Identification of genes involved in <i>Citif1</i> promoter activation and endoderm differentiation

In tadpole larva of the ascidian Ciona intestinalis (one of the most basal chordate) the endoderm is a simple tissue comprising about 500 cells. The commitment of endoderm occurs very early; in a 64-cells stage embryo five blastomeres pairs are restricted to endodermal fate. Although three genes, that exhibit an endoderm specific expression, Ci-alkaline phosphates, Cs-lhx3 and Cititf1 have been cloned, it remains to be elucidated how the first step of endoderm specification take place. As first strategy, I cloned a Ciona SoxE gene; this was found not to be endodermally expressed. In a second strategy, I focused on Cititf-1, a gene homologous to mammalian Nkx2.1 and the earliest zygotic endoderm specific marker isolated from Ciona thus far. Although its temporal and spatial expression seems to be finely regulated, the factors involved in its activation and likely involved in endoderm specification are unknown. The minimal region responsible for endodermal expression of Cititf1, has previously been located to a genomic fragment of about 200 base pairs upstream to putative TATA box sequence (Fanelli A. et al., 2003 Developmental Biology. 263, 12-23). Here I describe two imperfect repeats of eleven nucleotides, named R1 and R2, within this region and investigate their involvement in the transcriptional activation of Cititf1 promoter. Biochemical analyses showed that both repeats bind factors from ascidian embryos in a sequence-specific manner. The R2, however, is more important for endoderm specific expression and is able to drive the endodermal expression of a reporter gene. The R1 repeat cooperates with R2 to enhance this expression. A yeast one-hybrid screening was performed, leading to identification of a putative DNA-binding factor that could be involved in the regulation of Cititf1 in endoderm