This is a renewal proposal to investigate the mechanisms that underlie the timing of gene expression during early embryogenesis. Using the histone gene family of the sea urchin as a model system, my laboratory has demonstrated that the L 1 late H2B histone gene of the sea urchin Strongylocentrotus purpuratus is transcriptionally activated in late blastula-stage embryos by a mechanism that depends on an enhancer element located 3' of the gene. A nuclear factor, designated H2B activator binding protein 1 (H2B abp 1) specifically binds this element and increases in activity during development in parallel with the transcriptional activation of the L1 H2B gene. We now have evidence that H2B abp 1 is Hbox4, one of three embryonically expressed antennapedia (antp) class homeodomain proteins. Thus, the timing of L1 H2B gene expression apparently is regulated by a member of a gene family whose congeners in Drosophila play roles in region specification. We therefore propose to test the hypothesis that the stage-specificity of late H2B histone gene expression is a consequence of a cascade of gene expression involving antp-class homeobox genes, and that this transcriptional cascade plays an important part in embryonic development. We have three specific aims. First, we will confirm that the H2B abp 1/Hbox4 protein is responsible for the stage-specific activation of the Ll H2B gene. This will entail purifying H2B abp 1 to verify that it is encoded by Hbox4 or a similar gene, and examining the link between the expression of H2B abp 1/Hbox4 gene and the activity of the Ll late H2B histone gene.Second, we will determine whether the H2B abp 1/Hbox4 gene has a wider role in development. We will ask whether sea urchin embryonic development is altered by abnormally timed, ectopic, or reduced H2B abp 1/Hbox4 expression. The assay for such alterations will be perturbations in embryo morphology or in the activity of genes that serve as markers for the various embryonic territories of the sea urchin embryo. Finally, we will examine the mechanism of H2B abp I/Hbox4 regulation. To understand the processes that govern the timing of Ll H2B expression, and to understand how these processes might participate more generally in embryogenesis, it is necessary to know how the regulatory signal that activates the Ll H2B gene at the late blastula stage is propagated from fertilization onwards in development. Accordingly, we will examine mechanisms that govern the timing of H2B abp I/Hbox4 expression during development. The end result of this study will be an understanding of the key steps of the gene regulatory pathway that mediates the stage-specific expression of the Ll H2B histone gene--a pathway that may also have a more general role in development. Moreover, since the sea urchin occupies a key position in the evolution of Deuterostomes, its bilaterally symmetrical larva resembling the stem organism that gave rise to the Deuterostome lineage, the proposed studies will also contribute fundamental information about the evolution of developmental mechanisms.
