Study of cis transcriptional regulation of asymmetric expression of the SpHE gene along the sea urchin embryo animal-vegetal axis

In order to understand the molecular basis of the animal-vegetal axis of the sea urchin embryo, I have studied transcriptional regulation of the SpHE gene of Strongylocentrotus purpuratus, which is non-vegetally expressed at very early blastula stage. By using microinjection, reporter gene expression, electrophoresis mobility shift assay and in vitro footprinting, I have demonstrated that the high level output of the gene can be conferred by a 310 bp promoter, in which multiple cis elements were found to interact with nuclear proteins. I developed an assay to examine the spatial expression pattern of injected SpHE promoter-{dollar}beta{dollar}-galactosidase reporter gene constructs, which provided evidence that the transgenes are also correctly spatially regulated. By in vivo footprinting, I observed increased occupancy of the cis elements by proteins in embryos when the gene is active and decreased occupancy when the gene is inactive, correlating with the gene's switching on and off.; The non-vegetal expression of the SpHE gene could be regulated by one of two mechanisms: Vegetally localized negative transcription activity or non-vegetally positioned positive activity. Characterization of the individual cis elements revealed that functionally they are all positive, redundant in activity and spatial regulation ability. This result suggests that it is the positive, but not the negative activities that are responsible for the endogenous SpHE spatial expression. I proposed that the non-vegetal expression pattern of SpHE is activated by multiple non-vegetally localized positive transcription activities. This notion is supported by the activity of a chimeric construct in which the SpHE promoter sequence was linked upstream of the vegetal-specific SM50 gene promoter, which drove ubiquitous expression of the {dollar}beta{dollar}-galactosidase gene, without any obvious reciprocal repression. It is likely that specification of the non-vegetal and vegetal transcription activity domains is one of the early events to polarize the animal-vegetal axis in early sea urchin embryos. This research has provided clues to pursue the trans-acting factors of the SpHE promoter which might be molecular components that specify the animal-vegetal axis of the sea urchin embryo.