| The mechanistic basis for the differentiation of somatic cells during the development of multicellular lineages is not well understood. Volvox carteri is a simple multicellular organism with only two cell types: ca. 2,000 small somatic cells and up to 16 large reproductive cells. The terminal differentiation of somatic cells involves the expression of regA, a transcription factor thought to repress nuclear genes coding for chloroplast proteins. The expression of regA is restricted to somatic cells, and its induction is strictly dependent on the cell size at the end of embryogenesis. Several intronic elements are critical for the differential expression of regA, and thought to contain enhancer and silencer elements. However, the trans-acting factors that bind to these regulatory sequences, as well as the mechanism responsible for translating differences in cell size into differential regA expression are not known. To identify such trans-acting factors, we used a yeast
one-hybrid system: intronic sequences were cloned as "bait" upstream of a reporter gene, and a cDNA library of V. carteri was expressed in yeast as fusions to the GAL4 activation domain. Fusion proteins interacting with "bait" intronic sequences induced the expression of the reporter gene and enabled yeast cells to grow on a selection medium. Potential positive clones were further analyzed. To address the relationship between cell size and regA expression, we hypothesized that the increase in surface-to-volume ratio in smaller cells causes a change in chloroplast redox state during early cytodifferentiation (which is linked to the onset of daylight). This hypothesis predicts that regA expression is specifically induced by photosynthetic activity. A qRT-PCR approach was used to test this hypothesis. Data will be presented and interpreted in the general context of regA regulation. A better understanding of regA regulation should provide insights into the mechanistic basis of somatic cell differentiation in V. carteri. |