The goal of this project is to understand mechanisms underlying cell type specific gene activation during development using the sea urchin aboral ectoderm and the Spec/LpS1 genes as a model. Much is known about the cell lineages comprising the aboral ectoderm and the cell interactions required for differentiation. The Spec and the LpS1 genes serve as powerful markers for probing mechanisms involved in specifying aboral ectoderm.
The specific aims are: (1) To characterize the Spec2a promoter. Virtually all positive control of the Spec2a gene lies within a 188-bp enhancer. This enhancer is within a 600-bp repetitive sequence element termed RSR. The RSR enhancer contains multiple DNA-elements including a key element, the A/T palindrome. Additional elements in the Spec2a promoter will be identified, along with the corresponding DNA-binding proteins. Non-Spec genes having nearby RSR elements will be cloned to ask if their presence is linked to aboral ectoderm expression. (2) To clone and characterize the A/T palindrome binding protein (A/TBP). The A/T palindrome contains a binding site for a putative bicoid-class protein. attempts will be made to purify this protein and in addition to obtain a cDNA clone using oligonucleotides with bicoid-class sequences. Once cloned, it will be determined if the A/TBP has properties consistent with a role in activation of Spec2a and aboral ectoderm specification. (3) To characterize the LpS1-beta promoter and the ectoderm and endoderm/mesoderm G-string factors. The proximal G-string is a positive Ell-regulatory element on the LpS1-beta promoter that binds to ectoderm specific and endoderm/mesoderm specific factors. Other DNA-elements will be identified on this promoter and the G-string factors will be cloned by protein purification and homology with the mammalian factor, IFI. (4) To determine the function of the Spec/LpS1 proteins. Spec/LpS1 proteins are hypothesized to function in transport of calcium across the ectoderm. This will be tested using antisense procedures. Reduced levels of calcium should result in skeletal defects in the embryo. (5) To test if suppressive cellular interactions repress Spec gene expression in non-aboral ectoderm cells. In the absence of cell interactions, blastomeres exhibit greater developmental potential than in the intact embryo. It will be determined if the Veg2 tier is capable of activating Spec genes in isolation and if this activation is suppressed upon co-culture with animal blastomeres. These experiments should lead to an understanding of Spec/LpS1 gene activation and its relationship to aboral ectoderm specification.
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