts that are stable in unfertilized eggs and show significantly increased expression in 2-to-3 hour post-fertilization embryos. 358 of 395 Class-II genes require SMAUG for zygotic expression. Class-III genes produce maternal transcripts that are degraded in unfertilized eggs and show significantly increased expression in 2-to-3 hour post-fertilization embryos. 65 of 408 Class-III genes require SMAUG for expression in 2-to-3 hour embryos. mRNA was extracted from staged fertilized or unfertilized eggs, as well as stage 14 oocytes as described previously (Tadros et al., 2007a). To assay mRNA quality, known stable (rpA1) and unstable (Hsp83) transcripts were probed on Northern blots. Total RNA was then reverse transcribed with random primers (Tadros et al., 2007a) and labeled asÃ?described in the Indirect Labeling of Total RNA for Microarray Hybridization protocol at http://www.flyarrays.com. The fluorescently labeled cDNA probes were hybridized to 12Kv1 microarrays obtained from the Canadian Drosophila Microarray Centre (http://www.flyarrays.com; GEO platform accession number GPL1467: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL1467). Hybridization and scanning were performed using a PerkinElmer/GSI ScanArray 4000 scanner and the ScanArray software as previously described (Neal et al., 2003). The 16 bit TIFF image files were quantified using QuantArray Version 3 (PerkinElmer), using the adaptive quantification algorithm and analyzed using GeneTraffic Duo3.2 (Iobion Informatics/Stratagene). The 12Kv1 arrays were normalized using the rank invariant LOWESS extrapolation method (Schadt et al., 2001).

Project description:Postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs with include at least 2 determinants (Macho1 and PEM1) involved in muscle differentiation, unequal cleavage and posterior patterning of the embryo. Two main classes have been defined - type I (localized in oocytes) and type II (localizing in embryos). Based on the fact that Vasa RNA - the only type II recognized in Ciona - segregates into B8.12 (germ) and sister B8.11 cells, while many others (including Macho1 and PEM1) segregate only into B8.11 (somatic) cells a first attempts at re-classifying these RNAs has been made. We have analyzed the associations of postplasmic/PEM RNAs with cortical structures in 2 ascidians species Ciona intestinalis and Phallusia mammillata using: - very high resolution fluorescent in situ hybridization localization in eggs and embryos and in their cortical fragments, - comparisons of RNAs extracted from whole oocytes and from isolated cortices using microarray analysis – screening of the Ciona intestinalis genome for the presence in 3’ UTR regions of repeats containing the tri-nucleotide CAC motif (xCACx) followed by localization of xCACx rich RNAs using in situ hybridization. Based on our combined approaches we observe that: - 1) the vast majority of Ciona intestinalis postplasmic/PEM RNAs (including Ci-Vasa) are cortically localized in the eggs (type I) - 2) postplasmic/PEM RNAs are much more likely to contain xCACx motifs in their 3’UTR than other transcripts. Using this criterion we have found 2 new postplasmic/PEM RNAs: MnK and PSD - 3) some postplasmic/PEM RNAs are anchored to cER while others are located into granules. We therefore come to the conclusion that there are at least 2 distinct categories of postplasmic/PEM RNAs based on their cortical anchorage and cell fate: I) cER sub-domain associated postplasmic/PEM RNAs (Macho1 like) which segregate in the somatic B8.11 cells. II) postplasmic/PEM RNAs associated with (putative germ) granules (Vasa like) which in addition to B8.11 cells segregate into B8.12 germ cells.

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Comparative analysis of gene expression profiles between four kinds of blastomere at 8-cell stage embryos.

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