Unknown,Transcriptomics,Genomics,Proteomics

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Expression profile analysis of Drosophila melanogaster embryonic and larval MANF mutants together with paternal rescue and overexpression conditions

ABSTRACT: To find out possible pathways and processes influenced by DmMANF we compared the changes in MANF null and overexpressing animals. We used two developmental stages of Drosophila, st 17 embryos and 29-50 hrs after egg laying (AEL) larvae. The timing were decided according to the lethality of MANF mutants with or without maternal contribution of MANF. Maternal and zygotic MANF mutants (MANFmzD96) were compared to paternal rescue and wild type embryos of the same age. Zygotic MANF mutants (MANFD96) with maternal contribution were compared to wild type and MANF ectopic overexpression larvae harvested well before the average lethality occured (75 hrs AEL). The changes in expression profiles were more drastic in MANFmzD96 embryos than in MANFD96 larvae. Gene ontology annotations were analyzed and clustered by DAVID. The major result was alterations in membranes, changes in membrane transporter expressions and disturbed intracellular membrane traffic. The results were verified by qPCR and transmission electron microscopy. Two developmental stages - 21-24 hrs and 29-50 hrs AEL of MANFD96 mutants without and with maternal contribution were compared to wild type (Wmix means crossed w1118 x w- ) and with paternal rescue or ectopic overexpression of MANF respectively by Drosophila Agilent microarray expression platform.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Dario Greco

PROVIDER: E-GEOD-28820 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In zebrafish, vitellogenic oocytes can incorporate significant amounts of 17β-estradiol released from nearby granulosa cells according to a first-order kinetics, since the steroid low polarity ensures high permeability and affinity for yolk lipids. Estrogen bioactivity is likely, because the maternal mRNA for the estrogen receptor-β2 (ers2a) is highly expressed in ovulated oocytes. This transcript is available for translation in the embryo until its sharp decline from 4 to 8 hours post-fertilization (hpf), being replaced by low levels of zygotic ers2a mRNA from 24 hpf to hatching at 48 hpf, as determined by qRT-PCR. Estrogen receptors-α and -β1 are only expressed zygotically at low levels from 24 hpf onwards. To test the functional role of maternal ers2a mRNA, 1- or 2-cell embryos were injected with 10.3 ng each of morpholino to knockdown translation (MO2-ers2a) of both maternal and zygotic ers2a transcripts, missplicing morpholino (MO3-ers2a) to block post-transcriptionally the zygotic transcript alone, and a nonspecific morpholino (MO-control) as a control. Treatment with MO2-ers2a caused severe malformations in 63% of 1-5 dpf larvae, as compared to 10-11% in those treated with MO3-ers2a and MO2-control. Defects included body growth delay and curved shape, abnormal brain and splanchnocranium development, enlarged and hemorrhagic pericardial cavity, uninflated swim bladder and rudimentary caudal fin with aberrant circular motion. Affected larvae could survive for only 12-14 days. Co-injection of an anti-p53 MO failed to rescue the MO2-ers2a-phenotypes, eliminating the possibility of off-target effects. Pangenomic microarray analysis revealed that 240 and 219 significantly expressed transcripts were up- and down-regulated, respectively, by maternal Ers2a protein deficiency in 8-hpf MO2-ers2a-embryos. Also at 48 hpf, 162 and 120 presumably zygotic transcripts were up- and down-regulated, respectively, but only 18 were in common with each of the 8-hpf sets. Whole-mount in situ hybridization revealed an intensified expression of the genes six3.1 and emx1 in MO2-ers2a-embryos at 24-48 hpf, as compared to controls. These findings suggest the involvement of maternal ers2a mRNA in the epigenetic programming of zebrafish development. MO2-ers2a morphants were compared with MO-control at 8 hpf and 48 hpf. MO2-ers2a is a morpholinos selected to knockdown translation of ers2a mRNA

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Expression profile analysis of Drosophila melanogaster embryonic and larval MANF mutants together with paternal rescue and overexpression conditions

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