ARTICLE

Ectopic Expression of SPARC in Xenopus Embryos Interferes with Tissue Morphogenesis: Identification of a Bioactive Sequence in the C-terminal EF Hand

Correspondence to: Maurice J. Ringuette, Dept. of Zoology, Univ. of Toronto, Toronto, Ontario, Canada M5S 3G5.

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 morphogenesis, response to injury, and cyclic renewal and/or repair. In an earlier study with Xenopus embryos we had shown a highly specific and regulated pattern of SPARC expression. We now show that ectopic expression of SPARC before its normal embryonic activation produces severe anomalies, some of which are consistent with the functions of SPARC proposed from studies in vitro. Microinjection of SPARC RNA, protein, and peptides into Xenopus embryos before endogenous embryonic expression generated different but overlapping phenotypes. (a) Injection of SPARC RNA into one cell of a two-cell embryo resulted in a range of unilateral defects. (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 peptides containing follistatin-like and copper-binding sequences were without obvious effect, whereas SPARC peptide 4.2, corresponding to a disulfide-bonded, Ca²⁺-binding domain, was associated with a reduction in axial structures that led eventually to complete ventralization of the embryos. Histological analysis of ventralized embryos indicated that the morphogenetic events associated with gastrulation might have been inhibited. Microinjection of other Ca²⁺-binding glycoproteins, such as osteopontin and bone sialoprotein, resulted in phenotypes that were unique. We probed further the structural correlates of this region of SPARC in the context of tissue development. Co-injection of peptide 4.2 with 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, generated no axial defects. We have therefore shown for the first time in vivo that the temporally inappropriate presence of SPARC is associated with perturbations in tissue morphogenesis. Moreover, we have identified at least one bioactive region of SPARC as the C-terminal disulfide-bonded, Ca²⁺-binding loop that was previously shown to be both counteradhesive and growth-inhibitory. (J Histochem Cytochem 45:643-655, 1997)

Key Words: microinjection, ECM, metal binding, ventralization, counteradhesive

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This article has been cited by other articles:

				E. Busch, E. Hohenester, R. Timpl, M. Paulsson, and P. Maurer Calcium Affinity, Cooperativity, and Domain Interactions of Extracellular EF-hands Present in BM-40 J. Biol. Chem., August 11, 2000; 275(33): 25508 - 25515. [Abstract] [Full Text] [PDF]

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