Identification and characterization of a negative regulator required for spatial control of the territory-specific CyIIIa gene in the sea urchin embryo

Author: Wang, David Guo-Wei

Year: 1995

Degree: Dissertation (Ph.D.)

Advisor: Davidson, Eric H.

Committee Member: Unknown, Unknown

Option: Biology

DOI: 10.7907/tejh-wh40

Abstract

The Cyllla cytoskeletal actin gene of the sea urchin Strongylocentrotus purpuratus is activated in late cleavage and expressed exclusively in the aboral ectoderm territory of the embryo. Previous gene transfer studies defined a 2.3 kb cis-regulatory domain that is necessary and sufficient for correct temporal and spatial expression of a Cyllla-CAT fusion gene. In this study, a negative regulatory element within this region was identified that is required for repression of the Cyllla gene in skeletogenic mesenchyme cells. The repression mediated by this element takes place after initial territorial specification.

To study negative spatial control of the Cylila gene at the molecular level, a cDNA clone encoding a DNA-binding protein with twelve Zn fingers (SpZ12-1) was isolated by probing an expression library with this cis-regulatory element. Deletion analysis of the SpZ12-1 protein confirmed that a DNA-binding domain is located within the zinc finger region. SpZ12-1 is the only DNA-binding protein in embryo nuclear extract that interacts with the specific cis target sites required for repression of Cyllla-CAT in skeletogenic mesenchyme and is likely to be the trans factor that mediates this repression.

SpZ12-1 is present in significant quantities even in unfertilized egg cytoplasm, and in similar quantities in mesenchyme blastula-stage embryo cytoplasm. Taken together with earlier measurements of Calzone et al. (Genes & Dev. 2,1074-1088, 1988), our results indicate that SpZ12-1 enters the embryonic nuclei between late cleavage and mesenchyme blastula stages. A low prevalence of SpZ12-1 mRNA is also present throughout development. Translation of this mRNA could easily account for the complete complement of SpZ12-1 protein in the embryo, as estimated from its DNA binding activity. SpZ12-1 probably functions at several developmental stages, and is evidently of both maternal and embryonic provenance.

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