SPATIAL AND TEMPORAL INFORMATION-PROCESSING IN THE SEA-URCHIN EMBRYO - MODULAR AND INTRAMODULAR ORGANIZATION OF THE CYIIIA GENE CIS-REGULATORY SYSTEM

The CyIIIa cytoskeletal actin gene of Strongylocentrotus purpuratus is expressed specifically in the aboral ectoderm, In earlier work we ide ntified a 2.3 kb cis-regulatory region that is necessary and sufficien t for correct spatial and temporal expression of a CyIIIa . CAT gene, This region includes about 20 sites of specific protein-DNA interactio n, at which at least nine different transcription factors may be bound , All except two of these factors have been cloned, In this work we ha ve analyzed by deletion or mutagenesis each specific interaction, A sp ecific function was identified for every binding site examined, These individual functions include control of amplitude and timing of expres sion at different phases of embryogenesis, and control of spatial expr ession, We show that particular negative regulatory interactions are r equired to repress expression of the CyIIIa . CAT construct in oral ec toderm and in skeletogenic mesenchyme at different stages, In further experiments we determined the overall functional organization of the C yIIIa cis-regulatory system, and we show that this system is modular i n its regulatory structure. The 'proximal module' (with respect to the transcription start site) extends upstream for about 800 base pairs, and includes nine target sites serviced by six different transcription factors, Its major role is to establish CyIIIa expression in the abor al ectoderm territory as the blastomere founder cells are specified an d the oral-aboral axis is determined, and to activate the CyIIIa gene late in cleavage, The 'middle module,' which lies upstream of the prox imal module, acquires major control of CyIIIa function after the blast ula stage, It includes six target sites, serviced by four different fa ctors, The middle module is responsible for a sharp increase in expres sion occurring during gastrulation, mediated by the positively acting factors that bind within it, The middle module also includes sites at which two different negatively acting spatial control factors bind, th e functions of which are required for correct spatial expression late in embryogenesis, The 'distal module' contains a number of sites at wh ich a positively acting factor binds, but this module exercises no spa tial regulatory function, Interactions within the distal module are re quired for the normal levels of function of both the proximal and midd le modules.