Regulation of SPARC expression during early Xenopus development: evolutionary divergence and conservation of DNA regulatory elements between amphibians and mammals.

Abstract

SPARC (Secreted Protein, Acidic, Rich in Cysteine/osteonectin/BM-40) is a highly conserved metal-binding extracellular matrix (ECM) glycoprotein which is first expressed by Xenopus embryos during late gastrulation/early neurulation (stage 12/13), by presumptive notochord and somitic cells. When animal cap explants of stage 9 embryos were cultured in vitro, SPARC expression was not detected until sibling embryos reached late neurula stage (stage 19). Addition of activin, a potent dorsal mesoderm inducer, to animal caps resulted in SPARC being expressed by the time sibling embryos reached stage 16. While basic fibroblast growth factor (bFGF), a ventral mesoderm inducer, had modest effects on SPARC mRNA expression, the combination of both activin and bFGF was synergistic. The appearance, however, of SPARC transcripts 11 h after the addition of activin and bFGF, indicates that unknown intermediates were likely to be involved in activating SPARC expression. In order to identify the potential intermediate regulatory factors which may activate and control SPARC expression, we examined the genomic organization of the 5' end of the Xenopus SPARC gene. No significant homology to the equivalent region that is highly conserved in the mouse, bovine and human SPARC genes was observed. Thus, while mammalian SPARC promoters lack TATA or CAAT boxes, the Xenopus gene contains a consensus TATA box. Moreover, promoter-proximal GGA-box repeats necessary for high level expression of mammalian SPARC are absent in Xenopus. When reporter constructs containing the 5' flanking region of the Xenopus gene were microinjected into two-cell embryos, 868 bp of 5' flanking DNA was sufficient to mimic the temporal and tissue-specific pattern of SPARC expression observed in whole embryos. While a bovine SPARC promoter reporter construct containing 740 bp of the 5' flanking DNA was expressed at a significant level in Xenopus embryos, significant differences in the cell-type expression of the reporter genes were obtained between the bovine and Xenopus constructs. The data indicate that zygotic activation of SPARC mRNA is mediated by regulatory factors acting downstream of major mesoderm induction events. The high DNA sequence conservation at the 5' end of mammalian SPARC genes is not conserved in Xenopus. These differences led to differences in their ability to direct tissue-specific gene expression in early Xenopus embryos.