A calcium-binding motif in SPARC/osteonectin inhibits chordomesoderm cell migration during Xenopus laevis gastrulation: Evidence of counter-adhesive activity in vivo

Secreted protein, acidic, rich in cysteine (SPARC) is a Ca2+-binding, count er-adhesive, extracellular glycoprotein associated with major morphogenic e vents and tissue remodeling in vertebrates. In Xenopus laevis embryos, SPAR C is expressed first by dorsal mesoderm cells at the end of gastrulation an d undergoes complex, rapid changes in its pattern of expression during earl y organogenesis. Another study has reported that precocious expression of S PARC by injection of native protein into the blastocoele cavity of pregastr ula embryos leads to a concentration-dependent reduction in anterior develo pment. Thus, normal development requires that the timing, spatial distribut ion, and/or levels of SPARC be regulated precisely. In a previous study, we demonstrated that injection of a synthetic peptide corresponding to the C- terminal, Ca2+-binding, EF-hand domain of SPARC (peptide 4.2) mimicked the effects of native SPARC. In the present investigation, peptide 4.2 was used to examine the cellular and molecular bases of the phenotypes generated by the aberrant presence of SPARC. Exposure of late blastula embryos to LiCl also generated a concentration-dependent reduction in anterior development; therefore, injections of LiCl were carried out in parallel to highlight th e unique effects of peptide 4.2 on early development. At concentrations tha t caused a similar loss in anterior development (60-100 ng peptide 4.2 or 0 .25-0.4 mu g LiCl), LiCl had a greater inhibitory effect on the initial rat e of chordomesoderm cell involution, in comparison with peptide 4.2. Howeve r, as gastrulation progressed, peptide 4.2 had a greater inhibitory effect on prospective head mesoderm migration than that seen in the presence of Li Cl. Moreover, peptide 4.2 and LiCl had distinct influences on the expressio n pattern of dorso-anterior markers at the neural and tail-bud stages of de velopment. Scanning electron microscopy showed that peptide 4.2 inhibited s preading of migrating cells at the leading edge of the involuting chordomes oderm. While stili in close proximity to the blastocoele roof, many of the cells appeared rounded and lacked lamellipodia and filopodia extended in th e direction of migration. In contrast, LiCl had no effect on the spreading or shape of involuting cells. These data are the first evidence of a counte r-adhesive activity for peptide 4.2 in vivo, an activity demonstrated for b oth native SPARC and peptide 4.2 in vitro.