Journal of Histochemistry and Cytochemistry, Vol. 46, 1007-1016, September 1998, Copyright © 1998, The Histochemical Society, Inc.
The Distribution of the Matricellular Protein Thrombospondin 2 in Tissues of Embryonic and Adult Mice
Themis R. Kyriakidesa,
Yu-Hong Zhua,
Zhantao Yanga, and
Paul Bornsteina
a Department of Biochemistry, University of Washington, Seattle, Washington
Correspondence to:
Paul Bornstein, Department of Biochemistry, Box 357350, U. of Washington, Seattle, WA 98195..
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Summary |
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Mice that lack the matricellular protein thrombospondin 2 (TSP2) develop a pleiotropic phenotype characterized by morphological changes in connective tissues, an increase in vascular density, and a propensity for bleeding. Furthermore, dermal cells derived from TSP2-null mice display adhesion defects, a finding that implicates TSP2 in cellmatrix interactions. To gain a better understanding of the participation of TSP2 in the development and maturation of the mouse, we examined its distribution in embryonic and adult tissues. Special attention was paid to the presence of TSP2 in collagen fibers, because collagen fibrils in the TSP2-null mouse appear to be irregular in size and contour by electron microscopy. Immunohistochemical analysis of Day 15 and Day 18 embryos revealed TSP2 in areas of chondrogenesis, osteogenesis, and vasculogenesis, and in dermal and other connective tissue-forming cells. Distinctly different patterns of deposition of TSP2 were observed in areas of developing cartilage and bone at Days 15 and 18 of embryonic development. A survey of adult tissues revealed TSP2 in dermal fibroblasts, articular chondrocytes, Purkinje cells in the cerebellum, Leidig cells in the testis, and in the adrenal cortex. Dermal fibroblasts were also shown to synthesize TSP2 in vitro. The distribution of TSP2 during development is in keeping with its participation in the formation of a variety of connective tissues. In adult tissues, TSP2 is located in the pericellular environment, where it can potentially influence the cellmatrix interactions associated with cell movement and tissue repair. (J Histochem Cytochem 46:10071015, 1998)
Key Words:
immunohistochemistry, extracellular matrix, chondrogenesis, embryonic development, matricellular protein
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Introduction |
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Thrombospondin 2 (TSP2) is a member of a small family of secreted glycoproteins whose functions are poorly understood. The family can be divided into two branches on the basis of structural considerations. TSP1 and TSP2 are homotrimers composed of monomer chains of ~145,000 kD, whereas TSP3-5 are homopentamers with chains of lower molecular weight, because they lack several of the domains present in TSP1 and 2 (Bornstein and Sage 1994
; Adams 1995
). Virtually nothing is known of the functions of TSP3-5, except that mutations in the gene encoding TSP5/COMP have been identified in some patients with either pseudoachondroplasia or multiple epiphyseal dysplasia (Briggs et al. 1995
; Hecht et al. 1995
). Since its discovery as an
-granule protein that was released during platelet activation (Baenziger et al. 1971
), the structure, cellular synthesis, and function in vitro of TSP1 have been studied extensively but little consensus has been reached on its functions in vivo (Frazier 1991
; Bornstein 1995
; Tuszynski and Nicosia 1996
). TSP2 was the second member of the TSP family to be identified (Bornstein et al. 1991
; Laherty et al. 1992
). Although studies of its expression during murine and avian development have been performed at the mRNA level (Laherty et al. 1992
; Iruela-Arispe et al. 1993
; Tucker 1993
; Tucker et al. 1995
), the difficulty in obtaining the protein in pure form and in substantial quantities has hindered immunocytochemical and functional studies.
TSP1 and TSP2, together with functionally related proteins such as tenascin-C, SPARC, and osteopontin, have been termed matricellular proteins to highlight a postulated role for these extracellular proteins in modulating cellmatrix interactions, in preference to subserving more conventional structural functions (Bornstein 1995
). In an attempt to gain an understanding of the functional properties of TSP2 and to test the matricellular hypothesis, we generated a targeted disruption of the Thbs2 gene in mice (Kyriakides et al. 1998
). The phenotype of TSP2-null mice included abnormal collagen fibrillogenesis, which resulted in fragile skin and hyperflexible tendons and ligaments, increased cortical thickness of long bones, a bleeding tendency, and increased vascular density in many tissues. Skin fibroblasts from TSP2-null mice displayed defects in attachment on many different extracellular matrix (ECM) protein substrates. The unexpected complexity of the TSP2-null phenotype suggests that TSP2 plays a critical role in cellmatrix interactions and in the generation of ECMs, but the basis for these abnormalities remains unclear. In considering possible mechanisms by which a lack of TSP2 could cause the defects that are manifest in TSP2 knockout mice, it appears helpful to have a thorough understanding of the pattern of synthesis and deposition of TSP2, both during embryonic development and in the adult mouse. Therefore, as an example, in distinguishing a role for TSP2 as either an integral component of a collagen fibril, or as a modulator of fibroblastcollagen fibril interactions, a knowledge of the location of TSP2 in tissues would clearly be helpful.
We have therefore undertaken an immunocytochemical study of the distribution of TSP2 in both embryonic and adult mouse tissues. Two antibodies against mouse TSP2 were employed, one to the intact protein and another to the amino-terminal heparin-binding domain. The amino-terminal domain of TSP2 was selected because this region of the protein shows the maximal difference from the corresponding region of TSP1 (Laherty et al. 1992
; Bornstein and Sage 1994
). Both proteins were expressed with the use of the Baculovirus system and purified from serum-free conditioned medium of virus-infected insect cells (Chen et al. 1994
). One of the strengths of this study lies in its ability to distinguish whether immunoreactivity in wild-type tissues and cells was specific for TSP2, as judged by the absence of reaction product in corresponding preparations from mutant animals.
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Materials and Methods |
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Production of Baculovirus Transfer Vector and Recombinant Virus
The vector mTSP2183/pEV/35K, containing a full-length mouse TSP2 cDNA (Chen et al. 1994
), was a kind gift from Dr. D. Mosher, (University of Wisconsin). The construct was digested with EcoR I and Bgl II to release a 900-BP fragment encoding the amino-terminal domain of TSP2 (amino acids 1296). This fragment was then ligated into pBacPAK 9 (Clontech; Palo Alto, CA) to generate pBacPAK/mNT2. Bsu 36I-digested viral DNA (Clontech) and linearized pBacPAK/mNT2 were co-transfected into SF9 insect cells with transfection reagent (Clontech). Recombinant viruses were plaque-purified twice and titered according to the supplier's instructions.
Expression and Purification of Recombinant Protein
Production of the NH2-terminal fragment of TSP2 (NTSP2) was achieved by infection of serum-free SF9 cells at a multiplicity of infection of 10. Conditioned medium was supplemented with 100 ng/ml of phenylmethylsulfonyl fluoride and centrifuged at 1000 x g for 15 min at room temperature. To purify NTSP2, 200 ml of the centrifuged, conditioned medium was precipitated with ammonium sulfate. The 030% fraction was collected, resuspended in 0.1 M PBS, pH 7.4, containing 0.3 mM CaCl2 (PBSCa), and dialyzed at 4C for 24 hr against the same buffer, with one change of buffer at 12 hr. The solubilized protein was then applied to a heparinSepharose CL-6B column (Pharmacia; Piscataway, NJ) which had been equilibrated with PBSCa. After extensive washing, the heparin column was then eluted consecutively with PBSCa containing 250 mM, 400 mM, 600 mM, and 2 M NaCl. The 400 mM eluate contained the majority of the NTSP2 and the preparation appeared to be about 95% pure by SDS-PAGE. Further purification was achieved by application of the eluate to 1.5-mm-thick 12% SDS-polyacrylamide gels and excision of NTSP2 with the aid of guide lanes at the edges of the gel. The collected gel fragments were soaked in distilled H2O at 4C for 24 hr, minced into 1-mm2 pieces, and subjected to electroelution in a Biorad (Hercules, CA) electroeluter according to the manufacturer's instructions. Removal of SDS was achieved by electrodialysis during electroelution. The purified NTSP2 was shown to be pure by SDS-PAGE and migrated at the expected Mr of 32 kD. Purification of TSP2 was carried out essentially as described above, with the following modification. After elution from the heparinSepharose column with 400 mM NaCl, this TSP2-containing fraction was further purified by FPLC on a Superose-12 column (Pharmacia). The collected fractions were analyzed for the presence of TSP2 by Western blotting and were shown to be pure by SDS-PAGE.
Recombinant mouse TSP1, produced by insect cells, was a kind gift of Dr. D. Mosher. Human TSP1 was purified from platelets (Bornstein and Sage 1994
).
Production of Anti-NTSP-2 and Anti-TSP2 Antibodies
Polyclonal antibodies against mNTSP2 were produced in guinea pigs. Fifty µg of protein emulsified in complete Freund's adjuvant was administered by SC injection to each of two guinea pigs. After 3 weeks the guinea pigs were boosted with 50 µg of mNTSP2 emulsified in incomplete Freund's adjuvant, and after an additional week the animals were bled by cardiac puncture. The IgG fraction of serum was enriched by ammonium sulfate precipitation (040% fraction), followed by DEAE cellulose chromatography according to standard protocols. Small amounts of affinity-purified IgGs were generated by incubation of the antibody preparations with NTSP2 immobilized on nitrocellulose and elution with 0.1 M glycine-HCl, pH 2.6. The titers of antibody preparations were determined by Western blot analysis of known amounts of NTSP2 with differing antibody dilutions (Kyriakides et al. 1998
). Anti-TSP2 antibody, generated in rabbit, was a kind gift from Dr. D. Mosher. This antibody was titered as described above. The optimal titers for immunohistochemistry were determined empirically for each tissue.
Immunohistochemistry and Immunocytochemistry
Embryos and adult tissues were isolated, processed, and immunostained with biotin-conjugated secondary antibodies, ABC reagent, and DAB substrate, as previously described (Kyriakides et al. 1998
). Dilutions of primary antibodies varied and are indicated in the figure legends. Selected sections were also treated with TUF (tissue unmasking factor; Signet, Dedham, MA) for 10 min at 90C before blocking with 1% BSAPBS (see figure legends). In some cases this treatment permitted the use of higher primary antibody dilutions, thus reducing nonspecific background staining. For each specimen analyzed, controls included sections in which treatment with primary or secondary antibody, or both, was omitted. In addition, all specimens treated with anti-NTSP2 were also treated with preimmune serum instead of primary antibody. Preimmune serum from the same animal in which the anti-TSP2 antibody was produced was not available to us. To confirm the specificity of this antibody preparation, sections were therefore treated with primary antibody that was preincubated with purified TSP2 to block the specific activity of the antibody. The optimal ratio of protein to antibody was determined to be 5 µg of TSP2 per µg of antibody.
Skin fibroblasts were isolated by explant culture of biopsies taken from the backs of adult mice (Kyriakides et al. 1998
). Cells appeared to be purely fibroblastic after several passages. Cells obtained from confluent cultures were plated in four-chamber tissue culture slides (Nunc; Naperville, IL) at a concentration of 5 x 104 cells/ml. After 2 days in culture, the cells were fixed for 30 min in 10% formalin in saline. The slides were then incubated in 3% hydrogen peroxide/0.1% sodium azide in methanol to eliminate the activity of endogenous peroxidase, blocked with 1% BSAPBS, and sequentially incubated with anti-TSP-2 (1:1600), anti-rabbit IgGbiotin conjugate, ABC reagent, and DAB substrate. Three 5-min PBS washes were performed between each treatment. Controls were the same as described above for tissue sections. After visualization of the specific peroxidase acivity, the chamber slides were counterstained with methylene green for 30 sec and processed and mounted as described above. Examination was performed with the aid of a Nikon Eclipse 800 microscope. For protein extraction, confluent fibroblast cultures were rinsed extensively with PBS, trypsinized, centrifuged at 1000 rpm for 5 min, and washed three times with PBS. The pellets were dissolved in 1 x SDS-PAGE sample buffer, separated on a 7% polyacrylamide gel, and electroblotted to nitrocellulose for Western analysis.
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Results |
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Characterization of Antibodies
Purified mouse TSP2 and NTSP2 were subjected to electrophoresis on polyacrylamide gels and the gels stained with Coomassie Blue. Both proteins appeared to be pure by this criterion (Figure 1A). Western blot analysis indicated that anti-NTSP2 and anti-TSP2 reacted against NTSP2 and TSP2 but did not recognize TSP1 (Figure 1B and Figure 1C). The specificity of the anti-NTSP2 antibody was further shown by its failure to crossreact with other components in serum or in SF9 cell-conditioned medium (Figure 1B). Anti-TSP2 antibodies detected TSP2 synthesized by dermal fibroblasts cultured from skin of control mice but the protein could not be detected in fibroblasts derived from TSP2-null mice (Figure 1D). A similar observation was made with anti-NTSP2 antibodies (data not shown).

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Figure 1.
Purification of TSP2 and characterization of anti-TSP2 antibodies. (A) Coomassie Blue-stained acrylamide gel: Lane 1, TSP2 2.5 µg (7% gel); Lane 2, NTSP2 2.5 µg (12% gel). (B) Western blot of a 7% polyacrylamide gel with anti-NTSP2 (1:2000): Lane 1, hTSP1 1 µg; Lane 2, mouse serum 20 µg; Lane 3, SF9 cell-conditioned medium 10 µg; Lane 4, TSP2 0.5 µg; Lane 5, NTSP2 0.5 µg. (C) Western blot of a 7% polyacrylamide gel with anti-TSP2 (1:8000): Lane 1, TSP2 0.5 µg; Lane 2, mTSP1, 0.5 µg. (D) Western blot of a 7% polyacrylamide gel with anti-TSP2 (1:8000): Lane 1, total protein (50 µg) from confluent, passage-5 TSP2-null dermal cells; Lane 2, total protein (50 µg) from confluent, passage-5 WT dermal cells.
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Immunohistochemical Staining of Tissues from Day 15 Embryonic Mice
The distribution of TSP2 at Day 15 of murine embryonic development was compatible with its involvement in chondrogenesis and in the formation of other connective tissues. Immunoreactivity to TSP2 was evident in most proliferating chondrocytes and in some hypertrophic chondrocytes at this stage of development. TSP2 was observed in the majority of proliferating chondrocytes in the femoral head and acetabulum (Figure 2A). A similar section from a mutant embryo, treated similarly, confirmed the absence of TSP2 (Figure 2B). A cross-section through a developing rib showed the presence of TSP2 in hypertrophic chondrocytes and in the cells and connective tissue adjacent to the developing rib (Figure 2C). The perichondrium appeared devoid of TSP2 at this stage. Dense myotendinous tissue at the base of the skull of a wild-type embryo also stained positively for TSP2 (Figure 2D), whereas the same tissue was negative in a TSP2-null mouse (Figure 2E). No gross morphological abnormalities were observed in these tissues in TSP2-null mice. The reticular layer of fibroblast-like cells in the developing dermis in wild-type embryos was immunoreactive with anti-NTSP2 antibodies (Figure 2F). Corresponding sections from mutant embryos were negative (data not shown). Staining of the epidermis is nonspecific and was also observed in sections from mutant animals. Immunoreactivity to TSP2 was also evident in nasal septum, developing mandible and Meckel's cartilage, and heart. In addition, a weak signal was evident in the brain at this stage of development (data not shown).

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Figure 2.
Distribution of TSP2 in 15-day-old (AF) and 18-day-old (GO) mouse embryos. Paraffin sections of formalin-fixed embryos were exposed to either anti-NTSP2 or anti-TSP2 IgGs and specific immune complexes were visualized by the avidinbiotinperoxidase method. Methylene green was used as counterstain. Brown DAB reaction product indicates the presence of TSP2. (A) Proliferating chondrocytes in the femoral head and acetabulum of the coxal bone of a wild-type embryo; anti-NTSP2 (1:250). (B) The same region in a TSP2-null embryo shows no reaction product; anti-NTSP2 (1:250). (C) Hypertrophic chondrocytes (arrows) in a rib of a wild-type embryo; arrowheads denote the perichondrium; anti-TSP2 (1:800). (D) Developing myotendinous junction at the base of the skull of a wild-type embryo; anti-TSP2 (1:800). (E) The same tissue from a TSP2-null mouse; anti-TSP2 (1:800). (F) Cells of the reticular dermis (arrowheads) from a wild-type embryo are positive for TSP2; anti-NSTP2 (1:250). (G) Transverse section of a 18-day embryo. TSP2 is detected in the developing vertebrae (large arrowheads), iliac bone (small arrowhead), and mandible and Meckel's cartilage (arrow); anti-NTSP2 (1:500). (H) A similar section from a TSP2-null embryo is negative; anti-NTSP2 (1:500). (I) The intercellular junctions between chondrocytes within the basioccipital bone (arrowheads) are positive; anti-TSP2 (1:800). (J) TSP2 is detected in the perichondrium (arrowheads), and surrounding vasculature (arrows) of the basioccipital bone; anti-TSP2 (1:800). (K) A similar section from a TSP-2 null embryo shows only background immunoreactivity; anti-TSP-2 (1:800). (L,M) Developing scapula (L) and ulna (M) show TSP2 within the perichondrium/periosteum (arrows); anti-NTSP2 (1:500). (N) Nasal septum cartilage is strongly immunoreactive (arrowheads); anti-NTSP2 (1:250). (O) Cells of the developing reticular dermis (arrowheads), and perichondrium of developing rib (arrow) are also positive for TSP2; anti-TSP2 (1:1600). Sections in AK are sagittal. Sections in LO are transverse. Sections in C,G,H,L,M, and O were treated with TUF (see Materials and Methods). Bars: A,B,M = 100 µm; CF, JL, N,O = 50 µm; I = 50 µm; G,H = 2.5 mm.
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Immunohistochemical Staining of Tissues from Day 18 Embryonic Mice
By Day 18 the presence of TSP2 within developing cartilage, bone, and other connective tissues of wild-type embryos was evident (Figure 2G). Vertebral bodies displayed strong immunoreactivity in perichondria/periostea. Mutant embryos lacked specific immunoreactivity (Figure 2H). In areas of chondrogenesis, TSP2 was localized to the perichondrium, with relatively little staining in chondrocytes (Figure 2I, Figure 2L, and Figure 2M). Thus, the perichondria of developing bones such as basioccipital (Figure 2I), scapula (Figure 2L), and ulna (Figure 2M) stained positively for TSP2. As with Day 15 embryos, no morphological abnormalities were observed in these tissues. In the ulna, the reaction was concentrated in the perichondrium surrounding hypertrophic chondrocytes, whereas the membrane surrounding ossifying chondrocytes was negative (Figure 2M). On the contrary, in the scapula, immunoreactivity was increased in the membrane surrounding ossifying chondrocytes (Figure 2L). Hypertrophic chondrocytes appeared to stain weakly positive for TSP2 (data not shown). Low levels of immunoreactivity for TSP2 were also observed in areas of endochondral ossification (data not shown ). In the basioccipital bone, immunoreactivity for TSP2 also appeared at intercellular chondrocyte junctions (Figure 2I) and in adjacent blood vessels (Figure 2J). However, some blood vessels were not immunoreactive (Figure 2J); this observation was also made in several other tissues (data not shown). A section from the basioccipital bone of a TSP2-null embryo lacked immunoreactivity for TSP2 (Figure 2K). Forming cartilage, such as nasal septum, displayed strong immunoreactivity (Figure 2N), as did most cartilaginous regions of the head (data not shown). TSP2 was also evident in cells of the reticular dermis (Figure 2O). The stain observed in the epidermis is nonspecific and was observed in sections from mutant embryos (data not shown).
Immunohistochemical Staining of Tissues and Cells from Adult Mice
The distribution of TSP2 in tissues and cells from 3-month-old mice was investigated. In addition, skin sections from 3-week-old mice were examined. Of all the tissues tested, only skin, cartilage, testis, adrenal, and brain displayed specific immunoreactivity for TSP2. Several other tissues, such as kidney, heart, and lung, were immunoreactive for TSP2, but interpretation of the results was hampered by high background staining of mutant tissues. Tissues that required special processing, such as bone, were not examined. Several attempts were made to immunolocalize TSP2 to collagen fibers in connective tissues such as skin and tendon, but the presence of TSP2 as a constituent of these fibers could not be documented. As was reported previously (Kyriakides et al. 1998
), immunostaining of adult skin sections proved to be inconclusive. The staining was repeated with tissues from 3-week-old mice treated with TUF (see Materials and Methods), but immunoreactivity for TSP2 still appeared to be cell-associated (Figure 3A). In tail tendon, both cells and collagen fibers lacked immunoreactivity with anti-TSP2 antibodies (Figure 3B). The staining of sweat glands and hair follicles in Figure 3A is nonspecific. At an osteotendinous junction in tail, chondrocytes were weakly immunoreactive for TSP2 (Figure 3C). Cultured skin fibroblasts from wild-type animals were strongly positive for TSP2 (Figure 3D): TSP2-null fibroblasts, cultured and stained in the same manner, showed no immunoreactivity (Figure 3E). In the adrenal cortex, TSP2 was present exclusively within the cortex and mostly in the fasciculata and reticularis zones (Figure 3F). The presence of TSP2 in the testis was limited to interstitial Leidig cells (Figure 3G). In brain, TSP2 was detected in the Purkinje cells of the cerebellar cortex (Figure 3H). No staining was observed in Purkinje cells of TSP2-null animals (Figure 3J). With the exception of the dermis (Kyriakides et al. 1998
), no significant differences in morphology at the light microscopic level were observed in tissues of mutant animals.

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Figure 3.
Distribution of TSP2 in adult mouse tissues. Paraffin sections of formalin-fixed tissues were exposed to either anti-NTSP2 or anti-TSP2 IgGs. Sections were counterstained with methylene green. (A,B) TSP2 is present in dermal fibroblasts (arrowheads, A) but not in tendon fibroblasts (arrowhead, B); anti-NTSP-2 (1:250). (C) Chondrocytes in an osteotendinous junction show a positive reaction for TSP2 (arrowheads); anti-NTSP2 (1:250). (D,E) Cultured dermal fibroblasts from wild-type mice (D) are positive, whereas those from TSP2-null embryos (E) are negative for TSP2; anti-TSP2 (1:1600). (F) Sections of adrenal gland show a positive reaction for TSP2 in the cortex (c) but not in the medulla (m); anti-TSP2 (1:1600). (G) A strong reaction for TSP2 is observed in Leidig cells in the interstitium of the testis (arrowheads); anti-NTSP2 (1:500). (H,I) Purkinje cells in the cerebellum stain positively for TSP2 (arrowheads, H). The same cells in TSP-2-null embryos are negative (arrowheads, I); anti-NTSP2 (1:500). Sections shown in F,G,H, and I were treated with TUF (see Materials and Methods). Bars: A,D,E,H,I = 50 µm; B,C,G = 50 µm; F = 100 µm.
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Discussion |
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The studies presented here document the presence of TSP2 in developing dermis, cartilage, bone, and blood vessels of the mouse embryo, findings that are consistent with the phenotype of the TSP2 knockout mouse (Kyriakides et al. 1998
). In the adult mouse TSP2 was also detected in brain, testis, and adrenal gland. These tissues have thus far not been found to be affected in the TSP2-null mouse, and therefore the function of TSP2 in the latter organs, if any, remains uncertain. Similar distributions for TSP2 mRNA were described in the mouse and chick embryo, as shown by in situ hybridization with TSP2-specific probes (Iruela-Arispe et al. 1993
; Tucker 1993
; Tucker et al. 1995
). In addition, Iruela-Arispe et al. 1993
detected mRNA for TSP2 in meninges, brain, skeletal muscle, kidney, and gut. In keeping with our detection of TSP2 in the adrenal cortex, bovine adrenocortical cells were shown to express TSP2 in culture (Pellerin et al. 1993
). Furthermore, adrenocortical cells could be induced to overexpress TSP2 by treatment with ACTH (Lafeuillade et al. 1996
) or TGF-ß1 (Negoescu et al. 1995
).
The similarities among the TSPs in the amino acid sequence of some domains, especially between TSP1 and TSP2, complicate the preparation of monospeci-fic antibodies. Therefore, although antibodies against platelet TSP1 were employed in the analysis of the distribution of this protein during mouse embryonic development (O'Shea and Dixit 1988
), subsequent examination of the distribution of the transcripts for TSP1, 2, and 3 (Iruela-Arispe et al. 1993
) and the use of more specific antibodies to TSP1 (Corless et al. 1992
) suggested that the earlier immunohistochemical study had been unable to distinguish between TSP1 and TSP2 in some tissues. We utilized two different antibodies with specificity for TSP2 in the immunohistochemical analyses reported in this work, and each localization presented in Figure 2 and Figure 3 was verified by use of a second antibody. Similarly treated tissue sections derived from TSP2-null animals served as negative controls. In addition, we employed purified recombinant mouse TSP2 to preadsorb our antibodies and to determine that immunoreactivity was abolished. The pattern of deposition of TSP2 that we observed showed little or no overlap with the distribution of Thbs1 transcripts when both temporal and spatial differences were considered (Iruela-Arispe et al. 1993
). Taken together with our analysis of the specificity of our antibodies on Western blots, these controls validate our immunohistochemical data.
In mouse embryos, the most pronounced immunoreactivity towards TSP2 was observed in tissues of mesenchymal origin. At Day 15 the myotendinous junction was immunoreactive. TSP2 was associated with both cellular and fibrillar components. Areas of chondrogenesis were also immunoreactive at Days 15 and 18 of embryonic development. At Day 15 we observed TSP2 in chondrocytes and in the pericellular space. Chondrocytes at different stages of differentiation were immunoreactive. The perichondrium did not stain positively for TSP2, a finding that does not agree with the distribution of Thbs2 transcripts (Iruela-Arispe et al. 1993
). By Day 18, however, TSP2 appeared predominantly in the perichondrium/periosteum and in ossification centers, findings that are consistent with the previously described expression of transcripts. Staining of the areas of the perichondrium/periosteum that appeared immunoreactive for TSP2 with hematoxylineosin or with Verhoeffvan Gieson stains suggested that TSP2 was associated with an acellular collagenous matrix (unpublished observations). In the developing cartilage of the nasal septum, which is not programmed to undergo ossification, TSP2 was deposited in a bilaterally symmetrical linear array. Some Day 18 hypertrophic chondrocytes were faintly immunoreactive (data not shown). Interestingly, a weak signal for the transcript was observed in proliferating but not in hypertrophic chondrocytes in embryos of approximately the same age (Iruela-Arispe et al. 1993
). The basis for this difference is not known. Our inability to detect TSP2 in some areas that were shown to express TSP2 transcripts could be due to translational control or to the more limited sensitivity of protein detection. Alternatively, minor errors in assessing the age of mouse embryos may contribute to this apparent discrepancy. However, it should be emphasized that, despite such minor differences in the localization of protein and transcript, the overall spatial and temporal distribution of TSP2 is consistent with that of its transcript. Our observations suggest that the synthesis of TSP2 by chondrocytes is more closely correlated with the developmental stage of the tissue than with the state of differentiation of the cells.
The effect, if any, of a lack of TSP2 during chondrogenesis is not known. TSP2-null mice display increased cortical thickness and density of long bones, findings that may be the result of abnormal bone development (Kyriakides et al. 1998
). However, sections of developing cartilage from TSP2-null embryos appeared histologically normal. Therefore, postnatal bone formation may be abnormal in mice lacking TSP2.
Skin sections were also immunoreactive for TSP2 at Days 15 and 18, with the majority of the stain concentrated in the reticular dermis. Our observations are consistent with the expression of the transcript observed by Iruela-Arispe et al. 1993
. The reaction product appeared to be associated with cells that assumed an ordered arrangement in a more or less linear fashion; a similar pattern was observed for the transcript. Because dermal collagen fibers at these stages of development are not well defined, it was difficult to evaluate a possible association of TSP2 with the extracellular matrix. Previously, we had also attempted to immunolocalize TSP2 in adult (3-month-old) dermal collagen fibers without success (Kyriakides et al. 1998
). We therefore obtained dermis from 3-week-old mice to test for the possible presence of TSP2 in collagen fibers that are undergoing active remodeling. To improve the immunolocalization of TSP2, we employed a tissue unmasking factor that enabled us to use higher antibody dilutions with a reduced background. A similar antigen recovery method had been used to study the tissue distribution of TSP1 (Grossfeld et al. 1996
). Employing this method, we observed a marked improvement in the detection of cell-associated immunoreactivity. However, collagen fibers remained nonimmunoreactive for TSP2, suggesting either that the protein is not a structural component of the fibrils or that it is present at levels that are below the sensitivity of our detection method. Nevertheless, the overall distribution of TSP2 at Days 15 and 18 of embryonic development indicates that, although this protein is present in and around cells, it is also associated with extracellular matrices. Thus, immunoreactivity was present in acellular collagenous structures such as the perichondrium, as well as in developing cellular structures such as the nasal septum.
As in the case of collagen fibers in adult skin, fibers in adult tendon failed to display immunoreactivity for TSP2. In addition, tendon fibroblasts did not appear to be immunoreactive. The apparent lack of TSP2 in tendon fibroblasts may reflect the synthetic quiescence of these cells in the absence of appreciable growth or tissue repair. Embryonic tendons, which are more cellular and are growing rapidly, display high levels of Thbs2 transcript (Iruela-Arispe et al. 1993
; Tucker et al. 1995
). It is unclear at what stage in their development tendon fibroblasts cease their synthesis of TSP2. We are now analyzing the distribution of TSP2 in tendons of mice of various ages by both light and electron microscopy. The developmental program for expression of TSP2 in tendon is of interest because tendons in TSP2-null mice display irregular collagen fibrils. Interestingly, articular chondrocytes at tendonbone junctions in tail stained weakly for TSP2. These cells are known to persist throughout adulthood in a stable differentiated state, thus avoiding the fate of chondrocytes in ossifying cartilage (Pacifici 1995
). It is not yet known whether TSP2 is expressed in articular chondrocytes throughout development as well as in the mature tissue.
The inconsistent presence of TSP2 in the vascular endothelium, and in the vascular wall in general, is puzzling. During embryonic development, some but not all blood vessels are immunoreactive for TSP2. It has been shown that TSP2 can inhibit the bFGF-induced neovascularization of the rat cornea and block migration of capillary endothelial cells, findings suggesting that the protein has direct effects on the behavior of endothelial cells (Volpert et al. 1995
). We now know that TSP2-null mice have an increase in vascular density, a finding that implicates TSP2 as a potent inhibitor of angiogenesis (Kyriakides et al. 1998
). However, our observations of the distribution of TSP2 in blood vessels during murine development do not clarify the basis for the antiangiogenic properties of TSP2. Iruela-Arispe et al. 1993
were able to detect Thbs2 transcripts in meningeal capillaries at Day 12 of embryonic development and in the endothelium of larger blood vessels in neonatal animals. Perhaps analysis of younger embryos (Days 1014) or of neonatal animals may prove more informative in determining whether TSP2 acts directly on endothelial cells to regulate angiogenesis.
The location of TSP2 within adult tissues was shown to be predominantly cell-associated. Cell types such as dermal fibroblasts, adrenocortical cells, chondrocytes, and Purkinje and Liedig cells were immunoreactive. On the other hand, collagenous matrices in dermis, tendon, and cornea (data not shown) were examined and were shown to display no immunoreactivity towards TSP2. If TSP2 does not participate as a structural component in the formation of collagen fibrils, what then is the basis for the generation of irregular and abnormal collagen fibrils observed in TSP2-null mice? Our results demonstrate the ability of dermal fibroblasts to synthesize TSP2 in vitro and to be immunoreactive for TSP2 in vivo. We suggest, as one possibility, that the lack of TSP2 could influence the ability of collagen-synthesizing fibroblasts to interact appropriately with growing collagen fibrils and thus to properly compartmentalize the pericellular space in which these fibrils are believed to develop (Birk and Trelstad 1986
; Birk and Linsenmayer 1994
). This idea is supported by the adhesion defects that were observed in dermal fibroblasts derived from TSP2-null animals (Kyriakides et al. 1998
), and with the defective ability of these cells to contract a collagen gel (our unpublished observations). The consequences of a lack of TSP2 for the brain, testis, or adrenal glands remain to be determined. A role for TSP2 in the function of the adrenal gland is suggested by the observation that, in bovine adrenocortical cells in culture, exposure to ACTH leads to upregulation of both Thbs2 mRNA and TSP2 protein (Lafeuillade et al. 1996
).
In conclusion, our results are consistent with a role for TSP2 in the development of connective tissues during embryogenesis. In accord with a matricellular hypothesis for the function of TSP2 and functionally related proteins (Bornstein 1995
), the absence of TSP2 in TSP2-null mice does not appear to lead to serious structural defects during organogenesis. Rather, a primary role for TSP2 as a modulator of cellmatrix interactions is reflected in the largely cellular and pericellular distribution of the protein. TSP2 was found to be associated with collagenous matrices during the rapid growth of tissues that accompanies embryonic development. However, no clear structural function can be attributed to this matrix-associated protein, and it may serve as a reservoir for vicinal cellular activity. The lack of structural changes at the light microscopic level in embryonic tissues such as skin and tendon may simply reflect the rudimentary state of development of collagenous matrices in these tissues. In the adult animal, TSP2 appeared to be associated almost exclusively with cells and with their pericellular environment. In these locations TSP2 may serve the cellular functions required by the slow growth of mice, even during adulthood, and the small degree of tissue repair that follows minor injuries. However, the possibility that TSP2 does participate as a structural component of adult connective tissue matrices but in locations that are inaccessible to antibodies, or at levels below those that are detectable, cannot be excluded. We are currently analyzing a number of adult tissues by immunogold electron microscopy with anti-TSP2 antibodies to determine whether the protein can be detected by this more sensitive method.
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Acknowledgments |
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Supported by NIH grants P01 HL 18645 and P30 DK 17047 and by NSF grant ECC 9529161.
We thank Kim Yeargin for technical assistance, Dr Lynne Smith for advice on immunohistochemistry, and Dr Deane Mosher for providing critical reagents. The Diabetes and Endocrinology Research Center at the University of Washington provided assistance with embedding and sectioning of tissues.
Received for publication February 9, 1998; accepted May 5, 1998.
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