ABSTRACT: Expression profiling of Ciona intestinalis neurula and mid-tailbud stage embryos expressing altered forms of the transcription factor Ci-Tbx2/3 in the notochord
Project description:Despite being found in the notochord of several chordates, the roles of the Tbx2 subfamily of T-box transcription factors in the development of this tissue remain largely unknown. We explored the targets of the only Tbx2 subfamily member in Ciona intestinalis, Ci-Tbx2/3, by expressing mutant forms of the transcriptional regulator using the Ci-Bra promoter region. We produced a dominant interfering version of Ci-Tbx2/3 (Tbx2/3DBD) through expression of a truncated version consisting only of its DNA-binding domain (DBD) and a constitutive activator form by attaching the T-box of Ci-Tbx2/3 to the VP16 transactivation domain (Tbx2/3VP16). These constructs were introduced into 1-cell stage embryos and grown to the neurula (N) or mid-tailbud (mTb) stage to capture targets regulated throughout notochord morphogenesis.
Project description:Despite being found in the notochord of several chordates, the roles of the Tbx2 subfamily of T-box transcription factors in the development of this tissue remain largely unknown. We explored the targets of the only Tbx2 subfamily member in Ciona intestinalis, Ci-Tbx2/3, by expressing mutant forms of the transcriptional regulator using the Ci-Bra promoter region. We produced a dominant interfering version of Ci-Tbx2/3 (Tbx2/3DBD) through expression of a truncated version consisting only of its DNA-binding domain (DBD) and a constitutive activator form by attaching the T-box of Ci-Tbx2/3 to the VP16 transactivation domain (Tbx2/3VP16). These constructs were introduced into 1-cell stage embryos and grown to the neurula (N) or mid-tailbud (mTb) stage to capture targets regulated throughout notochord morphogenesis. Ci-Tbx2/3 targets were ascertained using whole-genome custom Affymetrix microarrays to compare the transcription levels of Tbx2/3DBD and Tbx2/3VP16 expressing embryos to wild-type controls (Bra>GFP) at the neurula or mTb stages.
Project description:Expression profiling of Ciona intestinalis initial tailbud stage embryos expressing mutant forms of the transcription factor Ci-XBP1 in the notochord.
Project description:XBP transcription factors are well-known regulators of the the unfolded protein response and are required for plasma cell differentiation. However, recent studies have shown that the XBP1 gene is also expressed in the developing notochord of Ciona and various vertebrates, and to date its role in the formation of this organ is largely uncharacterized. We identified putative targets of Ci-XBP1 through a microarray screen, using RNAs extracted from embryos expressing mutant forms of this transcriptional regulator in the notochord. We expressed in the Ciona notochord a dominant-negative version of Ci-XBP1 (XBP DBD::GFP) by cloning its DNA-binding domain (DBD) downstream of the Ci-Brachyury (Ci-Bra) promoter. We created a constitutive activator form of Ci-XBP by fusing its DBD to the VP16 transactivation domain (Bra>XBP DBD::VP16::GFP). These constructs were introduced into 1-cell stage embryos and grown to the initial tailbud (iTb) stage to identify Ci-XBP notochord target genes.
