Huynh MH et al. (1999), A calcium-binding motif in SPARC/osteonectin in...

XB-ART-12429

Dev Growth Differ 1999 Aug 01;414:407-18. doi: 10.1046/j.1440-169x.1999.00443.x.

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

Huynh MH , Sage EH , Ringuette M .

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Secreted protein, acidic, rich in cysteine (SPARC) is a Ca2+-binding, counter-adhesive, extracellular glycoprotein associated with major morphogenic events and tissue remodeling in vertebrates. In Xenopus laevis embryos, SPARC is expressed first by dorsal mesoderm cells at the end of gastrulation and undergoes complex, rapid changes in its pattern of expression during early organogenesis. Another study has reported that precocious expression of SPARC by injection of native protein into the blastocoele cavity of pregastrula embryos leads to a concentration-dependent reduction in anterior development. Thus, normal development requires that the timing, spatial distribution, 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 the unique effects of peptide 4.2 on early development. At concentrations that caused a similar loss in anterior development (60-100 ng peptide 4.2 or 0.25-0.4 microg LiCl), LiCl had a greater inhibitory effect on the initial rate of chordomesoderm cell involution, in comparison with peptide 4.2. However, as gastrulation progressed, peptide 4.2 had a greater inhibitory effect on prospective head mesoderm migration than that seen in the presence of LiCl. Moreover, peptide 4.2 and LiCl had distinct influences on the expression pattern of dorso-anterior markers at the neural and tail-bud stages of development. Scanning electron microscopy showed that peptide 4.2 inhibited spreading of migrating cells at the leading edge of the involuting chordomesoderm. While still in close proximity to the blastocoele roof, many of the cells appeared rounded and lacked lamellipodia and filopodia extended in the 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 counter-adhesive activity for peptide 4.2 in vivo, an activity demonstrated for both native SPARC and peptide 4.2 in vitro.

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Species referenced: Xenopus laevis
Genes referenced: bcr chrd fst sparc

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	Fig. 1. Schematic representation of the molecular organization and biological activities of SPARC (secreted protein, acidic, rich in cysteine). The counter-adhesive activity of SPARC on tissue culture cells is mimicked by synthetic peptides. However, only peptide 4.2, corresponding to the second EF-hand related motif of SPARC, mimicked the phenotypic effects of precociously expressed SPARC in Xenopus embryos. EC, extracellular Ca2+-binding; FS, follistatin.
	Fig. 2. Injection of low doses of peptide 4.2 and LiCl into the blastocoele cavity of stage 8/9 embryos results in different rates of blastopore ring closure and failure in the development of anterior structures. External view of embryos injected with 20% Steinberg’s solution (A,D), 60–100 ng of peptide 4.2 (B,E), and 0.25–0.4 μg of LiCl (C,F). (A–C) Embryos were allowed to develop after injection until Steinberg’s solution (control) embryos reached stage 12, as determined by the size of the blastopore (bp). (D–F) Embryos were allowed to develop until Steinberg’s solution-injected embryos reached stage 33. Control embryos had well-developed eyes and a prominent cement gland. For both peptide 4.2- and LiCl-injected embryos, there was an absence of eyes and cement gland. Moreover, peptide 4.2 embryos appeared more rotund, with remnants of a blastocoele cavity (arrow, bc).
	Fig. 3. Injection of peptide 4.2 results in incomplete gastrulation, with embryos lacking anterior structures. Sagittal sections of 20% Steinberg’s solution (A,D), 60–100 ng peptide 4.2- (B,E) and 0.25–0.4 μg LiCl (C,F)-injected embryos. Embryos were allowed to develop until control (Steinberg’s solution)-injected embryos reached stage 21 (A–C) or stage 33 (D–F). Steinberg’s solution-injected embryos show the internal structures expected of late neurula stage 21 embryos, for example, archenteron and brain anlage (panel A). In contrast, peptide 4.2-injected embryos were more spherical, with the prospective head (dorsal) mesoderm cells involuted approximately half the expected distance, and remnants of the blastocoele cavity (bc) still present (panel B). Although LiCl-injected embryos had a slower initial rate of blastopore closure than control embryos, by stage 21 there were few notable histological differences between 20% Steinberg’s solution- and LiCl-injected embryos (panel C). Steinberg’s solution-injected tail-bud stage embryos show the anterior structures expected of stage 33 embryos, for example, cement gland (cg), eye anlage (ea), foregut (fg) and otic vesicle (ov; panel D). Sections of peptide 4.2-injected embryos at stage 33 revealed a complete absence of anterior development (panel E). Compared to control sibling embryos, there was no evidence of a cement gland, brain anlage, foregut or eyes. Although LiCl-injected embryos also lacked eyes and cement glands, head mesenchymal tissue that normally gives rise to brain anlage was visible (panel F). Moreover, some degree of foregut development was also apparent. In contrast to head development, trunk dorsal development (e.g. somites, neural tube, notochord) was not significantly affected by injection of either peptide 4.2 or LiCl. bc, blastocoele cavity; bp, blastopore.
	Fig. 4. Scanning electron microscopy indicates that peptide 4.2 inhibits the spreading of cells at the leading edge of the involuting chordomesoderm. Steinberg’s solution (A,C)-, and peptide 4.2 (B,D)- injected embryos. Embryos were allowed to develop until Steinberg’s solution-injected embryos reached stage 11–12. For Steinberg’s solution-injected embryos, lamellipodia were visible at the leading edge of cells migrating along the blastocoele roof (A, black arrow). Cells exposed to peptide 4.2 had a rounded morphology, with fewer cells showing lamellipodia in the direction of migration (B, black arrow). Neither peptide 4.2 nor LiCl had any effect on the morphology of endodermal cells lining the bottom of the blastocoele cavity (D,E). BCR, blastocoele cavity roof; E, blastocoele floor endodermal cell; M, migrating mesodermal cell. White arrows indicate the direction of migration in A,B. Bar, 75 μm (A,B); 120 μm (C,D).
	Fig. 5. Peptide 4.2-injected embryos show decreased chordin RNA expression during gastrulation and in the chordoneural region of tail-bud embryos. Chordin mRNA expression in Steinberg’s solution-injected embryos is as reported by Sasai et al. (1994). Chordin mRNA was restricted to the notochord and prechordal plate (head mesoderm) at stages 13 and 21 (A,D). By stage 32, expression was restricted to the chordoneural region of the tail-bud (G). In peptide 4.2-injected embryos, the low levels of expression restricted to the dorsal lip of the blastopore (B) indicate that chordomesoderm involution has been severely compromised. In contrast, LiClinjected embryos showed chordin RNA expression throughout the notochord, but not in the chordomesoderm (C). By stage 21, the pattern of chordin expression for peptide 4.2 and LiCl were the same (E,F). Strong expression was visible within the notochord. However, as with stage 13 embryos, expression is absent from the anterior region. Staining of the lining of the archenteron is an artifact. Although histological analyses did not indicate any significant morphological differences in trunk development between Steinberg’s solution and peptide 4.2, expression of chordin was not observed in the chordoneural ridge in tail-bud embryos of peptide 4.2. Thus, the organizer potential of the tail might be compromised by injection of peptide 4.2 (H). As embryos develop from the late neurula stage, chordin expression in the notochord decreased and was eventually undetectable by the tail-bud stage, except for the chordoneural ridge (D,G). However, for LiCl-injected embryos, expression continued in the notochord after sibling control embryos no longer showed expression (I). Arrow indicates the anterior of the embryos.