Journal of Histochemistry and Cytochemistry, Vol. 45, 643-656, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

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

Sashko Damjanovskia, Xantha Karpa, Sarah Funkb, E. Helene Sagea,b, and Maurice J. Ringuettea,a
a Department of Zoology, University of Toronto, Toronto, Ontario, Canada, 98195
b Department of Biological Structure, University of Washington, Seattle, Washington, 98195

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


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Extracellular matrix glycoproteins make major morphoregulatory contributions to early embryonic development. Their complex, transient expression patterns, structural heterogeneity, and functional diversity indicate important roles in the modulation of a broad spectrum of cellular activities, such as adhesion, migration, communication, and/or proliferation. For example, the blastocoel roof of amphibian embryos is coated with an elaborate network of fibronectin fibers before the involution of mesodermal cells during gastrulation (Adams and Watt 1993 ; Boucaut et al. 1984 ). Microinjection of anti-fibronectin antibodies into the blastocoel cavity of newt embryos inhibited gastrulation, and the use of Xenopus cells and RGD peptides in vitro confirmed that fibronectin was an adhesive substrate that promoted cell migration (Smith et al. 1990 ; Boucaut et al. 1984 ). Tenascin has also been found to be associated with mesodermal cell migration during amphibian gastrulation in vivo (Riou et al. 1990 ) and promotes neural crest cell migration in avian embryos (Akitaya and Bronner-Fraser 1992 ). Because both tenascin and thrombospondin inhibit cell adhesion to fibronectin substrates in vitro, these two large ECM glycoproteins might have counter-adhesive potential in vivo (Bornstein 1995 ; Chiquet-Ehrismann et al. 1988 ).

It has become apparent that proteins such as tenascin C, thrombospondin 1, and SPARC (secreted protein, acidic, and rich in cysteine) primarily modulate cell-matrix interactions and do not subserve structural roles in the ECM per se. This group of proteins has been termed "matricellular" and is typified by the Ca2+ and Cu2+-binding glycoprotein SPARC, a prominent component of remodeling tissues (Bornstein 1995 ; Lane and Sage 1994 ; Sage and Bornstein 1991 ).

We recently demonstrated that SPARC expression begins at Stage 13 (late gastrulation) in Xenopus embryos. Whole-mount in situ hybridization and immunohistochemical analysis demonstrated staining within the notochord and somites from the onset of their development (Damjanovski et al. 1994 ). As development progressed, SPARC RNA and protein underwent marked and rapid changes in expression during neurulation and early organogenesis. In particular, SPARC transcripts and protein were found to be enriched in cells known to undergo Ca2+-dependent adhesion and that were electrically coupled via gap junctions. The highly regulated pattern of expression indicates that SPARC has important functions in early embryonic development. This hypothesis is based in part on experiments in which microinjection of anti-SPARC antibodies into the blastocoel cavity led to major trunk defects (Purcell et al. 1993 ). Ectopic expression of SPARC produced comparable muscle defects in C. elegans (Schwarzbauer and Spencer 1993 ). Although the precise functions of SPARC during early embryonic development are not known, a variety of potential functions have been described for SPARC in vitro, e.g., inhibition of cell-substratum adhesion and cell spreading (Sage et al. 1989 ), binding to and/or modulation of growth factors (Hasselaar and Sage 1992 ; Raines et al. 1992 ), regulation of cell cycle progression (Funk and Sage 1991 , Funk and Sage 1993 ), and augmentation of interendothelial cell permeability (Goldblum et al. 1994 ; reviewed in Lane and Sage 1994 ). Furthermore, SPARC interacts with several ECM macromolecules in vitro (Tremble et al. 1993 ; Kelm and Mann 1991 ).

In this study we examined the developmental consequences of the ectopic expression of SPARC before its normal embryonic activation. We report that microinjection of SPARC RNA, native protein, and synthetic peptides had distinct but nonexclusive developmental consequences. Our observations from a developmental system in vivo are consistent with some of the functions previously identified for SPARC in vitro.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
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Literature Cited

Xenopus embryos were fertilized and were raised according to standard techniques (Wu and Gerhart 1991 ). Staging was according to Nieuwkoop and Faber 1956 .

SPARC RNA and Protein
Full-length mouse SPARC cDNA (Mason et al. 1986 ) was subcloned, in both orientations, into the Pst 1 site of pGEM 4Z. Orientation was determined using restriction enzyme analysis. Both capped and poly(A)-tailed full-length sense and (control) anti-sense mouse RNAs were synthesized with Boehringer Mannheim cap-scribe and United States Biochemical poly(A) adenylation kits with the T7 plasmid promoter. Rat bone sialoprotein cDNA (a gift of Dr. Jaro Sodek, University of Toronto) was subcloned and transcribed similarly. Intact mouse SPARC protein was isolated from the conditioned medium of PYS-2 cells and was purified as previously described (Sage et al. 1989 ). This protein preparation was shown to disrupt barrier function in cultured endothelial cells, as described by Goldblum et al. 1994 , and to inhibit endothelial and smooth muscle cell proliferation in vitro, according to Funk and Sage 1993 . Porcine SPARC/osteonectin (Domenicucci et al. 1988 ) and porcine osteopontin (Zhang et al. 1990 ), obtained from Dr. Jaro Sodek, porcine bone sialoprotein (Goldberg et al. 1988 ), obtained from Dr. Harvey Goldberg (University of Western Ontario), bovine brain calmodulin (Calbiochem; La Jolla, CA), and bovine serum albumin (Pharmacia, fraction V; Uppsala, Sweden) were used as controls.

Immunoprecipitation
To confirm that SPARC RNA, injected 2 hr post fertilization (pf) into two-cell embryos, was translated, we co-injected [35S]-methionine. Embryos were lysed at 2 and 6 hr after injection [before the embryonic induction of SPARC at Stage 13 (Ringuette et al. 1992 ), 16 hr post fertilization], and protein was immunoprecipitated with a polyclonal anti-porcine SPARC antibody [previously demonstrated to bind to mouse and Xenopus SPARC (Ringuette et al. 1992 ; Bolander et al. 1989 )] with a BRL (Gaithersburg, MD) Immunoprecipitin kit. Protein precipitates were fractionated by SDS-PAGE; the gels were subsequently fixed, dried, and exposed to X-ray film.

Synthetic Peptides
Peptides representing various regions of mouse SPARC (Figure 1), synthesized by the Howard Hughes Institute (University of Washington) or by Zymogenetics (Seattle, WA), were purified by high-performance liquid chromatography (Lane and Sage 1990 ). All peptides were assayed in vitro according to Lane and Sage 1990 and Funk and Sage 1993 . In this study we injected peptide 2.1 [amino acids 54-73 (CQNHHCKHGKVCELDESNTP)] corresponding to the N-terminal end of the cysteine-rich Domain II; peptide 2.3 [amino acids 113-130 (TLEGTKKGHKLHLDYIGP)] representing the C-terminal end of Domain II, and peptide 4.2 [amino acids 254-273 (TCDLDNDKYIALEEWAGCFG)], representing the C-terminal EF-hand Domain IV of SPARC (Sage et al. 1995 ). Mutant peptide 4.2K (identical to peptide 4.2 except for an aspartic acid to lysine substitution at amino acid 258) and peptide 4.2AA (also identical to peptide 4.2 except for two cysteine to alanine substitutions at amino acids 255 and 271) were also microinjected into the embryos.



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Figure 1. Diagrammatic representation of SPARC domains and position of synthetic peptides used for microinjection. SPARC can be subdivided into four distinct structural domains. The N-terminal glutamic acid-rich Domain I can bind with low affinity up to eight Ca2+ ions (Kd 10-3-10-5 M); SPARC is therefore likely to be sensitive to changes in ECM Ca2+ levels. Domain II contains a follistatin-like module with 10 conserved Cys residues and two Cu2+-binding sites. Domains III and IV, representing the C-terminal half, both contain a series of {alpha}-helical segments. In addition, Domain IV has a unique disulfide bridge and a Ca2+-binding EF hand, which can bind one Ca2+ with a Kd 10-7 M. Peptide 2.1 [amino acids 54-73 (CQNHHCKHGKVCELDESNTP)] corresponds to the N-terminal end of Domain II. Peptide 2.3 [amino acids 113-130 (TLEGTKKGHKLHLDYIGP)] is C-terminal to peptide 2.1. Peptide 4.2 [amino acids 254-273 (TCDLDNDKYIALEEWAGCFG)] represents the EF-hand of Domain IV. Mutant peptides 4.2K and 4.2AA are identical to peptide 4.2 except for an Asp&Aelig;Lys substitution at amino acid 258 and two Cys&Aelig;Ala substitutions at amino acids 255 and 271, respectively.

Microinjection
Embryos were injected with glass needles prepared with a Narishige glass needle puller and a Narishige forced-gas system. Stage 2 (RNA injections, 2 hr pf) and Stage 8 (protein and peptide injections, 8 hr pf) Xenopus embryos in 100% Steinberg's solution with 3% Ficoll were injected with 5-200 pg of RNA (in 5-50 nl water) into one cell of a 2-cell embryo. Between 10 and 1000 ng (in 10-300 nl PBS) of protein was injected into the blastocoel cavity of Stage 8 embryos. Embryos were healed for several minutes in 100% Steinberg's solution before transfer into 20% Steinberg's solution.

Whole-mount Immunohistochemistry
Embryos were immersed for 2 hr in Dent's fixative (25% dimethylsulfoxide in methanol), washed several times in PBS, and incubated with a neural-specific 2G9 antibody (1:250 dilution) overnight at 4C (Jones and Woodland 1989). After several washes with PBS, the neural tissue distribution was visualized with an anti-mouse alkaline phosphatase-conjugated secondary antibody reacted with NBT and BCIP. Embryos were immersed in Bouin's fixative, dehydrated in methanol, and cleared in benzyl benzoate and benzoic acid before photography.

Histology
Embryos were fixed for 2 hr in 4% formalin, washed several times in PBS, and dehydrated in ethanol. Dehydrated embryos were embedded in Paraplast, sectioned (10 µm), and placed on collagen-coated slides. Sections were stained with hematoxylin and eosin according to standard protocols (Kelly et al. 1991 ) and were mounted with Permount for photography.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Generation of Unilateral Defects by Microinjection of SPARC RNA into Early Blastomeres
Microinjection of 5-25 ng of RNA had no effects on development, whereas microinjection of 200 ng of RNA resulted in death in greater than 75% of the embryos. Microinjection of 100 pg of capped and poly(A)-tailed mouse SPARC sense RNA into one cell of 2-cell embryos (Table 1) resulted in defects restricted to one side of some embryos (Figure 2A-D). Co-injection of rhodamine dextran sulfate revealed that the affected region corresponded to the progeny of the injected blastomere (data not shown). Eyes were either small and deformed (arrow, Figure 2A) or absent, on the left or right side (Figure 2B vs 2C), coincident with the blastomere injected. Defects were also observed within the trunk. The absence of somites on one side resulted in embryos with severely curved axes (triangles, Figure 2A and Figure 2D). Histological analysis revealed somite malformations that corresponded with the kinked axis (Figure 2E). More severe axial defects (Figure 2D) were associated with malformed axial features, such as notochords, identified histologically (Figure 2E). Control SPARC anti-sense RNA-injected embryos (Table 1) were normal, both morphologically (Figure 2F) and histologically (Figure 2G). Similarly, control BSP sense or anti-sense RNA-injected embryos were normal (Table 1).



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Figure 2. Injection of SPARC RNA into early blastomeres is associated with head and axis defects. Microinjection of 100 pg capped and poly(A)-tailed mouse SPARC sense RNA into one cell of a two-cell embryo resulted in the reduction of axial structures on the injected side. These defects included a reduced eye on one side of the embryo (A, arrow) , an eye present on one side (B) but missing from the other side of the embryo, and various axial kinks (A,D, triangles). Histological sections of sense RNA-injected embryos revealed asymmetric somites and poorly formed notochords (E). In contrast, microinjection of anti-sense RNA had no effects on development as seen both externally (F) and histologically (G). n, notochord; nt, neural tube; s, somites. Bars = 300 µm.


 
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Table 1. Summary of macromolecules injected and their phenotypic effectsa

To confirm that injected SPARC sense RNA was translated into SPARC protein, we immunoprecipitated labeled embryonic protein with SPARC antibody 2 and 6 hr after RNA injections and 4-8 hours before embryonic activation of SPARC RNA occurred. The Mr of SPARC on SDS-polyacrylamide gels is 43,000. [35S]-Methionine injected alone or with anti-sense RNA was not associated with a band of Mr 43,000 in embryos up to 6 hr after injection (Figure 3, Lanes A and B). After injection of [35S]-methionine with sense RNA, two bands were immunoprecipitated (at both 2 and 6 hr post injection) (Figure 3, Lanes C and D). The upper bands, with an estimated molecular weight of 43 kD, matched the molecular weight expected for SPARC. The lower molecular weight band (middle band; Figure 3, Lanes C and D) probably represents a degradation product of SPARC. An unidentified band present in all lanes corresponds to a nonspecific reactivity of the polyclonal SPARC antibody.



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Figure 3. Translation of microinjected SPARC sense RNA. An autoradiograph of [35S]-methionine-labeled protein extracts immunoprecipitated with anti-SPARC IgG and fractionated by SDS-PAGE. Lane A, control [35S]-methionine injected, and Lane B, [35S]-methio-nine, anti-sense SPARC RNA co-injected embryos did not express SPARC-like protein after 6 hr. In contrast, co-injection of [35S]-methionine with sense SPARC RNA resulted in the detection of a SPARC-like protein at 2 hr (Lane C) and 6 hr (Lane D), post injection. Molecular weight standards of 43 kD (ovalbumin) and 30 kD (carbonic anhydrase) are shown.

Microinjection of Mouse SPARC into the Blastocoel Cavity Causes a Broad Spectrum of Defects
Control uninjected embryos and those injected with protein initiated gastrulation at the same time and appeared to progress through gastrulation and early neurulation in a similar manner, as judged by the morphological criteria of blastopore and neural tube formation. However, shortly after neurulation a range of defects was apparent for the embryos injected with SPARC: (a) approximately 25% of the embryos (20 of 78) had minor abnormalities in head structure and defective truncated axes by stage 28 (Figure 4A); (b) 50% (40 of 78) appeared to have a broad range of severe head and axial defects (Figure 4B); and (c) 25% of injected embryos appeared unaffected (see also Table 1). Head defects included a lack of eyes and reduced cement glands (arrows, Figure 4A and Figure 4C). To confirm that observed effects were not unique to mouse SPARC, we also injected porcine SPARC. Amino acid analysis of SPARC, based on cDNA sequences, has demonstrated a 90% amino acid identity among mammals and 80% identity between Xenopus and mammals (Damjanovski et al. 1992 ). The effects of porcine SPARC (Figure 4C) were very similar to those of mouse SPARC (Table 1; Figure 3A and Figure 3B). Sagittal sections of embryos with defective axes confirmed the absence of anterior structures and a lack of tissue differentiation, with the exception of a stacked columnar cell aggregate with cement gland-like appearance (arrow, Figure 4D and Figure 4E). Within the trunk region of some of these embryos, a notochord-like structure could also be identified (Figure 4E).



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Figure 4. Microinjection of SPARC protein into the blastocoel cavity leads to severe axial defects. Injection of mouse SPARC into a Stage 8-9 blastocoel cavity resulted in reduction of head structures, including reduced cement glands (arrows in A,C) and severe axis defects (A-C). Defects in axes were confirmed with histological sections, which revealed cement gland-like structures (arrows in D,E), notochord-like structures (n, E), but few other discernible tissues (D,E). Effects of mouse SPARC were confirmed through the similar injection of porcine SPARC (C). Bars = 200 µm.

SPARC has been demonstrated to bind to a variety of macromolecules (Lane and Sage 1994 ). Therefore, developmental defects generated by precocious overexpression of SPARC might have been due to nonspecific chelation of macromolecules that play critical roles in early embryonic development. To address this concern, we injected equivalent amounts of two structurally-related Ca2+-binding glycoproteins into the blastocoel cavity. Embryos injected with porcine osteopontin were arrested in development before gastrulation, whereas no developmental defects were observed after injection of porcine bone sialoprotein (Table 1; and M. Ringuette, unpublished experiments).

Because the N-terminal, glutamic acid-rich region of SPARC can bind up to 8 Ca2+ with Kd ranging from 10-3-10-5 M, we were also concerned that the effects shown in Figure 2 might have been due to the chelation of extracellular Ca2+ by SPARC; both inter- and intracellular Ca2+-mediated pathways would thus be compromised. However, pre-incubation and co-injection of SPARC with 0.3-10 mM CaCl2 did not alter the results (Table 1). Because the blastocoel cavity of Xenopus embryos is known to contain 1.5 mM free Ca2+, we also tested for Ca2+-dependent effects of SPARC by the injection of SPARC with EGTA. Co-injection with 0.3-3 mM EGTA did not interfere with the phenotypic effects of SPARC. However, further increases in EGTA concentration (with or without SPARC) led to embryonic death as a result of the dissociation of blastomeres, which depend on cadherins for their cohesion (Angres et al. 1991 ).

Because the conformation of SPARC is labile (even at -70C), increasing doses of SPARC were required over time to obtain consistent results. Therefore, the data presented were generated by microinjection of 80-750 ng of SPARC. Similar results with respect to phenotypes were also obtained with 300 ng of porcine SPARC microinjected as described.

Microinjection of Synthetic Peptides
Synthetic peptides corresponding to Domains II and IV of SPARC (Figure 1) were found to mimic some of the major biological effects of native SPARC on tissue culture cells (Sage et al. 1995 ; Lane and Sage 1990 ). Therefore, we sought to determine whether these peptides could mimic the developmental changes associated with the microinjection of native SPARC.

Microinjection of Peptides 2.1 and 2.3 Does Not Interfere with Embryonic Development
Peptide 2.1, representing the N-terminal region of the cysteine-rich, follistatin-like Domain II (Figure 1), has been shown to arrest endothelial cells and fibroblasts at the G1 stage of the cell cycle (Lane and Sage 1994 ). Surprisingly, no effects on development were observed when 10-1000 ng of peptide 2.1 was injected into Xenopus embryos (Table 1; Figure 5C). Similar results (Figure 5D) were also obtained with peptide 2.3, a cationic peptide C-terminal to peptide 2.1 (Figure 1). Injection of up to 1000 ng of BSA (Figure 5A) or up to 300 nl of PBS (Figure 5B) had no apparent effects on development. Sections of control uninjected, PBS-, BSA-, peptide 2.1-, or peptide 2.3-injected embryos were indistinguishable (data not shown).



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Figure 5. Microinjection of SPARC cysteine domain peptides does not affect development. Control microinjections of 300 nl PBS (A) or 1000 ng BSA (B) had no apparent effect on development. Embryos injected with 1000 ng SPARC peptide 2.1 (C), representing the N-terminal region of the cysteine-rich, follistatin-like domain, also appeared normal. Similar results were also obtained with 1000 ng peptide 2.3 (D), representing a cationic region C-terminal to peptide 2.1 (see Figure 1).

Abnormalities Associated with Injection of Peptide 4.2
Microinjecting 60 ng (25 pM) of peptide 4.2, representing the Ca2+-binding EF-hand domain of SPARC (Figure 1), generated dorso-anterior defects. Virtually all embryos (63 of 65) had small heads, with abnormal eyes and cement glands by Stage 30 (Table 1; Figure 6A). Also visible were large ventral swellings, which sometimes collapsed to form highly convoluted surface folds. The trunk of the embryos appeared to have developed normally. Increasing the level of peptide 4.2 to 100 ng (40 pM) led to a complete absence of head development in 100% (58 out of 58) of the embryos (Table 1; Figure 6B). More than 90% of these embryos had relatively normal dorsal axes and continued to develop well into the late tailbud (Stage 50), and died shortly thereafter (Figure 6C). Histological sections revealed that otic vesicles (Figure 7D) were the first anterior tissues formed and thus confirm that anterior development was truncated (Figure 7A-G). The neural tube and notochord were always present (Figure 7F), but differed in their anterior progression. Use of the neural 2G9 antibody, however, revealed that neural tissue did indeed reach the extreme anterior of most embryos (Figure 8C, middle embryo).



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Figure 6. Abnormalities associated with microinjection of peptide 4.2. Microinjecting 60 ng of peptide 4.2 (A), representing the high-affinity, Ca2+-binding, EF-hand domain of SPARC, generated embryos with small heads and abnormal eyes and cement glands (A, single arrows). Large ventral swellings were visible, which sometimes collapsed to form highly convoluted surface folds (double arrows). Injection of 100 ng peptide 4.2 (B) was associated with a complete absence of head development in 100% of the embryos. As in A, swelling in the anterior ventral region or surface-convoluted folds (double arrows) were present. As a result of this swelling or excess tissue, the ventral endoderm and anterior regions of later-Stage 40-42 embryos (C) appeared malformed and swollen. Microinjection of 250 ng peptide 4.2 (D) was associated with embryos lacking heads, and with significantly shorter dorsal axes and a prominent collapsed ventral mass (double arrows). Microinjection of up to 1000 ng peptide 4.2 was associated with a broad range of axial defects. (E) Embryos were distorted or had nonexistent axes. (F) Embryos had formed prominent axial ridges (arrow), which spanned about one fifth of the circumference of the embryos. Results with peptide 4.2 were reproduced by peptide 4.2K, a non-Ca2+ binding mutant of peptide 4.2. Injection of 100 ng (G) or up to 1000 ng (H) peptide 4.2K produced results indistinguishable from 100 ng (B) or 1000 ng (E) peptide 4.2.



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Figure 7. Histological analysis of embryos injected with SPARC peptide 4.2. Transverse serial histological sections (anterior to posterior series, A-G) of eyeless embryos that were injected with 100 ng peptide 4.2 (shown in Figure 5B) revealed otic vesicles (o, D) to be the first discernible anterior structure. The ventral swellings or convoluted folds were continuous with the surface epidermis (arrows, E-G) and were coincident with the region containing the remnants of the blastocoel cavity (b, F). In embryos (shown in Figure 5D) injected with 250 ng peptide 4.2, transverse serial histological sections (anterior to posterior series, H-J) revealed that the somites (s) were the most anterior structures identified, followed by the neural tube (nt) and notochord (n)-like structures. Dorsal is top. Bar = 50 µm.



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Figure 8. Injection of a mutant SPARC Domain IV peptide results in different phenotypes. Microinjection of 1000 ng peptide 4.2AA resulted in embryos which underwent normal development until the tailbud stage, at which point ventral swelling became prominent (A, arrow). Head and axis structures in 4.2AA-injected embryos appeared normal (A). Microinjection of 500 ng calmodulin caused about 30% of the embryos to undergo an exogastrulation-like process, resulting in protruding endoderm (B). The external pigmented ectoderm of these embryos seemed to be turned inside-out, as the unpigmented internal cells protruded to form an elongated axis (B, arrows). Use of an anti-neural antibody (2G9) revealed that the ectoderm ridge of the calmodulin-injected embryos was neural (D, arrows). In normal and in peptide 4.2-injected headless embryos, 2G9 revealed a broad neural stripe (C, top two embryos), whereas embryos injected with peptide 4.2 (showing severely reduced axes) exhibited no neural tissues (C, bottom embryo).

The above results indicated that there was a concentration-dependent decrease in anterior to posterior development associated with the microinjection of peptide 4.2. This observation was confirmed by microinjection of 250 ng (100 pM) peptide 4.2. All of the 70 injected embryos lacked heads and had significantly shorter dorsal axes (Table 1; Figure 6D). Histological analysis revealed that the otic vesicles were also missing from the anterior region (Figure 7H-J). The somites, notochord, and neural tube were always present but often did not span the entire length of the dorsal axis as is characteristic of normal embryos. Frequently, transverse sections of the extreme anterior end of the axis revealed that the axial ridge was composed of only somite-like cells (Figure 7I). Posterior sections also revealed a loosely organized neural tube-like structure (Figure 7I and Figure 7J). Similarly, the notochord frequently failed to extend into the anterior region (Figure 7H and Figure 7I).

Increasing the concentration of microinjected peptide 4.2 from 250 ng to 500 ng (200 pM) or 1000 ng (400 pM) resulted in more severe axial defects (Figure 6E and Figure 6F; Table 1). The increased volume required to microinject larger amounts (due to constraints on solubility) caused significant swelling of the blastocoel. For many embryos the swelling caused by injection was so significant that external signs of gastrulation could not be seen. However, by the time sibling control embryos had reached Stage 35 (tailbud), these embryos had formed an axial ridge (Figure 6F). The ridges spanned about one fifth of the circumference of the embryos. Embryonic death occurred shortly thereafter. Antibody 2G9 revealed no neural tissues within these embryos (bottom embryo, Figure 8C). As we had observed with native SPARC, pre-incubation of peptide 4.2 with 0.3-10 mM CaCl2 or 0.3-3.0 mM EGTA did not alter these results (Table 1).

Effect of Peptide 4.2 Mutants
To determine whether binding of Ca2+ to the EF-hand contributed to the effect of peptide 4.2 on development, we injected peptide 4.2K in which an aspartic acid was substituted by a lysine (Sage et al. 1995 ). Peptide 4.2K displays a significantly reduced affinity for Ca2+ (Sage et al. 1995 ). No differences between the effects of peptides 4.2 and 4.2K were observed in intact or sectioned embryos. Injection of 100-1000 ng (40-400 pM) of peptide 4.2K (Figure 6G and Figure 6H) or wild-type peptide 4.2 (Figure 6B and Figure 6E) generated near by identical dose-dependent effects (see also Table 1).

An unusual feature of the EF hand of SPARC is that it is stabilized by a disulfide bridge. To determine whether this disulfide bond contributed to the biological activity of peptide 4.2, we microinjected peptide 4.2AA, in which the two cysteine residues of peptide 4.2 had been replaced by alanines. Microinjection of up to 500 ng peptide 4.2AA did not interfere with development. Embryos injected with 1000 ng underwent normal development until the tailbud stage, at which time ventral swellings became prominent (Table 1; Figure 8A). The effect (or cause) of this phenotype is unclear, because these embryos were otherwise morphologically normal and had intact head and axial structures.

As an additional control, a protein with four Ca2+-binding EF hands (bovine brain calmodulin) was also injected. Injection of up to 200 ng (10 pM) calmodulin did not interfere with development. Injection of 500-1000 ng (25-50 pM) led to embryonic death in 20 of 30 injected embryos by the time sibling control embryos reached late gastrulation. The remaining 10 injected embryos underwent an exogastrulation-like process (Table 1; Figure 8B), and appeared as endodermal masses that were capped by ectoderm (arrows, Figure 8B). Antibody 2G9 revealed that the ridge of the ectodermal cap was neural tissue (arrows, Figure 8D).


  Discussion
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Materials and Methods
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Discussion
Literature Cited

Whole-mount in situ hybridization and immunohistochemical analysis demonstrated that SPARC was expressed by Xenopus embryos at Stage 13, from the onset of the formation of the notochord and somites (Damjanovski et al. 1994 ). Rapid changes occurred in the level and spatial distribution of SPARC throughout the neurulation and tailbud stages of development. In this study we show that precocious ectopic expression of SPARC affects Xenopus development. Microinjection of capped and poly(A)-tailed full-length mouse SPARC RNA into one blastomere of 2-cell embryos (Stage 2, 14 hr before the expression of endogenous SPARC occurred) often generated unilateral defects. For example, eyes were usually absent or defective only on the injected side. In addition, some embryos had both dorso-anterior and trunk defects only on one side. Microinjection of SPARC peptides resulted in a similar phenotype, although the effects were bilateral.

In Xenopus, eye development (the first anterior organ formed) is affected by a wide variety of treatments, including injection of anti-gap junction antibodies into blastomeres of 8-cell embryos (Warner et al. 1984 ) and injection of reagents that affect dorsal-anterior development (e.g., retinoic acid, Xgsk, Xwnt-8, trypan blue, heparin, suramin) (reviewed in Kao and Danilchuk 1991 ). Embryos with defective eyes or lacking eyes have also been generated by microinjection of anti-SPARC antibodies into the blastocoel cavity (Purcell et al. 1993 ). Moreover, in situ and immunohistochemical studies have demonstrated that SPARC is a major component of developing retina in chicken, bovine, and monkey embryos (M. Ringuette, Q. Yan, H. Sage, A. Hendrickson, unpublished experiments). Although eye development in Xenopus is sensitive to a variety of experimental perturbations, several factors could account for the observations reported here. Lineage tracing, separation, and perturbation experiments indicate that the three body axes (anterior-posterior, dorsal-ventral, and left-right) are linked developmentally (reviewed in Almirantis 1995 ). It is therefore likely that their formation is in part influenced by common axis determinants. SPARC has been shown to modulate the activity of growth factors (Lane and Sage 1994 ; Raines et al. 1992 ). Therefore, binding of SPARC to inducing factors could interfere with the specification of embryonic tissues that would have occurred later in development. That the injection of other extracellular glycoproteins (osteopontin and bone sialoprotein) resulted in unique phenotypes indicates that nonspecific chelation of macromolecules by these acidic proteins was not a causal factor. However, it remains to be established if SPARC binds to and modulates the activity of inducing factors in early Xenopus embryos. We are now investigating whether or not SPARC has an affinity for, and/or interferes with, other known embryonic inducing factors.

Microinjecting SPARC into the blastocoel cavity (a region into which proteins can be injected within a large ECM cavity) before gastrulation enabled us to assess the impact of the precocious presence of SPARC (a) just before the start of major morphogenetic movements, (b) while major cell-cell inductive events were still occurring, and (c) 8-9 hr before endogenous expression of SPARC began. As with the microinjection of SPARC RNA, a variety of head and tail defects were observed, e.g., defective cement gland and eyes, somite misalignment, axis bending, and ventral swelling. However, in contrast to the microinjection of RNA, bilateral defects were observed. A plausible explanation is that the injected SPARC was distributed evenly within the blastocoel cavity. Because Xenopus embryos do not begin to express SPARC until mid-gastrulation and SPARC does not accumulate to any significant levels until neurulation (Damjanovski et al. 1994 ), the defects generated here were probably due to the precocious presence of SPARC before its endogenous activation. Of concern was that the effects could have been due to sequences and structures unique to murine SPARC (amphibian SPARC has not been purified nor characterized functionally). Because SPARC is a highly conserved protein [90% amino acid identity among mammalian species and 80% between Xenopus and mammals (Damjanovski et al. 1992 )], it is likely that common functions would be shared in different organisms. Indeed, when we microinjected porcine SPARC, the defects generated were very similar to those obtained with the murine protein.

Studies in vitro on endothelial cells, smooth muscle cells, and fibroblasts have demonstrated that SPARC delayed cell cycle progression and reduced cell-substratum interactions (reviewed in Lane and Sage 1994 ). However, SPARC had no obvious morphological effect on early cell division and movement associated with gastrulation and early neurulation in Xenopus. Nevertheless, the effects observed, at a time when control sibling embryos had reached the tailbud stage, indicate that SPARC did interfere with gastrulation events and at high doses resulted in the ventralization of embryos. However, we have recently observed that SPARC did not interfere with the induction of mesoderm in Xenopus animal cap explants by activin or bFGF (S. Damjanovski and M. Ringuette, unpublished observations), two factors that play important inductive roles in early development. One potential target of SPARC could have been PDGF, because SPARC binds to PDGF-AB and -BB with high affinity. Interference with PDGF signaling in Xenopus has also been shown to generate some axis and anterior defects (Ataliotis et al. 1995 ) that are comparable to those observed with microinjected SPARC. However, PDGF is only one of several factors known to participate in early development. It is conceivable that the more severe defects observed with SPARC injection were due to its binding to multiple factors. Moreover, any potential effects of SPARC on cell division and adhesion might have been subtle and therefore not obvious until later in development. Interestingly, the microinjection of two other acidic Ca2+-binding glycoproteins, bone sialoprotein and osteopontin, resulted in developmental effects that were distinct from each other and from those induced by SPARC; these results indicate that the phenotypes generated were protein-specific.

The effects of native SPARC on tissue culture cells have been recapitulated by synthetic peptides corresponding to discrete domains of SPARC. For example, peptides from the cysteine-rich, follistatin-like domain (2.1 ) and the C-terminal EF hand (4.2) inhibit cell cycle progression in mid-G1 (Funk and Sage 1993 ). In addition to inhibiting cell cycle progression, peptides 2.1 and 4.2 were found to compete for the binding of SPARC to collagen Types I and III and to endothelial cells. No effects on Xenopus development were observed when 60-1000 ng of peptide 2.1 or 2.3 was microinjected into the blastocoel cavity. However, injection of 60 ng of peptide 4.2 generated dorso-anterior defects. When the concentration of peptide 4.2 was increased to 100 ng, 100% of the embryos were headless. Further increases were associated with concentration-dependent defects in an anterior to posterior direction. The substantial effects of peptide 4.2 were surprising because, as discussed above, peptides 2.1 and 4.2 have similar effects on cultured cells in vitro. A potential explanation is that peptide 4.2 is a more potent inhibitor of cell cycle progression than is peptide 2.1 (4- to 100-fold) (Sage et al. 1995 ). It also inhibits cell spreading in vitro. The combination of cell-cycle modulation, cell surface receptor and collagen binding, and counteradhesion may account for the interference of peptide 4.2 with Xenopus development. Whereas gastrulation did not appear to be affected by peptide 4.2, as judged by closure of the blastopore, the absence of the dorso-anterior axis and structures indicates that it is very likely that interference with cell movement and/or inductive events occurred.

The inhibition of endothelial cell proliferation by peptide 4.2 was abrogated by two mutant peptides, 4.2AA and 4.2K. Peptide 4.2 K was associated with apparently the same developmental defects as were seen with peptide 4.2. In contrast, embryos injected with peptide 4.2AA appeared normal until the tailbud stage, with the exception of a subset that had ventral swelling. The effects in vivo of peptide 4.2 are therefore in part dependent on the formation of the disulfide bridge but are tolerant of a disruption of the Ca2+-binding consensus sequence. The potentially Ca2+-independent effect was confirmed by the observation that the co-injection of peptides 4.2, 4.2AA, or 4.2K with Ca2+ or EGTA did not alter the results seen with the peptide alone. Although direct comparisons between the effects observed in tissue culture vs whole embryos are difficult, the Ca2+-independent effects of peptide 4.2 in vivo might be consistent with data showing that SPARC does not act as a chelator of Ca2+ in the extracellular space, despite its ability to modulate endothelial cell shape (Sage 1992 ). The requirements of a disulfide bridge (an unusual feature of the EF-hand of SPARC) indicates that the secondary structure (and/or stability) of this region is critical. That microinjection of calmodulin (a protein containing four prototypic, nondisulfide-bonded EF hands) generated substantially different results indicated that the phenotypic effects of peptide 4.2 were particular to SPARC.

We have shown that precocious expression of SPARC led to a variety of developmental defects in Xenopus embryos. As a matricellular, multifunctional protein, SPARC has been shown to affect a variety of cell behaviors, including counteradhesion and regulation of the cell cycle. Although, the molecular basis of the phenotypes generated by precocious expression of SPARC, in particular with peptide 4.2, remains to be determined, the data indicate that cell migration and cell proliferation were probably affected during Xenopus gastrulation. Therefore, the counteradhesion and inhibition of cell cycle observed in vitro appear to be augmented in vivo by precocious overexpression of SPARC at a time during which rapid cell migration and proliferation are occurring.


  Footnotes

a Contribution equal by the last two authors.


  Acknowledgments

Supported in part by a Natural Sciences and Engineering Research Council grant (MJR) and by a National Institutes of Health grant GM40711 (EHS).

We wish to thank Drs Jaro Sodek and Harvey Goldberg for their kind gifts of reagents.

Received for publication October 8, 1996; accepted December 11, 1996.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Adams JC, Watt FM (1993) Regulation of development and differentiation by the extracellular matrix. Development 117:1183-1198[Free Full Text]

Akitaya T, Bronner-Fraser M (1992) Expression of cell adhesion molecules during initiation and cessation of neural crest migration. Dev Dyn 194:12-20[Medline]

Almirantis Y (1995) Left-right asymmetry in vertebrates. BioEssays 17:79-83[Medline]

Angres B, Måller AHJ, Kellerman J, Hausen P (1991) Differential expression of two cadherins in Xenopus laevis. Development 111:829-844[Abstract]

Ataliotis P, Symes K, Chou MM, Ho L, Mercola M (1995) PDGF signalling is required for gastrulation of Xenopus laevis embryos. Development 121:3099-3110[Abstract/Free Full Text]

Bolander ME, Robey PG, Fisher LW, Conn KM, Prabhakar BS, Termine JD (1989) Monoclonal antibodies against osteonectin show conservation of epitopes across species. Calcif Tissue Int 45:74-80[Medline]

Bornstein P (1995) Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J Cell Biol 130:503-506[Medline]

Boucaut J-C, Darribere T, Poole TJ, Aoyama H, Yamada KM, Thiery JP (1984) Biologically active synthetic peptides as probes of embryonic development: a competitive peptide inhibitory inhibition of fibronectin function inhibits gastrulation in amphibian embryos and neural crest cell migration in avian embryos. J Cell Biol 99:1822-1830[Abstract]

Chiquet-Ehrismann R, Kalla P, Pearson CA, Beck K, Chiquet M (1988) Tenascin interferes with fibronectin action. Cell 53:383-390[Medline]

Damjanovski S, Liu F, Ringuette MJ (1992) Molecular analysis of Xenopus laevis SPARC (Secreted Protein, Acidic, Rich in Cysteine): a highly conserved acidic calcium-binding extracellular-matrix protein. Biochem J 281:513-517[Medline]

Damjanovski S, Malaval L, Ringuette MJ (1994) Transient expression of SPARC in the dorsal axis of early Xenopus embryos: correlation with calcium-dependent adhesion and electrical coupling. Int J Dev Biol 38:439-446[Medline]

Domenicucci C, Goldberg HA, Hofmann T, Isenman D, Wasi S, Sodek J (1988) Characterization of porcine osteonectin extracted from fetal calvaria. Biochem J 253:139-151[Medline]

Funk SE, Sage EH (1993) Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts. J Cell Physiol 154:53-63[Medline]

Funk SE, Sage EH (1991) The Ca2+-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells. Proc Natl Acad Sci USA 88:2648-2652[Abstract]

Goldberg HA, Domenicucci C, Pringle GA, Sodek J (1988) Mineral-binding proteoglycans of fetal porcine calvarial bone. J Biol Chem 263:11191-11201

Goldblum SE, Ding X, Funk SE, Sage EH (1994) SPARC regulates endothelial cell shape and barrier function. Proc Natl Acad Sci USA 91:3448-3452[Abstract]

Hasselaar P, Sage EH (1992) SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothelial cells. J Cell Biochem 49:272-283[Medline]

Jones EA Woodland HR (1989) Spatial aspects of neural induction in Xenopus laevis. Development 107:785-791[Abstract]

Kao K, Danilchuk M (1991) Generation of body phenotypes in early embryogenesis. Methods Cell Biol 36:271-284[Medline]

Kelly GM, Eib DW, Moon RT (1991) Histological preparation of Xenopus laevis oocytes and embryos. Methods Cell Biol 36:389-417[Medline]

Kelm RJ, Mann KG (1991) The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation. J Biol Chem 266:9632-9639[Abstract/Free Full Text]

Lane TF, Sage EH (1994) The biology of SPARC, a protein that modulates cell-matrix interactions. FASEB J 8:163-172[Abstract/Free Full Text]

Lane TF, Sage EH (1990) Functional mapping of SPARC: peptides from two distinct Ca++-binding sites modulate cell shape. J Cell Biol 111:3065-3076[Abstract]

Mason IJ, Taylor A, Williams JG, Sage EH, Hogan BLM (1986) Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell "culture shock" glycoprotein of Mr 43000. EMBO J 5:1465-1472[Abstract]

Nieuwkoop PD, Faber J (1956) Normal Table of Xenopus laevis: A Systematic Chronological Survey of the Development of the Fertilized Egg Until the End of Metamorphosis. Amsterdam, Elsevier North-Holland

Purcell L, Gruia-Gray J, Scanga S, Ringuette M (1993) Developmental anomalies of Xenopus embryos following microinjection of SPARC antibodies. J Exp Zool 265:153-164[Medline]

Raines EW, Lane TF, Iruela-Arispe ML, Rose R, Sage EH (1992) The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors. Proc Natl Acad Sci USA 89:1281-1285[Abstract]

Ringuette M, Drysdale T, Liu F (1992) Expression and distribution of SPARC in early Xenopus laevis embryos. Rouxs Arch Dev Biol 202:4-9

Riou JL, Shi DL, Chiquet M, Boucaut JC (1990) Exogenous tenascin inhibits mesodermal cell migration during amphibian gastrulation. Dev Biol 137:305-317[Medline]

Sage EH (1992) Modulation of endothelial cell shape by SPARC does not involve chelation of extracellular Ca2+ and Mg2+. Biochem Cell Biol 70:56-62[Medline]

Sage EH, Bassuk JA, Yost JC, Folkman MJ, Lane TF (1995) Inhibition of endothelial cell proliferation is mediated through a Ca2+-binding EF-hand sequence. J Cell Biochem 57:127-140[Medline]

Sage EH, Bornstein P (1991) Extracellular proteins that modulate cell-matrix interaction: SPARC, tenascin, and thrombospondin. J Biol Chem 266:14831-14834[Free Full Text]

Sage EH, Vernon RB, Funk SE, Everitt EA, Angello J (1989) SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca+2 dependent binding to the extracellular matrix. J Cell Biol 109:341-356[Abstract]

Schwarzbauer JE, Spencer CS (1993) The Caenorhabditis elegans homolog of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility. Mol Biol Cell 4:941-952[Abstract]

Smith JC, Symes K, Hynes RO, DeSimone D (1990) Mesoderm induction and the control of gastrulation in Xenopus laevis: the roles of fibronectin and integrins. Development 108:229-238[Abstract]

Tremble PM, Lane TF, Sage EH, Werb Z (1993) SPARC, a secreted protein associated with morphogenesis and tissue remodelling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway. J Cell Biol 121:1433-1444[Abstract]

Warner AE, Guthrie SC, Gilula NB (1984) Antibodies to gap-junction protein selectively disrupt junctional communication in the early amphibian embryo. Nature 311:127-131[Medline]

Wu M, Gerhart J (1991) Raising Xenopus in the laboratory. Methods Cell Biol 36:3-18[Medline]

Zhang Q, Domenicucci C, Goldberg HA, Wrana JL, Sodek J (1990) Characterization of fetal porcine bone sialoproteins: secreted phosphoprotein 1 (spp1, osteopontin), bone sialoprotein and a 23 kDa glycoprotein. Demonstration that the 23 kDa glycoprotein is derived from the carboxyterminus of SPP1 J Biol Chem 265:7583-7589