Chondroitin sulfate proteoglycans are a diverse group of extracellular matrix (ECM) proteins proposed to have numerous roles in matrix organization, growth, migration, and differentiation during embryonic development. The importance of chondroitin sulfate proteoglycans during early developmental processes has been underscored by recent studies which identified an insertional mutation in the versican gene in the heart defect mouse (hdf) (Mjaatvedt et al., 1998). This defect was demonstrated to be embryonic-lethal in homozygous mutants, resulting in severe malformations in the developing heart as well as in other embryonic tissues (Yamamura et al., 1997). Many of the proposed functions of chondroitin sulfate proteoglycans have been attributed to their glycosaminoglycan (GAG) chains (Kjellen and Lindahl, 1991; Iozzo, 1998). The importance of GAGs to critical morphogenetic processes has been demonstrated recently in the zebrafish by the jekyll mutation, which results in severe disruption of cardiac valve formation (Walsh and Stanier, 2001). This mutation was caused by a defect in the gene encoding UDP-glucose dehydrogenase, an enzyme necessary for synthesis of the glucuronic acid moiety found in hyaluronic acid, heparan sulfate, and chondroitin sulfate GAGs (Lander and Selleck, 2000). In addition to important contributions to cardiac valve structure, morphological and biochemical studies in the teleost have suggested that GAGs perform an array of functions ranging from serving as a nutrition source (Pfeiler, 1996, 1998; Bishop et al., 2000), protecting the cardiac endocardium (Icardo et al., 1999), or assisting in buoyancy (Eastman et al., 1994) in the extreme environment of the Antarctic.
Although chondroitin sulfate GAGs have been implicated in numerous functions in the teleost during early development and in the adult, little is known about the precise localization of chondroitin sulfate GAGs at any age or in any tissue. In the present study we utilized immunofluorescence microscopy to evaluate the localization of chondroitin sulfate GAGs during embryonic and early larval development of a perciform fish, the striped bass (Morone saxatilis). The order Perciformes represents the most speciated vertebrate order, comprising nearly half of all fish species. This order is also closely allied with other recently evolved orders, including the Tetradontiformes and Gasterosteiformes. The striped bass is also an important commercial and recreational fishery resource of the Atlantic coast, and much effort has been devoted to management and maintenance of this species through both private and governmental efforts. Because of the important economic impact of this fishery to the Atlantic coastal region, aquacultural efforts directed toward the spawning and rearing of striped bass have increased during recent years. Surprisingly, despite this increased attention few developmental studies have been directed toward understanding important morphological events in the early development of the striped bass—an effort that could provide insight into improved strategies for aquacultural rearing of this important species.
In the present study three antibodies were utilized that recognize native epitopes on chondroitin sulfate GAG chains: 1) TC2 (Capehart et al., 1999) and 2) d1C4 (Capehart et al., 1997), which recognize, respectively, epitopes on GAGs enriched in chondroitin-4- and chondroitin-6-sulfate; and 3) CS-56 (Avnur and Geiger, 1984), which binds a different epitope on both chondroitin-4- and chondroitin-6-sulfate chains. Our results outline major sites of immunoreactivity with these three antibodies and demonstrate codistribution of chondroitin sulfate GAG epitopes in the ECM at some sites, but a striking heterogeneity of chondroitin sulfate GAG localization in several developing organs, including the eye and heart. This heterogeneity of chondroitin sulfate GAG expression correlated spatially and temporally with important events during organogenesis and maturation, suggesting that modulation of chondroitin sulfate GAGs in developing tissues is important in the regulation of proteoglycan function.
MATERIALS AND METHODS
Animals and Tissue Preparation
Morone saxatilis specimens were generously provided by the North Carolina Wildlife Resources Commission, Watha State Fish Hatchery, Watha, NC. Embryos and larvae were fixed at various time points in 3.7% formaldehyde in 85% ethanol with 5% glacial acetic acid (Wilkinson, 1992) and maintained in fixative at 4°C for 1–2 weeks. Embryos between 18–48 hr postfertilization were dechorionated. Dechorionated embryos and hatched larvae were transferred into methanol and stored at 4°C until they were processed for immunohistochemistry.
Monoclonal antibody (MAb) TC2 was produced by in vitro immunization of naïve mouse splenocytes with a peanut agglutinin-positive fraction from prechondrogenic micromass cultures of chick limb mesenchyme (Capehart et al., 1999). It recognizes a native epitope on GAGs enriched in chondroitin-4-sulfate (bovine trachea; Sigma Chemical Co., St. Louis, MO). MAb d1C4 was produced by in vitro immunization by direct exposure of naïve mouse splenocytes to unfixed, air-dried prechondrogenic micromass cultures of chick limb mesenchyme (Capehart et al., 1997). MAb d1C4 is reactive with a native epitope on chondroitin sulfate GAG chains enriched in chondroitin-6-sulfate (shark cartilage; Sigma). MAbs TC2 and d1C4 were utilized as hybridoma supernatants. MAb CS-56, which recognizes a native epitope on both chondroitin -4- and -6-sulfate chains (Avnur and Geiger, 1984), was obtained commercially as an ascites fluid (Sigma). All antibodies utilized were of the IgM class.
Fixed, dechorionated embryos were embedded in paraffin and sectioned at 7 μm. Deparaffinized sections were rehydrated through graded ethanols to phosphate-buffered saline (PBS) and blocked for 1 hr at room temperature with PBS containing 3% bovine serum albumin (BSA; Sigma) and 1% normal goat serum (NGS; Sigma). Sections were incubated with primary antibodies overnight at 4°C in a humidified chamber. Primary antibodies were diluted in blocking buffer as follows: TC2 1:50; d1C4 1:10; CS-56 1:4,000. Following primary antibody incubations, specimens were washed four times with PBS and incubated 2 hr at room temperature with fluorescein-conjugated goat anti-mouse IgM secondary antibody (Cappel, ICN Biomedicals, Aurora, OH) diluted 1:200 in blocking buffer. All samples were washed five times with PBS and postfixed in 80% and 50% ethanols (5 min each). Sections were equilibrated in PBS and mounted in 10% PBS-90% glycerol containing 100 mg/ml 1,4-diazabicyclo(2,2,2)octane (DABCO; Sigma) (Johnson et al., 1982).
Specimens utilized for whole-mount immunofluorescence labeling were rehydrated to PBS and blocked with PBS-3% BSA-1% NGS overnight at 4°C. Embryos were incubated with undiluted TC2 hybridoma supernatant containing 1% dimethylsulfoxide overnight at 4°C and washed thoroughly with PBS containing 1% BSA. Samples were incubated overnight at 4°C with FITC-conjugated goat anti-mouse IgM diluted 1:200 in blocking buffer, washed four times with PBS-1% BSA, once with PBS alone, and postfixed in 80% and 50% ethanols (30 min each). Samples were equilibrated in PBS and mounted in DABCO.
Controls for immunohistochemistry included omission of the primary antibody or use of an irrelevant IgM for the primary antibody incubation. To check for possible masking of epitopes by hyaluronic acid, in selected experiments deparaffinized tissue sections were incubated 1.5 hr at 37°C in 20 U/ml Streptomyces hyaluronidase (Sigma) in 30 mM sodium acetate, pH 5.2, containing 125 mM sodium chloride (Linhardt, 1994). Control sections were incubated in acetate buffer alone. Specimens were viewed and photographed with an Olympus BX-40 photomicroscope equipped with epifluorescence optics.
While genetic, biochemical, and morphological studies have suggested important functions for chondroitin sulfate GAGs in the teleost, little has been done to examine the precise localization of specific chondroitin sulfate GAG epitopes during their early development. Utilizing three antibodies that recognize different native epitopes on chondroitin sulfate GAG chains, the present study demonstrated a striking heterogeneity of chondroitin sulfate epitope localization during embryonic and early larval development of the striped bass (Morone saxatilis). Chondroitin sulfate GAGs are comprised of repeating disaccharide units consisting of a glucuronic acid-N-acetylgalactosamine backbone; they often vary in length and carbohydrate substitution, as well as in pattern and degree of sulfation. Many of the functions of proteoglycans are due to their associated GAG chains (Kjellen and Lindahl, 1991; Iozzo, 1998), and structural differences in individual chondroitin sulfate GAG chains may facilitate diverse activities for their associated proteoglycans. Such modifications of chondroitin sulfate GAGs are carefully regulated through tissue-specific or developmental mechanisms (Caterson et al., 1990; Sorrell et al., 1990, 1993; Fernandez-Teran et al., 1993; Nadanaka et al., 1998). Because of possible structural modifications in an individual CS-GAG chain, antigenic determinants are generated that may be quite different from the repetitive backbone disaccharide structure. While the precise epitopes recognized by the d1C4 and TC2 antibodies utilized in this study are not known at this time, it is clear from previous studies (Capehart et al., 1997, 1999) that they reside on native CS-GAG chains enriched in either chondroitin-4- (TC2) or chondroitin-6-sulfate chains (d1C4). The CS-56 epitope may be found on both chondroitin-4- and –6-sulfate chains (Avnur and Geiger, 1984), suggesting strongly that the determinant recognized by this antibody is a structural variation of the usual backbone of CS-GAG chains. Chondroitin sulfate chains on a given proteoglycan core protein may vary according to tissue type or temporal expression, hence it is important to note that immunoreactivity with the antibodies utilized in the present study at any specific site in developing striped bass does not imply exclusive association with any particular proteoglycan.
Hyaluronic acid may interact with and help organize other matrix components (such as the proteoglycans), hydrate the ECM, and assist other cellular functions (such as migration). In the present study, Streptomyces hyaluronidase pretreatment of tissue sections to remove hyaluronic acid from the ECM was often observed to enhance immunoreactivity slightly; however, the overall pattern of antibody localization remained unchanged. This observation suggests that hyaluronic acid is a component of the ECM in many tissues of the developing striped bass, but that differences in immunolocalization of the d1C4, TC2, and CS-56 epitopes are not due to masking by this specific molecule.
Immunolocalization of the CS-56 antigen is first noted during somitogenesis in the developing striped bass and is confined to the ECM in close proximity to the notochord. MAb d1C4 staining is also observed in the perinotochordal sheath at the hatching stage, by which time a full complement of somites (27) have formed and myotomes are evident (Mansueti, 1958) (Scemama et al., unpublished results). At the hatching stage, a period characterized by only sporadic swimming efforts (Mansueti, 1958), staining with both antibodies extends from the notochord into the fibrous myosepta. Myoseptal staining decreases by the early larval stage. In the early larva, striated skeletal muscle fibers are clearly evident (personal observation) and frequent swimming movements can be observed (Mansueti, 1958). A previous study of axial malformations in sea bream larvae (Santamaria et al., 1994) demonstrated that lordosis in the teleost often appears prior to development of the vertebral column, and is marked by irregularities in the notochord and disorganization of both muscle bundles and perinotochordal tissue. These authors also noted reduced interactions between collagen and proteoglycans in lordotic animals. Our results show that chondroitin sulfate GAGs are indeed present around the notochord and in the myosepta in a temporal sequence that suggests a possible role in maintenance of axial integrity and myomere organization during early development of the striped bass.
Differences in localization of CS-56 and d1C4 epitopes were evident around the otic vesicle by the hatching stage. CS-56 stained in an intermittent pattern around the otic vesicle, while d1C4 was localized within the discrete region of contact between the otic vesicle and neural tube. In the chick, the neural ectoderm becomes attached to the invaginating otic placode through fusion of basement membrane components. This interaction has been suggested to facilitate subsequent otic morphogenesis by providing a controlled anchorage point for the otic epithelium (Hilfer and Randolph, 1993). Chondroitin sulfate GAGs have also been demonstrated to play an important role during chick otocyst formation (Gerchman et al., 1995). In this study, depletion of chondroitin sulfate GAGs disrupted proper invagination of the otic primordium, suggesting that increasing levels of these GAGs in surrounding mesenchymal tissue assisted proper folding of the otic pit. Immunolocalization of the d1C4 epitope at sites of contact between the neural tube and otic vesicle noted in the present study suggests that chondroitin sulfate GAGs may also play a role in interaction between the neural tube and otic vesicle during otic development in the striped bass.
By the early larval stage, CS-56 and d1C4 antibody localization codistributed in nascent cartilage tissues of the developing splanchnocranium. Little or no TC2 staining was noted in early cartilages; however, TC2, d1C4, and CS-56 staining of more mature cartilages in 9-day-old larvae was observed (not shown). These results suggest that incipient cartilages of the splanchnocranium and chondrocranium in the striped bass are enriched in GAGs bearing chondroitin-6-sulfate moieties. Similarities in distribution of d1C4 and CS-56 epitopes were also apparent in basement membranes around the developing alimentary canal, but notable differences were observed in other basement membranes, particularly those associated with the branchial arches. CS-56 stained basement membranes of branchial arches at the hatching stage, while little d1C4 reactivity was observed at these sites. CS-56 staining was particularly intense in the epidermal basement membrane and underlying mesenchyme of the second arch in the vicinity of future opercular formation. In another location in the early larva, CS-56 was also reactive with the basement membrane underlying the apical ectodermal ridge of the newly forming pectoral fin bud. As chondroitin sulfate GAGs have been implicated in the sequestration and presentation of various growth factors, it is interesting to speculate that the CS-56 antigen might participate in regulation of molecular traffic between the epidermis and mesenchyme at these sites of important epithelial-mesenchymal interactions.
Heterogeneous expression of d1C4 and CS-56 epitopes was also noted in the eye of hatching embryos and early larvae. CS-56 localized primarily around the lens capsule and staining was most intense between the lens and neural retina. MAb d1C4 stained the lens, but also localized as distinctly immunoreactive bands within the neural retina. With further retinal development at the early larval stage, the d1C4-positive regions could be identified as inner and outer plexiform layers. Chondroitin sulfate proteoglycans have been localized to plexiform layers during development of the eye in both chick (Zako et al., 1997; Li et al., 2000) and rat (Inatani et al., 2000). It has been proposed that these proteoglycans function in regulation of neurite outgrowth and cellular organization within the retina. Molecules bearing GAG epitopes have also been hypothesized to facilitate other functions during ocular development, such as separation of the lens vesicle from the prospective corneal epithelium (Mizuno et al., 1995) and stabilization of the ciliary body and retina (Uusitalo and Kivela, 2001). Interestingly, chondroitin-6-sulfotransferase is expressed strongly in the mouse eye (Uchimura et al., 1998), suggesting that chondroitin-6-sulfate GAGs are important constituents of proteoglycans in the eye in that species. Localization of the d1C4 epitope, which is found on GAG chains enriched in chondroitin-6-sulfate, in the plexiform layers in the neural retina of the developing striped bass suggests that this antigen may also assist neurite patterning and organization in this teleost.
At the hatching embryo and early larval stages, the TC2 antibody localized primarily in tissues of the developing heart. The developing teleost heart is comprised of segments including the sinus venosus, atrium, ventricle, and conus arteriosus (conotruncus). Because similar structures are conserved in the primitive heart tube of higher vertebrates, the fish heart is considered a useful model for the study of vertebrate heart development (Munoz- Chapuli et al., 1994; Thisse and Zon, 2002). Like the bird and mammal, the fish heart develops initially as an outer myocardial epithelium surrounding an inner endocardium (Munoz-Chapuli et al., 1994), and is separated from it by an expanded ECM, the cardiac jelly (Davis, 1924). The cardiac jelly contains many molecules typically associated with basement membranes and is derived in large part from contributions from both the myocardium and endocardium (Kitten et al., 1987). In the inlet region of the heart, TC2 immunoreactivity was observed within the cardiac jelly lining the forming sinoatrial valve leaflets. In the elasmobranch, these valves form from lateral infoldings of the myocardial wall and, unlike their counterparts in many other species, remain an important mechanism for direction of blood flow into the atrium (Gallegro et al., 1997). A relatively thin cardiac jelly matrix lines the sinoatrial valves, and histochemical evidence suggests that sulfated glycosaminoglycans are localized in this region (Gallegro et al., 1997). Our results also illustrate heterogeneity in cardiac matrix organization in this region of the developing heart, and suggest that the TC2 antigen is one component of the matrix associated with the forming sinoatrial valves. Little or no staining with d1C4 or CS-56 antibodies was observed in this sinoatrial location.
As noted in the developing chick heart tube (Capehart et al., 1999), TC2 stains the cardiac jelly of the striped bass continuously from the atrioventricular through the conotruncal segments. Formation of the vertebrate heart tube involves recruitment of the primitive segments (de la Cruz, 1989), but questions remain about the origin of the conotruncus. Markwald et al. (1998) and Mjaatvedt et al. (1998) suggested that this portion of the primitive heart tube is derived from incorporation of additional anterior extracardiac splanchnic mesoderm. Strong localization of TC2 staining in this region of the heart tube suggests that this chondroitin sulfate GAG epitope is an important component of the cardiac matrix during formation and maturation of the conotruncal segment. In the chick, the atrioventricular and conal cushions form from regional expansions of the cardiac ECM that serve as primitive valves and are later populated by cells arising from an epithelial to mesenchymal transformation (Markwald et al., 1975). In the fish, the atrioventricular and conal cushions form by a similar mechanism and also contribute to the structure of the mature valves (Munoz-Chapuli et al., 1994).
While chondroitin sulfate GAGs have long been recognized as constituents of the cardiac matrix in the vertebrate heart tube, little has been done to examine the heterogeneity of chondroitin sulfate GAG expression in the different cardiac segments. In agreement with previous findings in the early heart tube of the chick (Capehart et al., 1999), in the present study chondroitin sulfate GAGs were localized differently and a similar localization of TC2 epitopes was conserved. The precise function(s) of proteoglycans bearing chondroitin sulfate GAG chains in the developing vertebrate heart is not known, but numerous roles have been suggested, including organization and hydration of the ECM, sequestration and presentation of growth factors, and facilitation or inhibition of cellular migration (Markwald et al., 1978; Funderburg and Markwald, 1986; Mjaatvedt et al., 1998; Capehart et al., 1999; Zanin et al., 1999). Characterization of the jekyll mutation in zebrafish as a defect in UDP-glucose dehydrogenase provided direct evidence that GAGs are required for proper formation of atrioventricular cushion tissues in the teleost (Walsh and Stanier, 2001). The importance of the chondroitin sulfate proteoglycans to cardiac morphogenesis was also demonstrated recently by a transgenic mouse model, hdf (Mjaatvedt et al., 1998). The hdf mouse bears an insertional mutation of the gene encoding the core protein of the large chondroitin sulfate proteoglycan, versican, and homozygous hdf embryos die of cardiac defects in utero at approximately 10.5 days postcoitum (Yamamura et al., 1997). The conotruncus and endocardial cushions are absent in homozygous hdf embryos, emphasizing the importance of the versican proteoglycan in morphogenesis of these heart segments. Interestingly, TC2 stained cardiac jelly of the heart tube in wild-type mice at 8.5–9.0 days postcoitum, but was absent from the heart tube of homozygous hdf mice, suggesting that the TC2 epitope may be found on versican in the embryonic mouse heart (Mjaatvedt et al., 1998). Colocalization of TC2 and anti-versican immunoreactivity was also observed during early stages of chick heart development (Capehart et al., 1999). Both the hdf and jekyll mutant models provide dramatic evidence that proteoglycans such as versican and their associated GAGs are critical to vertebrate cardiac development. Conservation of TC2 epitope localization in the cardiac jelly of specific segments of the developing heart in the bird, mammal, and fish suggests that this chondroitin sulfate antigen plays an important role in vertebrate heart development.
The present study has demonstrated a striking heterogeneity in localization of chondroitin sulfate GAG epitopes during early development of the striped bass. The expression of specific GAG sequences detected by the TC2, d1C4, and CS-56 antibodies in specific regions of the embryo and larva during organogenesis lends support to the hypothesis that modifications of the chondroitin sulfate GAG backbone structure are utilized by the teleost to modulate proteoglycan function during morphogenesis. At the present time it is unknown whether the native epitopes recognized by the three MAbs are found on multiple proteoglycans or whether there are changes in GAG substitution on a single chondroitin sulfate proteoglycan. Colocalization of CS-56 and d1C4 reactivity at several embryonic sites at least suggests the possibility that these epitopes may be found on the same GAG chain in some tissues. Experiments are under way to identify the proteoglycan species that bear these chondroitin sulfate GAG epitopes during development of the striped bass.