Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression data of Xrx1 gain and loss of function experiments from early Xenopus laevis embryos (stage 13)

ABSTRACT: Eye development is a multistep process that requires specific inductive signals and precise morphogenetic movements, starting early during development in the eye-field, a well-definite region of the anterior neural plate. It has been demonstrated that a gene network of eye field transcription factors (EFTFs) contributes to specify the neural and retinal fate of the eye field. Among these EFTFs, Xrx1 is involved in proliferation and neurogenesis in the eye field and is necessary for the correct development of the retina. By means of Affymetrix microarrays, a high throughput screening was performed, looking for genes that can mediate the function of Xrx1, by comparison of the expression profiles of whole embryos in which the Xrx1 function was either overexpressed or abolished by use of morpholino antisense oligonucleotides. Xenopus laevis embryos were harvested soon after fertilization. 50ng of capped Xrx1 mRNA or 0.5pmol of MoXrx1 antisense oligonucleotides were coinjected with 400ng of GFP synthetic mRNA as a tracer, in both dorsal blastomeres of 4-cells stage embryos. Embryos were let to develop until stage 13, in which the eye field induction steps are active, and selected for correct injection site by fluorescence topographic assessment. RNA was extracted from each single embryo and control PCRs were performed to assess the correct molecular signature of Xrx1 loss- and gain- of function.

ORGANISM(S): Xenopus laevis

SUBMITTER: Massimiliano Andreazzoli

PROVIDER: E-GEOD-35918 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Brief report: Rx1 defines retinal precursor identity by repressing alternative fates through the activation of TLE2 and Hes4.

Giannaccini Martina M Giudetti Guido G Biasci Daniele D Mariotti Sara S Martini Davide D Barsacchi Giuseppina G Andreazzoli Massimiliano M

Stem cells (Dayton, Ohio) 20131201 12

The molecular mechanisms underlying the acquisition of retinal precursor identity are scarcely defined. Although the homeobox gene Rx1 (also known as Rax) plays a major role in specifying retinal precursors and maintaining their multipotent state, the involved mechanisms remain to be largely deciphered. Here, following a highthroughput screen for genes regulated by Rx1, we found that this transcription factor specifies the fate of retinal progenitors by repressing genes normally activated in adj ...[more]

PMID: 24038725

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Project description:Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as direct target of xSu4-20h enzyme-mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4- 20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibian and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state. Total RNA samples from Xenopus laevis embryos. Transcript levels were analyzed after injection of control or Suv420 morpholinos into blastomeres.

Project description:Exostosin 1 (Ext1) is a glycosyltransferase involved in the biosynthesis of the extracellular matrix Heparan Sulfate Proteoglycan (HSPG). Knockdown of Ext1 caused gastrulation defects and formation of an abnormal body axis. Since ext1 has been implicated as an indirect contributor to multiple signaling pathways in vertebrate development, microarray was used to identify genes expressed in gastrulae that would be affected by a reduction in ext1 expression. Microarray-based comparisons of gene expression in control vs. Ext1 MO embryos showed that Ext1 is involved in regulating genes that are related to metabolic process, development and signaling pathways. Half of the hits from the microarray are uncharacterized genes. Approximately forty-five percent of genes are related to metabolic process and thirty percent of genes are belonged to signaling and developmental process categories. Ten percent of each up-regulated and down-regulated gene set is predicted to function in establishment of localization by GO, which is consistent with EXT1 being involved in the movement of extracellular substances. The transcription factors or signaling protein, Isl1, Pitx2, TBX5A, Wnt5A, Wnt7A, WT1, Pax3, Wnt1, and Xbra were identified as Ext1 regulated genes. This analysis investigating the role of Ext1 during gastrulation and provide the information that EXT1 plays an important role in Xenopus early development. Exostosin 1 (EXT1) is a glycosyltransferase involved in the biosynthesis of the extracellular matrix Heparan Sulfate Proteoglycan (HSPG). Knockdown of EXT1 caused gastrulation defects and formation of an abnormal body axis. Since ext1 has been implicated as an indirect contributor to multiple signaling pathways in vertebrate development, microarray was used to identify genes expressed in gastrulae that would be affected by a reduction in ext1 expression. Microarray-based comparisons of gene expression in control vs. EXT1 MO embryos showed that EXT1 is involved in regulating genes that are related to metabolic process, development and signaling pathways. Half of the hits from the microarray are uncharacterized genes. Approximately forty-five percent of genes are related to metabolic process and thirty percent of genes are belonged to signaling and developmental process categories. Ten percent of each up-regulated and down-regulated gene set is predicted to function in establishment of localization by GO, which is consistent with EXT1 being involved in the movement of extracellular substances. The transcription factors or signaling protein, Isl1, Pitx2, TBX5A, Wnt5A, Wnt7A, WT1, Pax3, Wnt1, and Xbra were identified as EXT1 regulated genes. This analysis investigating the role of EXT1 during gastrulation and provide the information that EXT1 plays an important role in Xenopus early development. The embryos were injected with mispair or Ext1 MO were collected at St. 10.5. The RNA was extracted and for probe synthesis and the hybridization was performed on Affymetrix Xenopus laevis genome v2.0 array. The experiments were repeated for three times. Results of paired control and experimental samples were compared.

Dataset Information

Expression data of Xrx1 gain and loss of function experiments from early Xenopus laevis embryos (stage 13)

Publications

Brief report: Rx1 defines retinal precursor identity by repressing alternative fates through the activation of TLE2 and Hes4.

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