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Spatial and temporal expression of syndecan--2 (fibroglycan) in chick heart development.

Syndecan-2 is but one member of the syndecan family of heparan sulfate proteoglycans. Four syndecan isoforms occur in higher vertebrates and a single isoform in invertebrates including Drosophila and the nematode Caenorhabditis elegans. Regardless of species or isoform, all syndecans share structural homology. In addition to a hydrophobic transmembrane domain, the cytoplasmic domain is organized into a membrane proximal constant domain (C1), a variable domain (V) with shared homology only between species, and a COOH terminal constant domain (C2) ending with a PDZ-binding motif (Lopes et al. 2006). The extracellular domain (aka. ectodomain) is the most variable of the regions and is thought to contribute to the distinct functions of the different syndecan isoforms (Fears & Woods 2006). Recent evidence indicates that the ectodomain core protein sequences are not only critical for the specification and display of glycosaminoglycans (Zhang et al. 1995), but are also essential for mediating cellular activity (McFall & Rapraeger 1998; Langford et al. 2005).

Syndecans are present on a wide variety of cell types and commonly exhibit a developmentally regulated pattern (Rapraeger 2001). Functional studies demonstrate that syndecans mediate cellular activities including cell adhesion, binding and modulation of growth factor activity, interacting with and arranging the cytoskeleton and organizing of the extracellular matrix. These processes are critical during morphogenesis and become reiterated in tissues undergoing wound healing following injury.

During cardiac morphogenesis, many of the biological activities mediated by syndecans are active in shaping cardiac tissue into a functioning complex organ. Cellular proliferation and differentiation are likely regulated to a large degree by the array of growth factors present, including basic fibroblast growth factor (bFGF) (Sheikh et al. 2004), transforming growth factor beta (TGF[beta]) (Akhurst et al. 1990) bone morphogenetic protein (BMP) (Neuhaus et al. 1999) and thrombospondin (Corless et al. 1992). Construction of the extracellular environment and directed cell migration are additional processes that require coordinated regulation among all the cells within the developing heart. Clearly, growth factors and adhesion molecules such as the integrins play a role in shaping the heart (Kim et al. 1999); however, the presence of the syndecans suggests that they are active participants in cardiac morphogenesis as well.

Proteoglycans are common constituents present within the developing heart (Handler et al. 1997; Litwack et al. 1998; Zanin et al. 1999) including syndecan-2 in avian and rodent cardiac tissue. Northern blot analysis has been used to show that late embryonic stage chick (Chen et al. 2002) and rat hearts (Asundi et al. 1997) contain syndecan-2 mRNA. The latter study extended these findings by suggesting that cardiomyocytes possess the potential to express syndecan-2. Cultures of cardiomyocytes were found to express low levels of syndecan-2 mRNA. While studies such as these demonstrate the ability of cardiac tissue to express syndecan-2, the pattern of syndecan-2 expression during early stages of cardiac morphogenesis has not been elucidated.

This study seeks to describe the pattern of expression within the developing chick heart using a polyclonal antibody shown to recognize chicken syndecan-2 (Chen et al. 2002). The data presented here demonstrate that syndecan-2 is expressed during Hamburger and Hamilton (HH) stage 15-25 within the developing myocardium of the heart. Earlier stages of HH 12-14 showed no detectable syndecan-2. Additionally, during no stage assayed was syndecan-2 detectable within the endothelium or the cardiac mesenchyme. These findings suggest that syndecan-2, expressed during these early stages of cardiac morphogenesis, may play a role in cardiac development mediated through growth factor attenuation, cellular adhesion, extracellular matrix organization or another cellular activity mediated by a member of the syndecan family.

MATERIALS AND METHODS

Collection of embryonic chick tissues. -- Fertilized chicken eggs were incubated for variable periods of time at 38[degrees]C in a forced air incubator with a humidified atmosphere. Embryos were staged according to the method of Hamburger and Hamilton (HH) (Hamburger & Hamilton 1992). Hearts from appropriately staged embryos were removed and placed in cold PBS (pH 7.2) and prepared for histological processing.

Tissue fixation and immunohistochemistry. -- Embryonic chick hearts (HH stage 12-25) were frozen at -196P[degrees]C in liquid nitrogen cooled 1,1,1,2-tetrafluoroethane (R-134a; Kitten et al. 1987) and transferred to vials containing frozen n,n-dimethylformamide. The water content of the tissue was gradually substituted with n,n-dimethylformamide by increasing the temperature to 5[degrees]C over 48 hours. The samples, once equilibrated at room temperature, were routinely processed through a series of graded ethanol solutions (25%-100%), infiltrated with the transition solvent Safeclear (Fisher Scientific, Pittsburg, PA), embedded in paraffin and sectioned at a thickness of 5 [micro]m.

After routine removal of the paraffin and rehydration of the tissue sections, this tissue was incubated in a blocking solution of 3% Carnation Instant Milk in PBS (pH 7.2) at room temperature for one hour. Primary and secondary antibodies were each diluted in a buffer of PBS containing 3% Carnation Instant Milk (pH 7.2). The primary antibody, (R1891, obtained from Dr. Anne Woods; Chen et al. 2002) was used at a dilution of 1:500. Sections were then incubated with a FITC conjugated donkey anti-rabbit IgG (Jackson Immunologicals, West Grove, PA) at a dilution of 1:100. Sections were examined using an Olympus BX50F compound light microscope equipped with ultraviolet epi-illumination. Images were photographed using a Nikon Coolpix 5000 digital camera.

RESULTS

During early stages of cardiac development, the heart begins as a muscular tube (i.e., myocardium) with an internal lining of endothelial cells (i.e., endocardium). An acellular milieu of proteins and carbohydrates, forming the cardiac jelly, separates the endothelium from the surrounding layer of myocardial cells. Throughout these stages (HH 12-14), the heart is being transformed from this "simple tube" into a multi-chambered organ via a process defined as looping. This dextral curvature of the heart initially creates two enlarged areas of the heart: the primitive ventricle and smaller, primitive atrium. Neither the myocardium nor the endocardium exhibit any detectable syndecan-2 staining (Figs. 1a & b).

As cardiogenesis proceeds through HH 15-16, the myocardial layer begins to expresses syndecan-2 in detectable levels (Figs. 1c & d). Myocardial cells in all regions of the developing heart exhibited weak staining for syndecan-2 suggesting that there is no regional difference in the expression of this proteoglycan during cardiac development. Interestingly, the outer surface of the myocardium consistently expresses syndecan-2 (Fig. 1d). During these stages, the epicardium begins to invest the outer surface of the heart. Thus, the localization of syndecan-2 to the myocardial surface suggests that this proteoglycan may play a role in epicardial cell migration. Also, the endocardium continues to lack detectable syndecan-2 (Fig. 1d, arrowhead).

[FIGURE 1 OMITTED]

During the stages HH 18-25, syndecan-2 becomes increasingly expressed throughout the myocardium (Fig. 2). Syndecan-2 expression continues to show no regional differences within the myocardium (i.e., atrium, ventricle, or outflow tract). In HH 18 hearts, a punctate staining pattern for syndecan-2 becomes evident throughout the heart including the ventricle (Fig. 2a) and outflow tract (Fig. 2b). This pattern is consistent with that observed on the surface of cultured fibroblasts using the same antibody (Chen et al. 2002). As cardiac morphogenesis proceeds through stage HH 21, valvulogenesis and septation begins and thus, an epithelial--mesenchymal transition occurs within the endocardial cushions of the atrioventricular region and the outflow tract. No detectable syndecan-2 correlated with cardiac mesenchyme in the atrioventricular cushions of stage HH 21 hearts (Fig. 2c; asterisk). Additionally, the endocardium lacked detectable levels of syndecan-2 (Figs. 2b & c).

[FIGURE 2 OMITTED]

The pattern of syndecan-2 expression, observed in earlier stages, continues and is more prominent in HH 25 hearts (Fig. 3). Syndecan-2 is heavily expressed in the myocardial layers throughout the heart (Figs. 3a-3d). While the staining is denser in these stages, the punctate nature of the expression pattern remains. In these later stages, cardiac morphogenesis has continued and through the epithelial-mesenchymal transition has fully populated the endocardial cushions with mesenchyme. However, no detectable syndecan-2 was observed in HH 25 endocardial cushion tissue (Figs. 3a, 3c-3d).

[FIGURE 3 OMITTED]

DISCUSSION

Using an antibody shown to be specific for chicken syndecan-2 core protein (Chen et al. 2002), these data demonstrate that syndecan-2 begins to be expressed by cardiomyocytes immediately prior to the formation of the epicardium. These results are consistent with previous Northern blot analysis studies that demonstrate syndecan-2 mRNA production by cardiac tissue. Chick hearts from day 9-15 were shown to produce syndecan-2 mRNA throughout this developmental period (Chen et al. 2002). Syndecan-2 mRNA also was detected in late stage (day 18) rat hearts, although at much lower levels than that of syndecan-3 and glypican (Asundi et al. 1997). Using isolated and cultured cardiomyocytes or cardiac non-myocytes, syndecan-3 was shown to be expressed by the later while syndecan-2 was expressed in cells from both cultures. While syndecan-2 expression in cardiac non-myocytes is in apparent contrast to the strict cardiomyocyte expression observed in the current study, a direct comparison of data obtained from an immunohistochemical analysis of early stage chick cardiac tissue (day 3-5) with molecular analysis of later stage rat cardiac mRNA is difficult due to the species difference and the tremendous morphogenetic activity that occurs between early and late stages of embryogenesis. Additionally, syndecans have demonstrated a tremendous ability to be spatially and temporally regulated during development. Syndecan-2, in particular, has been suggested to be the predominant syndecan expressed during development (Tkachenko et al. 2005) and has shown alteration in its expression pattern in response to other syndecan isoforms or other heparan sulfate proteoglycans as embryogenesis proceeds. This is exemplified by the pattern of expression of syndecan-4 in cultured cardiomyocytes. In cultures of neonatal cardiomyocytes, syndecan-4 localizes to the perinuclear region; however, it is expressed in focal adhesions in cultures of adult cardiomyocytes (VanWinkle et al. 2002). Thus, syndecan-2 likely is expressed early in cardiac morphogenesis and possibly downregulated later in embryogenesis as other syndecan isoforms are expressed in cardiac tissue.

During cardiac morphogenesis, regulation of cell proliferation, extracellular matrix deposition and organization are critical biological activities that occur and may be potentiated by members of the syndecan family. Fibroblast growth factor has long been known to modulate cardiac morphogenesis and more recently to regulate cardiomyocyte differentiation (Sheikh et al. 2004; Rosenblatt-Velin et al. 2005; Kruithof et al. 2006). This potent mitogenic factor requires the presence of heparan sulfate (as found on syndecans) as coreceptors for FGF receptor binding and signaling (Bernfield & Hooper 1991; Mundhenke et al. 2002). Transforming growth factor beta (TGF[beta]) is still another potent regulator of cellular activity that may regulate cardiac morphogenesis (Ross et al. 1993). Recent studies have demonstrated the influence of TGF[beta] on cardiomyocyte differentiation (Sheikh et al. 2004; McKoy et al. 2007). Interestingly, syndecan-2 has been demonstrated to be required for TGF[beta] signaling in fibroblasts (Chen et al. 2004). Thus, syndecan-2 may be functioning in the heart to regulate TGF[beta] mediated signaling of cardiomyocytes in vivo. Yet still another factor demonstrated to play a role in shaping the developing heart is bone morphogenetic protein (BMP) (Somi et al. 2004a). Similar to the other factors discussed, the activity of BMP is modulated by syndecans (Fisher et al. 2006) and appears to regulate cardiomyocytes activity (Somi et al. 2004b; Sugi et al. 2004; Ma et al. 2005; Kruithof et al. 2006).

Another role for syndecan-2 within the myocardium may be that of a matrix organizer. The cardiac jelly lying between the myocardium and endocardium is largely synthesized by myocardial cells. These cells not only produce many of the morphogens that will lead to the epithelial-mesenchymal-transition in the endocardial cushions, but also produce many of the matrix components that the mesenchymal cells will utilize as a migratory substratum. In addition to the collagens present, fibronectin and laminin are also found within the endocardial extracellular matrix (Bouchey et al. 1996; Nakajima et al. 1997). Recently, syndecan-2 has been shown to organize both of these adhesion molecules into a functional fibrillar network (Klass et al. 2000). In addition to organizing matrix components, syndecans facilitate cellular adhesion to matrix components. Studies of syndecan-4 have provided the bulk of evidence for the role of syndecans in mediating cell adhesion. The ability of fibroblasts to spread on fibronectin have been shown to require integrins and syndecan-4 (Saoncella et al. 1999), while carcinoma cell spreading on vitronectin requires syndecan-1 (Beauvais et al. 2004). With syndecan-2 as the primary syndecan expressed during development, this proteoglycan may aid in the adhesion of cardiomyocytes early in cardiac morphogenesis.

Additional evidence for the role of syndecans in mediating cellular activity has been attained using models of wound healing and tissue repair. Increased cellular proliferation in response to released growth factors, altered cellular adhesion and matrix remodeling are all processes shared between developmental events and wound healing in adult tissues. Just as syndecans have diverse patterns of expression and activities during development, they have also been implicated in the mediation of cellular processes leading to proper wound healing in many tissue types (Worapamorn et al. 2002; Elenius et al. 2004; Fears & Woods 2006). Specific to cardiac tissue, evidence is accumulating that suggests syndecans play a role in cardiac repair following myocardial infarcts. All syndecan isoforms were shown to be upregulated following a myocardial infarct in mice (Finsen et al. 2004). Additionally, specific upregulation of syndecan-1 in the infarct tissue appears to decrease the risk of dilation and dysfunction in the experimental model system (Vanhoutte et al. 2007). Taken together, all evidence points to a dynamic role for the syndecan family of proteoglycans during cardiac morphogenesis and during repair following cardiac injury. Cardiomyocyte expression of syndecan-2 during early stages of cardiac morphogenesis illustrates how this proteoglycan could be facilitating multiple roles during this critical period of embryogenesis.

ACKNOWLEDGMENTS

We would like to thank Dr. Anne Woods for her generous gift of the immunological reagents used in this study.

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JKL at: klangford@sfasu.edu

Ashley Gordon and J. Kevin Langford

Department of Biology, Stephen F. Austin State University

Box 13006 SFA Station, Nacogdoches, Texas 75962
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Date:Nov 1, 2007
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