CIS-REGULATION DOWNSTREAM OF CELL-TYPE SPECIFICATION - A SINGLE COMPACT ELEMENT CONTROLS THE COMPLEX EXPRESSION OF THE CYLLA GENE IN SEA-URCHIN EMBRYOS

CyIIa, a cytoskeletal actin gene of Strongylocentrotus purpuratus, is expressed specifically though transiently in the embryonic skeletogeni c and secondary mesenchyme and, later in development, is permanently a ctivated in the hindgut and midgut, CyIIa transcription follows, and i s therefore downstream of, the initial specification of these embryoni c domains. A detailed functional analysis of the cis-regulatory system governing the rate and the location of CyIIa expression during develo pment was carried out using GFP expression constructs. About 4.4 kb of CyIIa sequence including a leader intron were examined for cis-regula tory function. Distal elements scattered over several kb account for 6 0% of the quantitative output of the expression construct and a strong amplifier of expression is located within the leader intron, However, the complex spatial pattern of CyIIa expression is completely reprodu ced by a compact upstream regulatory element <450 bp in length, We fou nd no evidence anywhere in the 4.4 kb sequence examined for negative r egulators required to repress ectopic expression. The specific site th at mediates CyIIa expression in the midgut in late embryos and larvae was identified. This site is the same as that necessary and sufficient for midgut expression of the Endo16 gene late in development, and was shown to bind the same transcription factor. Except for some temporal and quantitative features, the S. purpuratus expression construct is expressed accurately and specifically in the same diverse cell types w hen introduced into embryos of Lytechinus pictus, which belongs to a d ifferent echinoid order. No ectopic expression was observed, in contra st to the result of a similar interspecific gene transfer experiment c arried out earlier on a different cytoskeletal actin gene that is expr essed much earlier in development. Presentation of the set of transcri ption factors that activate CyIIa in the differentiated cells in which it is expressed is apparently a conserved feature of these cell types .