REGULATION OF GENE-EXPRESSION IN THE SEA-URCHIN EMBRYO

In the undisturbed sea urchin embryo, cleavage of the blastomeres is i nvariant and gives rise to five polyclonal territories that are each d efined by the larval structures to which they give rise, and by unique programmes of gene expression. These territories are the aboral ectod erm, the oral ectoderm, the vegetal plate, the skeletogenic mesenchyme , and the small micromeres. Structural gene markers for four of these territories (all except the small micromeres, which participate in lat er development) have been cloned and characterized, and the regulatory domains of several of these genes have been mapped to the level of sp ecific protein-binding sites. The best characterized territory-specifi c gene is CyIIIa, which encodes a cytoskeletal actin expressed exclusi vely in the aboral ectoderm. Transcription of this gene is regulated b y between 8 and 12 different DNA-binding proteins that interact with a pproximately 20 different sites in the 2.3 kb regulatory domain of thi s gene. We have developed an automated method for purifying multiple D NA-binding proteins from nuclear extract, using specific oligonucleoti de binding sites linked to a tandem series of affinity columns; with t his method, most of the CyIIIa transcription factors have been isolate d in sufficient quantity for amino add sequence analysis. Amino acid s equence obtained from several of the affinity purified CyIIIa transcri ption factors has been used to make nucleic acid probes for cloning th e corresponding cDNAs. These cDNAs, and the recombinant proteins they encode, are being used to analyse the expression, regulation, and func tion of the regulatory proteins. We have shown previously, through chr omatographic enrichment and two-dimensional electrophoretic mapping, t hat the complexity of nuclear proteins which are candidates for being transcriptional regulators is of order 100. In addition, a recent stud y indicates that most if not all of the CyIIIa transcription factors a re present in the unfertilized egg cytoplasm, suggesting that localize d activation of a subset of maternal factors is responsible for initia l specification of the aboral ectoderm territory. These studies indica te that it is now possible, with the approach and techniques discussed here, to clone and characterize all the transcriptional regulatory mo lecules involved in early cell-fate specification in the sea urchin em bryo.