Ci-TCF Gene Function and Its Involvement in Ciona intestinalis Pigment Cell Differentiation

Parveen, Fateema (2010). Ci-TCF Gene Function and Its Involvement in Ciona intestinalis Pigment Cell Differentiation. PhD thesis The Open University.



Transcription factors of T cell factor (TCF) family have been identified in a wide range of organisms, from hydra to mammals, where they play important roles during embryonic development. As a terminal nuclear component they mediate the canonical Wnt signaling cascade by their context dependent transcriptional regulatory properties. The TCF protein family in Ciona intestinalis is represented by a single member, named Ci-TCF, whose zygotic expression has been observed in pigment precursor cells as well as in palp precursors. Ascidians occupy a strategic position in the phylogeny and thus provide a useful model system to study developmental processes difficult to approach in more complex organisms.

I aimed to better clarify the role played by Ci-TCF specifically in pigment cell differentiation in Ciona. To this end I exploited targeted interference with Ci-TCF function. I used an enhancer specific for pigment cell lineage to drive the expression of a dominant negative form of Ci-TCF. From this study it was possible to deduce that Ci-TCF is effectively involved in the terminal differentiation of pigment cells in sensory organs.

Simultaneously, my project was also focused on the regulatory factors controlling the precise spatio-temporal activation of Ciona TCF gene in pigment lineage. Analysis of the cis-regulatory region of Ci-TCF gene was performed through a series of constructs containing various Ci-TCF 5’ promoter fragments fused to a reporter gene. This study allowed me to characterize a region of 400 bp in Ci-TCF enhancer element which was able to drive reporter gene expression in pigment cells of Ciona. In silico analysis of this cis-regulatory region revealed the possible involvement of Ets transcription factors governing the tissue specific expression of Ci-TCF at the right time. The data was further confirmed by detailed in vivo and in vitro studies, through mutational assay and EMSA (Electrophoretic mobility shift assay), respectively. My study therefore led to the identification of Ets factors as the first upstream regulator of a TCF representative. Since Ets transcription factors are activated via MAP kinase pathway which is triggered by FGF signaling, the present study provides clues for a crosstalk between the Wnt and FGF signaling cascades during pigment cell differentiation in Ciona intestinalis. This finding is noteworthy, since up to now all the data on TCFs have concerned their function as effectors of Wnt signaling. No explanations have been reported so far as to why and how TCF factors are present at the right time and in the right place to play their role downstream from Wnt. So, my study gives a first answer to this question and represents a first step toward a better understanding of the regulation of TCF’s expression.

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