S. Damjanovski et al., ECTOPIC EXPRESSION OF SPARC IN XENOPUS EMBRYOS INTERFERES WITH TISSUEMORPHOGENESIS - IDENTIFICATION OF A BIOACTIVE SEQUENCE IN THE C-TERMINAL EF HAND, The Journal of histochemistry and cytochemistry, 45(5), 1997, pp. 643-655
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.