Regulatory elements controlling CisFrp1/5 expression in Ciona intestinalis during embryogenesis

D'Ambrosio, Palmira (2006). Regulatory elements controlling CisFrp1/5 expression in Ciona intestinalis during embryogenesis. PhD thesis The Open University.



In the ascidian, endodermal progenitor cells have the capacity to differentiate autonomously from the earliest stages of embryogenesis. Their fate is restricted at the 64/110 cell stage when, after 5-6 cell divisions, the progeny of five pairs of vegetal blastomeres give rise to all endodermal cells (almost 500). The endodermal cells in the larval stage are present in the antero-ventral region of the trunk and in a strand lying on the ventral side of the tail. Ci-titfl, a gene homologous to mammalian Titifl, is a marker for early endoderm specification, during Ciona intestinalis embryogenesis, and for endostyle differentiation during metamorphosis.
Several studies are currently focused on markers for early specification of organs of endodermal lineage, such as liver, pancreas, pharynx and digestive tube, given their physiological importance.
My project has focused on the identification of putative Ci-titfldownstream genes that could represent useful markers for early regionalization of endodermal tissues in Ciona intestinalis embryogenesis. For this purpose, through a subtractive hybridization screen between Ci-titfl overexpressing embryos and control embryos, I isolated and characterized a cDNA clone that, by sequence similarity analysis, appears to code for a factor belonging to the secreted frizzled related protein family (sFRp) and that has been called CisFrpl/5. sFRp proteins are modulators of the Wnt pathway, a genetic cascade that influences such diverse biological processes as developmental fate, cell polarity, adhesion, tumorigenesis and apoptosis. The expression data of CisFrpl/5, in the anterior region of the embryo (included the endoderm) from the neurula stage, indicate a putative involvement of this gene in late endoderm differentiation. In order to analyse the regulatory elements controlling CisFrpl/5 tissue specific expression during development, I used the electroporation technique to introduce reporter constructs, containing progressively deleted fragments of CisFrpl/5 5’ promoter region fused to the LacZ reporter gene into fertilised Ciona eggs.
This strategy led to the identification of a key fragment in the promoter sequence of the gene, essential for CisFrpl/5 activation in the areas where it is expressed. I gradually narrowed the extent of my investigation up to the identification of a 130 bp (130bp CisFrpl/5) element necessary for CisFrpl/5 endoderm activation. In addition, Cititfloverexpression and underexpression experiments indicated that Cititfl could influence the transactivation of the reporter gene downstream from 13 Op CisFrp 1/5, suggesting a correlation between Cititfl and the expression of CisFrpl/5.

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