ECTOPIC EXPRESSION OF SPARC IN XENOPUS EMBRYOS INTERFERES WITH TISSUEMORPHOGENESIS - IDENTIFICATION OF A BIOACTIVE SEQUENCE IN THE C-TERMINAL EF HAND

SPARC is a matricellular Ca2+-binding glycoprotein that exhibits both counteradhesive and antiproliferative effects on cultured cells. It is secreted by cells of various tissues as a consequence of morphogenesi s, response to injury, and cyclic renewal and/or repair. In an earlier study with Xenopus embryos we had shown a highly specific and regulat ed pattern of SPARC expression. We now show that ectopic expression of SPARC before its normal embryonic activation produces severe anomalie s, some of which are consistent with the functions of SPARC proposed f rom studies in vitro. Microinjection of SPARC RNA, protein, and peptid es into Xenopus embryos before endogenous embryonic expression generat ed different but overlapping phenotypes. (a) injection of SPARC RNA in to one cell of a two-cell embryo resulted in a range of unilateral def ects. (b) Precocious exposure of embryos to SPARC by microinjection of protein into the blastocoel cavity was associated with certain axial defects comparable to those obtained with SPARC RNA. (c) SPARC peptide s containing follistatin-like and copper-binding sequences were withou t obvious effect, whereas SPARC peptide 4.2, corresponding to a disulf ide-bonded, Ca2+-binding domain, was associated with a reduction in ax ial structures that led eventually to complete ventralization of the e mbryos. Histological analysis of ventralized embryos indicated that th e morphogenetic events associated with gastrulation might have been in hibited. Microinjection of other Ca2+-binding glycoproteins, such as o steopontin and bone sialoprotein, resulted in phenotypes that were uni que. We probed further the structural correlates of this region of SPA RC in the context of tissue development. Go-injection of peptide 4.2 w ith Ca2+ or EGTA, and injection of peptide 4.2K (containing a mutated consensus Ca2+-binding sequence), demonstrated that the developmental defects associated with peptide 4.2 were independent of Ca2+. However, the disulfide bridge in this region of SPARC was found to be critical , as injection of peptide 4.2AA, a mutant lacking the cystine, generat ed no axial defects. We have therefore shown for the first time in viv o that the temporally inappropriate presence of SPARC is associated wi th perturbations in tissue morphogenesis. Moreover, we have identified at least one bioactive region of SPARC as the C-terminal disulfide-bo nded, Ca2+-binding loop that was previously shown to be both counterad hesive and growth-inhibitory.