Heterogeneity of chondroitin sulfate glycosaminoglycan localization during early development of the striped bass (Morone saxatilis)

Authors


Abstract

Recent studies have suggested important functions for proteoglycan-associated chondroitin sulfate glycosaminoglycans (GAGs) during embryonic and larval development in numerous organisms, including the teleost. Little is known, however, about the specific distribution of different chondroitin sulfate GAGs during early development. The present study utilized immunohistochemistry to localize chondroitin sulfate GAG antigens during development of the striped bass (Morone saxatilis). Immunoreagents utilized were monoclonal antibodies (MAbs) TC2, d1C4, and CS-56, which recognize, respectively, native epitopes on glycosaminoglycan chains enriched in chondroitin-4-, chondroitin-6-, and both chondroitin-4- and -6-sulfate. Little or no immunoreactivity was observed in gastrulating embryos at 18 hr postfertilization with any MAb tested. By 24 hr (8 somites), the CS-56 epitope was localized around the notochord. At hatching (48 hr) and early larval (72 hr) stages, d1C4 and CS-56 antigens codistributed in some sites (e.g., the notochord and myosepta), but a striking heterogeneity of chondroitin sulfate GAG localization was observed in other developing tissues, including the eye and specific subsets of basement membrane. At these latter time points, TC2 reacted primarily with the extracellular matrix of the developing heart, particularly the ventricular and conotruncal segments. Heterogeneous patterning of these chondroitin sulfate GAG epitopes suggests dynamic regulation of proteoglycan function during critical morphogenetic events in early development of the striped bass. Anat Rec 268:47–58, 2002. © 2002 Wiley-Liss, Inc.

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 Antibodies

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.

Immunohistochemistry

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.

RESULTS

Morone saxatilis Specimens

Hatchery rearing of Morone saxatilis at the Watha State Fish Hatchery is routinely carried out at 18°C. The ages of the Morone saxatilis specimens examined in the present study were selected to localize chondroitin sulfate GAGs during the following key developmental stages: 18 hr, mid to late gastrula (approximately 90% epiboly); 24 hr, 8 somites; 48 hr, hatching embryo; and 72 hr, early larva (Mansueti, 1958) (Scemama et al., unpublished results). Immunohistochemical staining with chondroitin sulfate GAG antibodies was performed on a minimum of eight to 10 samples from each of the above developmental stages.

Gastrulation and 8-Somite Stages

Mid- to late-gastrula-stage embryos at 18 hr of development were sectioned in several planes in an attempt to immunolocalize TC2, d1C4, and CS-56 epitopes; however, at 18 hr little or no reactivity was observed with any MAb tested. In the 8-somite embryo at 24 hr, CS-56 immunostaining was observed surrounding the notochord (Fig. 1). In sectioned specimens at this time point, little or no CS-56 reactivity was observed at other embryonic sites, nor was immunostaining with TC2 or d1C4 noted in the notochord or elsewhere in the embryo. Similar results were obtained with whole-mount preparations (not shown).

Figure 1.

CS-56 localization in the striped bass embryo at 24 hr of development. A: CS-56 immunoreactivity is observed around the notochord (asterisk) in cross-section. B: Phase contrast image of A. Nt, neural tube; S, somite. Scale bar = 50 μm.

Hatching Embryo and Early Larval Stages

Notochord and somites.

In the trunk of the hatching embryo at 48 hr, CS-56 epitopes were again observed surrounding the notochord and extending into surrounding myosepta (Fig. 2A and B). MAb d1C4 immunostaining codistributed with CS-56 localization around the notochord and along myosepta at this stage (Fig. 2C and D). MAb d1C4 also stained the trunk neural tube at 48 hr. In the early larva at 72 hr, d1C4 immunoreactivity decreased notably in myosepta with further maturation of the myomeres. Removal of hyaluronic acid by Streptomyces hyaluronidase pretreatment of tissue sections did not reveal additional d1C4 epitopes in myosepta of the 72-hr larva (Fig. 2E and F).

Figure 2.

CS-56 and d1C4 epitopes are localized in myosepta in sagittal sections of the hatching striped bass embryo at 48 hr of development. A: CS-56 staining extends from the notochord (asterisk) into the fibrous myosepta (arrows) outlining myomeres in the trunk region. B: Phase contrast image of A. C: d1C4 also stains myosepta at this stage of development. D: Phase contrast image of C. E: Pretreatment with 20 U/ml Streptomyces hyaluronidase does not unmask additional d1C4 epitopes in myosepta of the 72-hr striped bass larva. F: Phase contrast image of E. Nt, neural tube. Scale bar = 100 μm for all panels.

Neural tube and otic vesicle.

At the level of the hindbrain in the hatching embryo at 48 hr, strong CS-56 reactivity extended into the parachordal mesoderm (Fig. 3A and B). CS-56 antigen was also localized discontinuously in the external limiting membrane along the ventral neural tube and was associated with the basement membrane of the otic vesicle. Intense CS-56 reactivity was also found along the epidermal basement membrane of the second pharyngeal arch in the region of the developing operculum. MAb d1C4 stained the notochordal sheath strongly, but reactivity did not extend into the parachordal mesenchyme at this stage (Fig. 3C and D). Immunostaining was also noted in the discrete region of contact between the otic vesicle and neural tube. At the early larval stage, d1C4 reactivity persisted around the notochord and between the neural tube and otic vesicle (Fig. 3E and F). TC2 staining was not observed in these sites at either the hatching or early larval stages (not shown).

Figure 3.

CS-56 and d1C4 immunoreactivity in cross-section through the hindbrain region of the striped bass at (A–D) 48 and (E and F) 72 hr of development. A: Intense CS-56 staining extends from the notochord (asterisk) into the matrix surrounding the parachordal mesenchyme. CS-56 staining is also observed around the otic vesicle (Ov) and developing operculum (large arrow). B: Phase contrast image of A. C: d1C4 reactivity (small arrow) is found in regions of contact between the otic vesicle and the neural tube (Nt). D: Phase contrast image of C. E: d1C4 reactivity persists between the neural tube and otic vesicle in the early larva. F: Phase contrast image of E. Scale bars = (A–D) 100 μm,; (E and F) 50 μm.

Eye and splanchnocranium.

In the developing eye of the hatching embryo, CS-56 reactivity was very pronounced around the lens, particularly in the interval between the lens and neural retina (Fig. 4A and B). CS-56 was again noted to react with subsets of basement membrane of the second and third pharyngeal arches. In the sagittal plane, CS-56 stained the otic vesicle only slightly. At 48 hr, d1C4 reacted with developing lens tissue and was strongly immunoreactive in distinct strata within the neural retina (Fig. 4C and D). Low-level d1C4 reactivity was noted within the neural tube, particularly in brain vesicles through the metencephalon. MAb d1C4 staining was again observed at sites of contact between the otic vesicle and neural tube. By 72 hr, the d1C4 immunoreactive sites in the neural retina could be resolved as inner and outer plexiform layers (Fig. 4E and F). At the early larval stage, nascent cartilages of the forming splanchnocranium were also stained by d1C4 and CS-56 (Figs. 4 and 5). In the present study, pretreatment of tissue sections with Streptomyces hyaluronidase was routinely employed to determine whether chondroitin sulfate GAG epitopes were masked by hyaluronic acid. Hyaluronate lyase pretreatment enhanced staining intensity slightly; however, CS-56 and d1C4 staining patterns overall were not altered appreciably (Fig. 5).

Figure 4.

CS-56 and d1C4 display heterogeneous localization in a sagittal section through the head region in the striped bass at (A–D) 48 and (E and F) 72 hr of development. A: CS-56 stains intensely around the developing lens (L) and pharyngeal arches (arrows). B: Phase contrast image of A. C: d1C4 stains discrete regions of the neural retina (arrowheads). Note reactivity in the neural tube predominantly through the metencephalon (Met). D: Phase contrast image of C. E: Higher magnification of d1C4 reactivity in inner (I) and outer (O) plexiform layers of the neural retina. Note also d1C4 staining surrounding the lens and in developing cartilage tissue (Ct). Ov, otic vesicle. Scale bars = (A–D) 100 μm, (E and F) 50 μm.

Figure 5.

(A and B) d1C4 and (C and D) CS-56 staining of developing cartilage (arrows) in the striped bass larva at 72 hr. Pretreatment of sections with enzyme vehicle alone is shown in panels A and C. Pretreatment of sections with 20 U/ml Streptomyces hyaluronidase (B and D) increases immunoreactivity slightly, but overall tissue distribution of CS-56 and d1C4 reactivity remains similar. Scale bar = 50 μm for all panels.

Pectoral fin bud.

Differential d1C4 and CS-56 localization was also observed in the developing pectoral fin bud in the 72-hr larva. MAb d1C4 stained pericellularly in aggregating mesenchyme during initial fin outgrowth (Fig. 6A and B), while CS-56 reactivity was noted along the basement membrane subjacent to the apical ectodermal ridge (Fig. 6C and D).

Figure 6.

d1C4 and CS-56 reactivity in the developing pectoral fin of the striped bass. Cross-section through the 72-hr larva. A: d1C4 reactivity is localized pericellularly in the mesenchyme of the incipient fin bud (arrow). B: Phase contrast image of A. C: CS-56 staining is noted intermittently in the subridge basement membrane (arrowhead). D: Phase contrast image of C. Aer, apical ectodermal ridge; Asterisk, notochord. Scale bar = 50 μm, A and B; 25 μm, C and D.

Alimentary canal.

In the early larva, d1C4 and CS-56 staining overlapped in the basement membrane around the intestine ranging from the foregut through the hindgut, but both epitopes were absent from the basement membrane surrounding the pronephros (Fig. 7A–C). At this same stage, the TC2 epitope was preferentially localized along the ventral aspect of the foregut (Fig. 7D–F). As noted for d1C4 and CS-56 reactivities, Streptomyces hyaluronidase pretreatment of tissue sections enhanced TC2 staining only slightly, and antigen localization overall was unchanged.

Figure 7.

d1C4, CS-56, and TC2 immunostaining in the digestive tract of the striped bass larva at 72 hr. (A–C) Cross and (D–F) sagittal sections through the gut tube. A: d1C4 stains regions of the basement membrane around the hindgut (Hg), but not the pronephros (P). B: CS-56 staining is also noted in the basement membrane and subjacent mesenchyme of the hindgut. C: Phase contrast image of B. D: TC2 immunoreactivity is observed along the ventral aspect of the foregut (arrow). E: Streptomyces hyaluronidase (20 U/ml) pretreatment of tissue sections increases TC2 staining intensity slightly, but overall tissue localization of TC2 reactivity is similar. F: Phase contrast image of E. Scale bar = 50 μm for all panels.

Heart tube and branchial arches.

In the 48-hr hatching embryo neither d1C4 nor CS-56 epitopes were observed in the heart; however, as noted in the whole-mount preparations (Fig. 8), TC2 reacted predominantly with developing cardiac tissue. In sectioned specimens at 48 hr, TC2 displayed discrete regions of reactivity along the heart tube. As shown in Figure 9A and B, only weak staining was noted along the ventral aspect of the sinus venosus, but reactivity increased markedly in the cardiac jelly lining the developing sinoatrial valve. The atrial segment of the heart at this stage did not exhibit TC2-positive staining, but strong TC2 immunoreactivity was observed consistently in the cardiac jelly of the ventricle and along the endocardium of conotruncal cushions (Fig. 9C and D), continuing throughout the cardiac jelly of the conotruncus. At the 72-hr larval stage, TC2 reacted strongly with cardiac jelly in both the atrioventricular region and the ventricle (Fig. 10A and B). In adjacent sections, CS-56 reactivity was most pronounced in the atrioventricular region (Fig. 10C and D). MAb d1C4 exhibited only weak immunostaining along the cardiac jelly of the ventricular myocardium (Fig. 10E and F). Extracardiac TC2, CS-56, and d1C4 staining was observed in scattered sites within the branchial arches, while CS-56 and d1C4 staining was again noted to codistribute around the anterior extent of the notochord and in dorsal elements of the branchial arch cartilages (Fig. 10C–F). In the conotruncus of the early larva TC2 and CS-56, immunoreactivities were again heterogeneously localized. As observed in the hatching embryo, TC2 staining within the cardiac jelly extended to the aortic sac (Fig. 11A and E). CS-56 localization was distinctly different from that of TC2, beginning in the cardiac jelly of the distal conotruncus and extending well into the ventral aorta (Fig. 11C and G).

Figure 8.

Whole-mount TC2 immunoreactivity in the hatching striped bass embryo at 48 hr of development. TC2 shows intense staining of the heart tube (arrow). Scale bar = 500 μm.

Figure 9.

TC2 immunostaining in sagittal sections through the heart tube of the hatching striped bass embryo at 48 hr of development. A: TC2 staining is associated with cardiac jelly surrounding a leaflet of the sinoatrial valve (arrow). B: Phase contrast image of A. C: TC2 stains cardiac jelly of the ventricle (V) and conotruncal cushions (arrowhead). D: Phase contrast image of C. At, atrium; SV, sinus venosus. Scale bar = 50 μm for all panels.

Figure 10.

TC2, CS-56, and d1C4 evidence heterogeneous staining in sagittal sections through the heart tube of the striped bass larva at 72 hr. A: Intense TC2 reactivity is noted from the atrioventricular region (Av) through the ventricle (V). B: Phase contrast image of A. C: CS-56 reacts strongly with the atrioventricular segment of the heart. D: Phase contrast image of C. E: d1C4 stains weakly in the atrioventricular and ventricular segments. F: Phase contrast image of E. Asterisks, pharyngeal arches; No, notochord; Ct, cartilage tissue. Scale bar = 50 μm for all panels.

Figure 11.

TC2 and CS-56 epitopes are localized differently in sagittal sections through the conotruncus of the striped bass larva at 72 hr. A: TC2 stains cardiac jelly strongly throughout the conotruncus (arrow). B: Phase contrast image of A. C: CS-56 reactivity is localized to more distal segments of the conotruncus and extends from the aortic sac into the ventral aorta (arrowheads). D: Phase contrast image of D. E: Higher magnification of TC2 staining in A. F: Phase contrast image of G. G: Higher magnification of CS-56 reactivity in C. Asterisk, notochord; Di, diencephalon; Ov, otic vesicle. Scale bars = (A–D) 100 μm; (E–G) 50 μm.

DISCUSSION

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.

Acknowledgements

The authors thank Jeff Evans, Hatchery Manager, Watha State Fish Hatchery, NC Wildlife Resources Commission, for the generous contribution of striped bass samples utilized in this study. We also acknowledge the technical assistance in immunohistochemistry provided by Patricia Fox.

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