The Endo16 gene of Strongylocentrotus purpuratus is expressed at the blastula stage of embryogenesis throughout the vegetal plate, at the gastrula stage in the whole of the archenteron and in postgastrular stages only in the midgut. We showed earlier that a 2300 bp upstream sequence suffices to faithfully recreate this pattern of expression when fused to a CAT reporter gene. Here we define the functional organization of this cis-regulatory domain, which includes over thirty high specificity binding sites, serviced by at least thirteen different putative transcription factors, in addition to >20 sites for a factor commonly found in the regulatory sequences of other sea urchin genes as well (SpGCF1). The Endo16 cis-regulatory domain consists of several different functional elements, or modules, each containing one or two unique DNA-binding factor target sites, plus sites for factors binding in other modules as well. Modular regulatory function was defined in experiments in which regions of the cis-regulatory DNA containing specific clusters of sites were tested in isolation, combined with one another, or by selective deletion, and the effects on expression of the CAT reporter were determined by whole-mount in situ hybridization or CAT enzyme activity measurements. The most proximal module (A) is mainly responsible for early embryonic expression, and module A alone suffices to locate expression in the vegetal plate and archenteron. The adjacent module (B) is responsible for a steep postgastrular rise in expression, when the gene is transcribed only in the midgut and, prior to this module B alone also suffices to promote expression in the vegetal plate and archenteron. The most distal module, G, acts as a booster for either A or B modules. However, no combination of A, B and G modules generates vegetal plate or gut expression exclusively. Ectopic expression of A-, B- and G-CAT fusion constructs occurs in the adjacent (veg1-derived) ectoderm and in skeletogenic mesenchyme cells. For expression to be confined to endoderm requires negative regulatory functions mediated by modules E, F and DC. Modules E and F each repress ectopic expression specifically in veg1 ectoderm. Module DC represses ectopic expression specifically in skeletogenic mesenchyme. Expression of some Endo16 constructs is dramatically increased by treatment with LiCl, which expands the territory in which the endogenous Endo16 gene is expressed at the expense of veg1 ectoderm. The same modules that act to repress ectopic expression in untreated embryos are required for enhanced expression of constructs after LiC1 treatment. Furthermore, both the negative spatial control functions and response to LiC1 require the presence of module A. The total regulatory requirements of the Endo16 gene during embryogenesis can be expressed in terms of the positive and negative functions of the individual modules and the interactions between modules that are identified in this study.

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