Role of heparan sulfate in dextral heart looping in chick

Xinping Yue1,3,4, Thomas M. Schultheiss3, Edward A. McKenzie5 and Robert D. Rosenberg2,3,4

3 Molecular Medicine Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; 4 Department of Biology, Massachusetts Institute of Technology, Bldg. 68–480, 77 Massachusetts Avenue, Cambridge, MA 02139; and 5 School of Biological Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester, M13 9PT, England

Received on January 17, 2004; revised on March 20, 2004; accepted on March 23, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Heparan sulfate (HS) has been shown to be involved in left–right asymmetry formation, including the process of dextral heart looping during embryonic development. The structural features of HS required in this process, however, have not been explored. In this study, we examined the structure of HS from the heart-forming regions (or heart fields) of Hamburger and Hamilton stage 5–9 chick embryos. No significant differences were found in HS to chondroitin sulfate (CS) ratio, HS chain length, or [35S] sulfate incorporation at HS disaccharide level between the left and the right heart fields. Compared to other parts of the embryo, however, lower ratio of HS to CS, shorter HS chain length, and higher [35S] sulfate incorporation at 6-O position of the glucosamine residue in the HS chains were observed in the heart-forming regions. Moreover, HS from the left and the right heart fields exhibit differential cleavage by heparanase, an endo-ß-D- glucuronidase that cleaves specific sequences within the HS chain. In embryo culture, microinjection of the active human heparanase enzyme into the right but not the left pericardial cavity at stage 7–8+ resulted in reversed heart looping in a dose-dependent manner. Heart reversal following microinjection of heparin or heparin derivatives suggests the involvement of N- and 6-O-sulfation but not 2-O-sulfation in the heart looping process.

Key words: 6-O-sulfation / chick / heart looping / heparan sulfate / heparanase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The establishment of left–right (L–R) asymmetry is an integral part of the vertebral body plan, and dextral heart looping is the first morphological asymmetry established during embryonic development (Capdevila et al., 2000Go; Hamada et al., 2002Go; Mercola and Levin, 2001Go). Heparan sulfate (HS) has been implicated in L–R asymmetry formation in Xenopus laevis: inhibition of proteoglycan synthesis with ß-xyloside from late gastrula to early neurula stage results in unlooped, tubular hearts (Yost, 1990Go); microinjection of bacterial heparinase into the blastocel at early gastrula stages randomizes the L–R orientation of the heart and gut (Yost, 1992Go); more recently, it was shown that disruption of syndecan-2, a transmembrane HS proteoglycan (HSPG), randomizes cardiac situs and alters the expression of left-sided markers (Kramer and Barnette, 2002Go; Kramer and Yost, 2002Go). Despite these findings, whether HS is involved in L–R asymmetry formation in species other than X. laevis and the structural features of HS required in this process are not clear.

HS is the glycosaminoglycan (GAG) moiety of HSPGs, the ubiquitous macromolecules associated with the cell surface, extracellular matrix (ECM), and basement membrane (Esko and Lindahl, 2001Go; Rapraeger, 2002Go; Rosenberg et al., 1997Go). The basic HSPG structure consists of a protein core to which several linear HS chains are covalently attached. HS polysaccharide chains are typically composed of repeating hexuronic acid (glucuronic or iduronic acid) and D-glucosamine disaccharide units. Concurrent with polymerization of the HS chain, a series of enzymatic modifications occur that generate diverse domain structures and sulfation patterns. These modifications include N-deacetylation/N-sulfation of GlcNAc, C5 epimerization of GlcA to IdoA, 2-O-sulfation of IdoA or GlcA, and 6-O- and 3-O-sulfation of D-glucosamine. Thus structural heterogeneity is generated through specific HS chain modifications in addition to the diverse nature of their core proteins. Mutations in either core proteins or HS biosynthetic enzymes result in severe developmental defects, such as arrested gastrulation, kidney agenesis, somatic overgrowth, and lung dysfunction, indicating the important functions of these molecules during embryonic development (Forsberg and Kjellen, 2001Go; Perrimon and Bernfield, 2000Go).

To gain insights into the structural features of HS required in the formation of L–R asymmetry, we concentrated on the heart looping process in developing chick embryos. In chick heart morphogenesis, the presumptive cardiac primordia arise early in gastrulation (starting at stage 3) as a pair of loose mesodermal sheets migrating in an anterior/lateral direction (Garcia-Martinez and Schoenwolf, 1993Go). By stage 6, the heart precursors have come to occupy a crescent in the anterior lateral region of the embryo. Starting at stage 6 with the formation of the foregut, the heart precursors in the left and right lateral plates are brought together on the ventral surface of the forming foregut. By stage 10, the left and the right heart primordia have fused to form a single tubular heart, which in almost all cases loops to the embryo's right-hand side (dextral heart looping) by stage 12 (Stalsberg and DeHaan, 1969Go). As suggested by experiments involving transpositions of the left and the right heart-forming regions, the directionality of the heart is specified before the fusion of the heart primordia (Hoyle et al., 1992Go; Salazar del Rio, 1974Go). An intrinsic change occurs in the precardiac mesoderm between stages 5 and 6 that defines the difference between the left and the right sides and later influences the direction of looping of the heart tube (Hoyle et al., 1992Go).

Using metabolic labeling and high-performance liquid chromatography (HPLC) analyses, we studied the HS structure in stage 5–9 chick embryos. Differential cleavage by heparanase was observed between HS from the left and the right heart fields, which correlated with heparanase's effect on the direction of heart looping in chick embryo culture. Microinjection of heparin or selectively desulfated heparin further demonstrated the requirement of HS N- and 6-O-sulfation but not 2-O-sulfation in the heart looping process.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Structural analysis of HS from the left and the right heart fields
To explore the structural features of HS required in the heart looping process, we took advantage of the easy accessibility of the developing chick embryos in modified New culture (Alsan and Schultheiss, 2002Go; Schultheiss and Lassar, 1997Go). We metabolically labeled sulfated GAG with [35S] sodium sulfate and analyzed labeled material by HPLC. At the stages examined (5–9), equal incorporations of [35S] sulfate were observed in the left and the right heart fields, and no significant differences were found in sulfated GAG composition, HS chain length, or [35S] sulfate incorporation at HS disaccharide level (data not shown). The absolute amount of HS present in the left and right heart fields also appeared to be equal, as determined by HPLC the glucosamine level after hydrolyzing HS for 3 h with 6 N HCl and 0.1% phenol (v/v) at 100°C (data not shown). It is worth mentioning that sulfated GAG in the heart-forming regions is composed of keratan sulfate, HS, and chondroitin sulfate (CS) in the ratio of 1:3:6. This result is different from what has been shown in X. laevis in which HS appeared to be the predominant form of sulfated GAG in the proteoglycans synthesized at comparable stages (Yost, 1990Go).

We further compared HS structure between the heart-forming regions and other parts of the embryo at the same developmental stages. Interestingly, significant differences were found at all three aspects examined (Figure 1 and Table I). Compared to other parts of the embryo and especially regions posterior to the heart fields, lower HS to CS ratio, shorter HS chain length, and higher [35S] sulfate incorporation at 6-O position of the glucosamine residues in the HS chains were observed in the heart-forming regions. The increase in sulfate incorporation at the 6-O position occurs in all 6-O-sulfate-containing disaccharides, including {Delta}UA-GlcNAc6S, {Delta}UA-GlcNS6S, and {Delta}UA2S-GlcNS6S, although the increase in {Delta}UA2S-GlcNS6S was not statistically significant. Corresponding decrease in sulfate incorporation was observed in {Delta}UA-GlcNS and {Delta}UA2S-GlcNS. Although the biological significance of these differences are not clear, these data suggest that HS biosynthesis is tissue- or region-specifically regulated at the developmental stages examined and structural features such as 6-O-sulfation could be important in L–R asymmetry formation initiated and/or propagated at these stages.



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Fig. 1. Structural analysis of HS from stage 7–8 chick embryos. (A) Stage 7–8 chick embryos (a stage 8 chick embryo is shown) were divided from anterior to posterior into four regions for structural analysis: region 1 (green), region 2 (red), region 3 (light blue), and region 4 (dark blue). (B) After removal of keratan sulfate from total sulfated GAG by keratanase digestion (digestion was carried out with 100 mU keratanase in 200 µl 50 mM Tris–HCl, pH 7.4, at 37°C for 4 h), remaining HS and CS from different regions of the embryos were analyzed by anion exchange HPLC. Due to differences in chain length and the amount of negative charge, HS (peak a) was eluted at lower NaCl concentration than CS (peak b). HS to CS ratios are also shown. (C) Chain lengths of HS from the four regions were analyzed by gel filtration chromatography. The elution position of blue dextran 2000 is 11.05 min; the elution position of {Delta}UA-GlcNS is 24.4 min. (D) [35S] sulfate incorporations at HS disaccharide level were analyzed by ion-pairing reverse-phase HPLC on a protein and peptide C18 column. Data from region 2 (red) and region 4 (dark blue) are shown. The elution positions of the disaccharide standards are: (1) {Delta}UA-GlcNS; (2) {Delta}UA-GlcNAc6S; (3) {Delta}UA-GlcNS6S; (4) {Delta}UA2S-GlcNS; (5) {Delta}UA2S-GlcNS6S. Arrows mark 6-O-sulfate-containing disaccharides.

 

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Table I. [35S] sulfate incorporation at HS disaccharide level in stage 7–8 chick embryos

 
HS from the left and the right heart fields exhibit differential cleavage by heparanase
Due to the heterogeneous nature of HS, subtle sequence differences may not be revealed simply by chain length or [35S] sulfate incorporation. To detect the possible subtle differences in HS structure, we subjected HS from the left and the right heart fields to heparanase cleavage. Heparanase is an endo-ß-D-glucuronidase that plays important roles in the pathogenesis of inflammation, tumor angiogenesis, and metastasis (Vlodavsky and Friedmann, 2001Go). Recent molecular cloning of this enzyme furthered its biochemical characterization (Fairbanks et al., 1999Go; Hulett et al., 1999Go; Kussie et al., 1999Go; Toyoshima and Nakajima, 1999Go; Vlodavsky et al., 1999Go). Heparanase is initially synthesized as a latent ~65-kDa proenzyme that undergoes proteolytic processing to the active heterodimer form of the enzyme, composed of an 8-kDa N-terminal subunit and a 50-kDa C-terminal subunit (Fairbanks et al., 1999Go; McKenzie et al., 2003Go). Heparanase cleaves the glycosidic bond within a HS chain through a hydrolase mechanism (Hulett et al., 2000Go), which differs from the bacterial heparin lyases that depolymerize HS through eliminative cleavage. Moreover, heparanase recognizes and cleaves specific sequences within the HS chain, yielding HS fragments of still appreciable size (10–20 sugar units) (Freeman and Parish, 1998Go; Pikas et al., 1998Go). It appears that highly sulfated structure in the immediate vicinity of the targeted GlcUA residue is critical for the enzyme action, and the preference of heparanase for the GlcN(NS) structure on the reducing side and the GlcN(6S) on the nonreducing side was recently demonstrated using purified recombinant human heparanase (Okada et al., 2002Go).

Using the recombinant human active heparanase protein, we found that HS from the left and the right heart fields exhibit differential cleavage by heparanase (Figure 2). This difference is subtle in that the difference in the gel filtration profile of the heparanase cleavage products can not be resolved by a single TSK-Gel G4000 gel filtration chromatography column. Using linked Biosep-SEC-S2000 and Biosep-SEC-S3000 columns, however, a shift to lower-molecular-weight (longer elution time) of the heparanase cleavage products from the left heart field is clearly seen (Figure 2B). There are two possible explanations. First, the difference in the gel filtration profile represents true differences in the chain length of the heparanase cleavage products, which implies that HS from the left heart field is more susceptible to heparanase cleavage than HS from the right heart field; second, this difference could be the result of differences in the spacing of sites (sulfate, carboxyl, and hydroxyl groups) along the HS chain. Although more extensive structural analyses are required to resolve this issue, both scenarios would support that microsequence differences indeed exist in HS between the left and the right heart fields.



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Fig. 2. In vitro digestion of HS from the left and the right heart fields by heparanase. HS (0.5–1 x 105 cpm) from the left and the right heart fields were incubated with heparanase (0.73 ng) in 100 µl of 100 mM sodium acetate/5 mM CaCl2, pH 5.6, at 37°C for 24 h before analysis by gel filtration chromatography. (A) HS from the left and the right heart fields incubated without (R, red dashed line; L, blue dashed line) or with heparanase (R + Hpa, red solid line; L + Hpa, blue solid line) were analyzed on a TSK-GEL G4000 column. The elution position of blue dextran 2000 is 11.1 min; the elution position of {Delta}UA-GlcNS is 24.4 min. (B) Heparanase cleavage products from the left (L + Hpa, blue) or the right (R + Hpa, red) heart field were also analyzed on linked Biosep-SEC-S2000 (600 x 7.8 mm) and Biosep-SEC-S3000 (600 x 7.8 mm) columns in 100 mM NH4HCO3 at a flow rate of 0.4 ml/min. The elution position of blue dextran 2000 is 54.4 min; the elution position of free sulfate is 101.8 min.

 
Microinjection of heparanase into the right but not the left pericardial cavity results in increased reversal of heart looping
To test whether the differential cleavage of the left and the right heart field HS by heparanase influences the direction of heart looping, we microinjected the active form of recombinant human heparanase into either the right or the left pericardial cavity of stage 7–10 chick embryos. These stages are comparable to neurula stages in X. laevis, during which the cardiac primordia migrate to the ventral midline, fuse into a single heart tube, and later loop to the right side of the body.

As shown in Figure 3 and Figure 4, microinjection of heparanase into the right but not the left pericardial cavity at stage 7–8+ resulted in reversed heart looping. This effect is dose-dependent, reaching 100% (complete reversal) at 0.73 µg/ml. In contrast, microinjection of the active heparanase into the left pericardial cavity at the same dosages had no effect on the direction of heart looping, and the heart tubes in these embryos looped normally as control embryos injected with buffer. These results are consistent with the scenario that HS from the left heart field is more susceptible to heparanase cleavage than HS from the right heart field. It is conceivable that microinjection of the active heparanase into the right pericardial cavity reversed the situation in vivo and resulted in more heparanase cleavage products on the right side rather than on the left. The dose-dependent effect of heparanase on the direction of heart looping (Figure 4), and the dose-dependent HS cleavage by the injected enzyme (data not shown) support this hypothesis. At the amount and concentrations used, the digestion was localized and limited to the injected side; leakage and digestion of HS on the opposite site was not observed. Buffer without enzyme was injected into the opposite side to ensure the same volume change. Control embryos injected with buffer into both the left and the right pericardial cavities developed normally as embryos without any manipulation (Figure 3C). Except of the direction of heart looping, embryos developed normally following enzyme treatment (Figure 3D and 3E).



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Fig. 3. Microinjection of heparanase into the right but not the left pericardial cavity results in reversed heart looping. Ventral views are shown, and embryonic right (R) and left (L) are as indicated. (A) Illustration of microinjection into the pericardial cavity. Dye (9.2 nl) was injected into the right pericardial cavity of a stage 8 chick embryo (for illustration only, dye was not injected in the experiments). Heart-forming region for the isolation of total GAG was outlined, reaching anteriorly at the anterior end of cardiac fusion and posteriorly at the level of the first somites. At the ventral midline region, only mesoderm and endoderm were isolated (neural tube was excluded), whereas in the lateral plate region, all three germ layers were included. Scale bar, 0.25 mm. (B) The purity of the active human heparanase protein was shown by 4–20% gradient SDS–PAGE followed by Coomassie brilliant blue staining. 1, molecular weight standards; 2, purified active heparanase. The active form of heparanase is a heterodimer composed of an 8-kDa N-terminal subunit and a 50-kDa C-terminal subunit (arrows). Arrowhead, minor degradation products. (C) Normal (rightward) heart looping in a control embryo injected with buffer. (D) Reversed (leftward) heart looping in an embryo injected with 9.2 nl 0.55 µg/ml heparanase into the right pericardial cavity (HpaR). (E) Normal heart looping in an embryo injected with 9.2 nl 0.55 µg/ml heparanase into the left pericardial cavity (HpaL). CE are at the same magnification; scale bar, 0.25 mm.

 


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Fig. 4. Dose-dependent effects of heparanase on the direction of heart looping at stage 7–8+. Microinjection of heparanase into the right (heparanase R), but not the left (heparanase L) pericardial cavity at stage 7–8+ results in reversed heart looping in a dose-dependent manner (1 to 4 are in increasing concentrations), reaching 100% reversal at 0.73 µg/ml. The dosages used are: 1, 0.05–0.1 µg/ml; 2, 0.12–0.14 µg/ml; 3, 0.55 µg/ml; 4, 0.73 µg/ml. Enzyme injected embryos were compared with control embryos using Mann-Whitney rank sum test; *p < 0.001. Numbers in parentheses represent number of embryos in each group.

 
The influence of heparanase on the direction of heart looping appeared to be restricted to stage 7–8+; embryos injected with heparanase at stage 9–10 developed normal looped hearts (data not shown). The effects of heparanase also appeared to be restricted to the heart in the L–R asymmetry formation, as the expression pattern of Nodal and Pitx2, asymmetric markers in the lateral plate mesoderm, were not altered by the enzyme treatment (data not shown). These results are not surprising because the injection of heparanase was localized to the heart-forming regions, and global influences on L–R asymmetry were not expected.

To test whether the effects of heparanase on the direction of heart looping is due to the partial degradation of HS, we injected heparin lyases, which would cause more extensive breakdown of the polymer, and compared the results to those of heparanase injection. In vitro, more than 85% of heart field HS were digested to di- and tetrasaccharides by heparitinase I (data not shown), compared to the larger fragments generated by specific scission of rare sequences by heparanase (Figure 2). Injection of heparitinase I at 1–2 mU/µl into the right pericardial cavity resulted in 50% reversal of heart looping (8 of the 16 embryos injected exhibited reversed heart looping), whereas injection into the left pericardial cavity had no effects (none of the 15 embryos injected exhibited looping reversal). Similar results were obtained with injection of heparitinase III (heparinase, data not shown). These data suggest that degradation of HS by heparanase is at least partly responsible for its effects on cardiac looping.

Heparanase is ubiquitously expressed in early chick embryos
The effects of heparanase on the direction of heart looping prompted us to examine the expression of this enzyme in the developing chick embryos. Reverse transcription polymerase chain reaction (PCR) was performed on total RNA isolated from stage 4–8 chick embryos using chick heparanase-specific primers (see Materials and methods). PCR fragments of expected sizes were generated (data not shown), indicating the expression of heparanase mRNA during these developmental stages. The expression of heparanase was further studied in chick embryos from stage 3 through heart looping stages by whole mount in situ hybridization. An overall ubiquitous pattern of expression was revealed by two RNA probes generated from vectors containing heparanase PCR fragments of different sizes (data not shown). This result is not surprising considering the involvement of heparanase in the cellular turnover of HS (Bai et al., 1997Go). In situ hybridization cannot reveal, however, the activity or the cellular localization of this enzyme (intracellular, cell surface, or secreted extracellular forms) that have been shown to be important in its function in inflammation, tumor angiogenesis, and metastasis (Vlodavsky and Friedmann, 2001Go).

Changes in fibronectin deposition pattern following heparanase treatment
HS binds to and assemble ECM components (including fibronectin, laminins, and interstitial collagens) and plays important roles in cell–cell and cell–ECM interactions (Bernfield et al., 1999Go). Among these ECM molecules, fibronectin has been shown to be involved in the migration of the presumptive cardiac mesoderm in chick (Linask and Lash, 1986Go, 1988aGo,bGo), and disruption of fibronectin function by RGD (Arg-Gly-Asp) containing peptides results in partial cardiabifida in chick (Linask and Lash, 1988aGo,bGo) and global randomization of L–R asymmetry in X. laevis (Yost, 1992Go). Fibronectin-null mice display a wide variety of defects, including variable deformed heart and embryonic vessels, and die during gastrulation (George et al., 1993Go). Considering the involvement of heparanase in the organization and stability of ECM and the known function of fibronectin in heart development, we examined fibronectin deposition pattern following heparanase treatment. As shown in Figure 5, compared to the buffer-injected control side, where a fine particulate fibronectin network was observed between the cardiogenic mesoderm and the adjacent endoderm (Figure 5E), a dense fibrillar pattern of expression was seen following heparanase treatment (Figure 5D). This suggests that heparanase could be involved in fibronectin fibrillogenesis. The change in fibronectin deposition pattern from fine particulate to fibrillar network could potentially increase the concentration of fibronectin, provide a higher driving force for the migration of the cardiogenic precursors of the enzyme treated side, and eventually result in heart looping to the side of less migratory potential.



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Fig. 5. Changes in fibronectin deposition pattern following heparanase treatment. (A) Hematoxylin and eosin staining of a cross-section of the heart-forming region from an embryo fixed 3 h after injection of 9.2 nl 0.55 µg/ml heparanase into the right pericardial cavity at stage 8. Embryonic right (R) and left (L) are as indicated. Scale bar, 100 mm. (B and D) Cross-sections of the right heart-forming region (enzyme-injected side). (C and E) Cross-sections of the left heart-forming region (buffer-injected control side). (B and C) HS immunostaining following heparanase treatment. The distribution of HS was detected by monoclonal antibody against HS 10E4 epitope. Intense staining was observed along the neural tube, underneath the ectoderm, and between the cardiogenic mesoderm and the adjacent endoderm. No significant differences were observed between the enzyme treated side (B) and the control side (C). (D and E) Changes in fibronectin deposition following heparanase treatment. A dense fibrillar pattern of fibronectin deposition (D) between the cardiogenic mesoderm and the adjacent endoderm was observed following heparanase injection, in comparison to the fine particulate pattern on the contralateral control side (E). BE are at the same magnification; scale bar, 20 mm. cm, cardiogenic mesoderm; en, endoderm; et, ectoderm; nt, neural tube.

 
We also examined HS distribution by immunostaining with the anti-HS monoclonal antibody 10E4 (David et al., 1992Go). No significant differences were observed between the enzyme-treated side (3 h after heparanase injection) and the control side (Figure 5B and C). This differs from the results in cell culture (McKenzie et al., 2003Go) where a reduction in 10E4 staining was found. Some potential explanations for this apparent discrepancy include that the heparanase cleavage products in embryo culture could remain bound to growth factors or other components of the ECM and subsequently are still recognized by the 10E4 antibody, whereas the cleavage products in cell culture would be washed away from the cell surface. This result could also be a reflection of the rapid HS turnover in the developing chick embryos.

Injection of heparin and heparin derivatives
To further test the structural requirements of HS in the heart looping process, heparin or heparin derivatives that lack specific sulfations were injected into the pericardial cavity, and their influences on the heart looping direction were studied. As shown in Figure 6, injection of heparin into the right (but not the left) pericardial cavity resulted in increased reversal of heart looping, mirroring the effect of heparanase injection. The phenomenon of heparin-induced looping reversal is also dose-dependent; concentration of 0.5 mg/ml (4.6 ng in 9.2 nl buffer per embryo) or higher (1 mg/ml) caused maximum increased reversal of heart looping with no significant differences observed between embryos injected with 0.05 mg/ml heparin and control embryos injected with buffer alone.



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Fig. 6. Heart looping reversal following microinjection of heparin or heparin derivatives. Control embryos (C) were injected with PBS into both the left and the right pericardial cavities. For embryos injected with heparin or heparin derivatives into either the right or the left pericardial cavity (as shown), PBS was injected into the opposite side. Injection of heparin into the right pericardial cavity resulted in increased reversal of heart looping in a dose-dependent manner. The concentrations used are: 1, 0.05 mg/ml; 2, 0.5 mg/ml; 3, 1 mg/ml. Heparin derivatives injected include CDSNAc, NDSNAc, 6-ODS, and 2-ODS at concentrations of 0.5 mg/ml. Heparin or heparin derivative injected embryos were compared with control embryos using Mann-Whitney rank sum test; *p < 0.05. Numbers in parentheses represent the number of embryos in each group. The relative potencies of heparin and heparin derivatives are summarized.

 
We further compared the effects of heparin and selectively desulfated heparin at the concentration of 0.5 mg/ml (Figure 6). Compared to the native heparin, injection of completely desulfated heparin had no effect on heart looping, indicating the importance of HS sulfation in this process. The effect of heparin on the rate of heart reversal was significantly reduced following desulfation at either N- or 6-O-position of the glucosamine residue (no significant differences were observed between N- or 6-O-desulfated heparin injected embryos and control embryos injected with buffer), indicating the requirement of both N- and 6-O-sulfation in cardiac looping. Surprisingly, 2-O-desulfated heparin caused reversal of heart looping to the same extent as native heparin, indicating that unlike N- and 6-O-sulfation, 2-O-sulfation is not important in the process of cardiac looping.

The quality of the modified heparins have been confirmed previously (all modified heparins except 2-O-desulfated heparin [ODS]) (Wu et al., 2003Go) and by mass spectrometer (2-ODS, data not shown). The native heparin contains 16% {Delta}UA-GlcNS6S and 66% {Delta}UA2S-GlcNS6S. The major disaccharides found in completely desulfated and N-acetylated heparin (CDSNAc) and N-desulfated/N-acetylated heparin (NDSNAc) heparins are {Delta}UA-GlcNAc and {Delta}UA2S-GlcNAc6S, respectively. The 6-ODS contains 19% {Delta}UA-GlcNS and 59% {Delta}UA2S-GlcNS, as a result of the selective 6-O-desulfation of {Delta}UA-GlcNS6S and {Delta}UA2S-GlcNS6S. Tested by mass spectrometer, the complete disappearance of {Delta}UA2S-GlcNS6S was observed in 2-ODS, with {Delta}UA-GlcNS6S being the major disaccharide found. Because the trisulfated disaccharide {Delta}UA2S-GlcNS6S was not found in 2-ODS and yet it caused looping reversal to the same extent as the native heparin, it is likely that the trisulfated disaccharide is not important for cardiac looping.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Previous evidence from X. laevis suggested the involvement of HS in the formation of L–R asymmetry, including the dextral heart looping process. We show in the present study that (1) HS is involved in dextral heart looping in chick, as in X. laevis; (2) heparanase, a physiological player in HSPG function, plays a role in cardiac looping; (3) and N- and 6-O-sulfation but not 2-O-sulfation are involved in the heart looping process.

The developmental stages during which microinjection of either heparanase or heparin influences the direction of heart looping are stage 7 to 8+. These stages in chick are comparable to the neurula stages in X. laevis, when inhibition of proteoglycan synthesis specifically eliminates the looping of the cardiac tube. This critical period is coincident with the migration of cardiac primordia to the ventral midline in both chick and X. laevis. Thus HSPG is likely involved in the transduction of L–R axial information to the cardiac primordia during migration in both species.

Interestingly, heparanase, the HS degradation enzyme involved in cell migration and tumor metastasis, influences the direction of cardiac looping in chick. HS from the left and the right heart fields exhibit differential cleavage by heparanase; microinjection of active heparanase into the right but not the left pericardial cavity results in reversed heart looping in chick embryo culture. Although in situ hybridization did not reveal any differences in the expression level of heparanase between the left and the right, the differential cleavage of HS in the left and the right heart fields by heparanase could result in differences in the amount and/or the properties of the heparanase cleavage products, the putative active component in the heart looping process. In fact, limited heparanase cleavage of HS is believed to affect a variety of biological processes. Cleavage of HS chain by heparanase converts the soluble syndecan-1 ectodomain from an inhibitor to a potent activator of fibroblast growth factor-2 (FGF-2) (Kato et al., 1998Go). In tumor metastasis and angiogenesis, heparanase cleavage releases growth factors such as basic FGF and vascular endothelial growth factor from the ECM, and the active HS fragments released by heparanase stimulates basic FGF/FGF receptor-1 signaling (Elkin et al., 2001Go). Although the identity of the HS-interacting proteins in the heart looping process are unknown, heparanase may modulate the functions of these proteins in laterality decisions by regulating cell adhesion and cell migration differences between the left and the right sides of the heart primordia.

Our finding that heparanase at maximum dose completely reverses the direction of heart looping, whereas heparin lyases or heparin (or its derivatives) only cause as high as 58% looping reversal (randomization) suggests the involvement of distinct mechanisms. It was recently shown that expression of heparanase in nonadherent lymphoma cells induces cell adhesion and augments cell invasion regardless of whether the transfected heparanase is in its active form or point-mutated inactive form (Goldshmidt et al., 2003Go), which suggests that heparanase can directly affect cell adhesion independent from its endoglycosidase role. It is likely that heparanase not only releases bioactive HS fragments (along with the complexed growth factors or morphogens) but also acts as an adhesion molecule in the heart looping process, whereas heparin lyases or free heparin would only displace tethered growth factors but not possess the adhesion promoting properties. The changes in fibronectin deposition pattern observed following heparanase injection support this hypothesis.

Through microinjection of heparin or selectively desulfated heparin, we further demonstrated the requirement of N- and 6-O-sulfation but not 2-O-sulfation in the heart looping process. A recent study using purified recombinant human heparanase suggests that heparanase cleaves in principle the glucuronidic linkage in the -GlcNAc(6S)- GlcUA-GlcN(NS)- sequence, although this sequence is not sufficient, and an additional sulfate group on this or adjacent sequence appears to be required (Okada et al., 2002Go). Considering the substrate preference of heparanase, it is likely that N- and 6-O-sulfates are differentially positioned in the HS chains between the left and the right heart fields, which results in their differential cleavage by heparanase.

It is not surprising that N-sulfation is required in the heart looping process because it constitutes the initial modification of the HS chain, which defines the overall design of the sulfation pattern (Kjellen, 2003Go). Our finding that 6-O-sulfation but not 2-O-sulfation is required in dextral heart looping is intriguing considering the importance of 6-O-sulfation in other biological processes. In murine neuroepithelial cells between days 10–12 of gestation, alterations in the patterns of 6-O-sulfation, total chain length, and the number of sulfated domains are observed (Brickman et al., 1998Go; Nurcombe et al., 1993Go; Walz et al., 1997Go). In Drosophila, 6-O-sulfotransferase has been shown to be involved in the formation of the tracheal system, and its spatial and temporal expression in tracheal cells resembles that of the FGF receptor, breathless (Kamimura et al., 2001Go). Although mice homozygous for the HS 2-O-sulfotransferase mutation exhibit bilateral renal agenesis and defects of the eye and the skeleton, heart development appears to be normal (Bullock et al., 1998Go). Recently Allen and Rapraeger (2003)Go showed that FGF8b and FGF receptor 3c binding and signaling specifically requires 6-O-sulfation but not 2-O-sulfation in the HS chain. Interestingly, FGF8 is not only involved in cardiac induction (Alsan and Schultheiss, 2002Go) but also plays important roles in L–R determination in the chick (Boettger et al., 1999Go), although direct involvement of FGF8 in cardiac looping has not been examined specifically.

In summary, we have shown in this study the role of HS in the process of dextral heart looping in chick. Our data suggest that heparanase plays a role in cardiac looping; HS N- and 6-O-sulfation but not 2-O-sulfation are involved in the heart looping process. It remains to be determined whether a specific HS structure or sequence is involved in cardiac looping, and the availability of new technologies should allow this issue to be resolved (Kuberan et al., 2002Go, 2003Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Chick embryos
Fertilized chicken eggs (Charles River SPAFAS) were incubated at 38°C in a humidified incubator and staged according to Hamburger and Hamilton (1951)Go. Embryos of desired stages were cultured in modified New culture as previously described (Alsan and Schultheiss, 2002Go; Schultheiss and Lassar, 1997Go).

Metabolic labeling and purification of sulfated GAG for structural analysis
Sulfated GAG was labeled with 50 mCi [35S] sodium sulfate (ICN, Irvine, CA) in 5 µl Tyrode's buffer by dropping the solution directly onto the ventral surface of stage 3–7 embryos in New culture. Labeling was carried out at 38°C for 4 h until they reach stage 5–9. To examine the changes in HS after heparanase injection, labeling was carried out initially at 38°C for 3 h, enzyme or buffer were then injected, and embryos returned to the incubator for another hour before harvest. The right and the left heart fields (Figure 3A) or different parts of the embryos (Figure 1A) were dissected out using microsurgical knifes (World Precision Instruments, Sarasota, FL). Same regions of at least eight embryos from each group were pooled and homogenized in 0.7 ml pronase (Sigma, St. Louis, MO) solution (40 mM NaAc, 0.32 M NaCl, 0.167 mg/ml pronase, 0.2 mg/ml CS C, pH 6.5) and digested at 37°C overnight. Total GAG was then affinity purified by anion exchange chromatography with 0.5 ml DEAE-Sepharose (Pharmacia, Uppsala, Sweden) packed in Poly-Prep columns (Bio-Rad, Hercules, CA). The columns were washed with 20 mM sodium acetate, 0.25 M NaCl, pH 6.0, and eluted with 20 mM sodium acetate, 1 M NaCl, pH 6.0. After ß-elimination with 1 M sodium borohydride in 0.5 M NaOH and ethanol precipitation, total GAG was dissolved in water for further analysis. All structural analyses described were performed at least twice with similar results.

HPLC analysis of total GAG, HS, and CS
Total GAG composition was analyzed by anion exchange HPLC (TSK DEAE-3SW, 7.5 cm x 7.5 mm ID, Tosoh, Grove City, OH). Samples (0.5–1 x 105 cpm) were eluted with a linear gradient of 0.2 to 1 M NaCl in 10 mM KH2PO4, pH 6.0, containing 0.2% CHAPS at a flow rate of 1 ml/min, and the radioactivity in the effluent was determined by in-line liquid scintillation spectrometry (Packard, Meriden, CT) with sampling rate of every 12 s. The labeled total GAG is composed of keratan sulfate, HS, and CS, which was confirmed by sequential digestion with keratanase, chondroitinase ABC, and a mixture of heparitinase I, heparitinase II, and heparitinase III (also called heparinase) (Seikagaku, Tokyo).

The chain lengths of HS or CS (after removal of other components with respective bacterial lyases) were analyzed by gel filtration HPLC (TSK-GEL G4000SWXL, 60 cm x 7.5 mm ID, Tosoh). The column was equilibrated with 100 mM KH2PO4, 0.4 M NaCl, pH 6.0, and run in the same buffer at a flow rate of 0.5 ml/min. Blue dextran 2000 (Pharmacia) and [35S] sodium sulfate or standard disaccharide were used to determine the V0 and Vt of the column, respectively. HS cleavage products following heparanase digestion were first analyzed using TSK-GEL G4000SWXL as described. To separate the low-molecular-weight cleavage products further, samples were also analyzed using Biosep-SEC-S2000 (600 x 7.8 mm) coupled with Biosep-SEC-S3000 (600 x 7.8 mm) in 100 mM NH4HCO3 at a flow rate of 0.4 or 0.5 ml/min.

HS disaccharide composition was studied as described previously (Zhang et al., 2001Go). Briefly, 0.5–1 x 105 cpm [35S] HS was digested with a mixture of bacterial heparitinase I, II, and III (1 mU each) in 100 µl 40 mM ammonium acetate containing 3.3 mM CaCl2 (pH 7.0) overnight at 37°C. Heparitinase I recognizes the sequences GlcNAc/NS ± 6S(3S?)-{downarrow}GlcUA/IdoUA-GlcNAc/NS ± 6S. The arrow indicates the cleavage site. Heparitinase II has broad sequence recognition: GlcNAc/NS ± 6S(3S?)-{downarrow}GlcUA/IdoUA ± 2S-GlcNAc/NS ± 6S. Heparitinase III (heparinase) recognizes the sequence GlcNS ± 6S ± 3S-{downarrow} IdoUA2S/ GlcUA2S-GlcNS ± 6S. The reaction products and references can be found in Seikagaku's catalog. Virtually all HS was digested into disaccharides under the conditions just described. The resulting disaccharides were purified by Bio-Gel P2 chromatography (0.75 x 200 cm) and resolved by ion-pairing reverse-phase HPLC (Protein & Peptide C18, Vydac, Hesperia, CA) with appropriate disaccharide standards (Seikagaku). Samples were eluted with step gradients of acetonitrile in a solution containing 38 mM ammonium phosphate monobasic, pH 6.0, 1 mM tetrabutylammonium phosphate monobasic at a flow rate of 0.5 ml/min.

In vitro digestion of HS by heparanase
Active human heparanase protein was expressed in insect cells and purified by heparin-sepharose chromatography to >90% purity as described (McKenzie et al., 2003Go). The purified protein was resolved on a 4–20% gradient sodium dodecyl sulfate–polyacrylamide gel and stained with Coomassie brilliant blue (Figure 3B). For in vitro digestion, 0.5–1 x 105 cpm HS from the left or the right heart field were incubated with heparanase (0.73 ng) in 100 µl 100 mM sodium acetate/5 mM CaCl2, pH 5.6 at 37°C for 24 h before analysis by gel filtration chromatography.

Microinjection
Microinjection of 9.2 nl materials was performed with a Drummond Nanoject II into either the right or the left pericardial cavity (embryonic celom) at stage 7–10. Injected materials include the active form of the human heparanase (in 25 mM Tris–HCl, 150 mM NaCl, pH 7.4), bacterial heparitinase I and heparitinase III (in phosphate buffered saline [PBS]), unmodified or modified porcine intestinal mucosal heparin in PBS including CDSNAc (Seikagaku), NDSNAc (Seikagaku), selectively 6-ODS, and selectively 2-ODS (Neoparin, San Leandro, CA). The same amount of corresponding buffer was injected into the contralateral control side. Control embryos were injected with buffer alone into both the left and the right pericardial cavities. An illustration of injection is shown in Figure 3A. Embryos were then returned to the incubator and cultured for additional 3 h for in situ hybridization, histology, and immunohistochemistry, or overnight until they reach stage 11–12 for the examination of the heart looping direction.

In situ hybridization
Whole-mount in situ hybridization was performed essentially as described (Schultheiss et al., 1995Go), using digoxigenin-labeled RNA probes to chick heparanase (two probes were used, which were 645 and 1360 bases in length, respectively), chick Nodal (Levin et al., 1995Go) and chick Pitx2 (St Amand et al., 1998Go). The 645- and 1360- base-pair fragments of chick heparanase were generated by reverse transcription PCR of total RNA isolated from stage 4–8 chick embryos, using forward primer 5'-GTGGCACCAGTACAGATTTCCT-3', reverse primer1 5'-AATCCTCCCTCGTTGCACTT-3', and reverse primer2 5'-ACTCTGCGTGCTCAAATGCAAG-3'. PCR fragments were then gel purified and blunt ligated into pPCR-Script Amp SK(+)(Stratagene, La Jolla, CA) and sequenced. To generate heparanase RNA probes, plasmid was digested with EcoRI and transcribed with T3 RNA polymerase (645-base probe), or digested with NotI and transcribed with T7 RNA polymerase (1360-base probe). Embryos were photographed through a Nikon SMZ stereoscopic zoom microscope using either a Nikon FX-35DX regular film camera or a Carl Zeiss AxioCam MR color digital camera. Selected embryos were then embedded in gelatin and sectioned as previously described (Schultheiss et al., 1995Go). Sections were examined and photographed through a Nikon Microphot-SA microscope.

Histology and immunohistochemistry
Normal embryos or embryos treated with heparanase for 3 h were rinsed in PBS and fixed in 4% paraformaldehyde/PBS for 30 min at room temperature. After rinsing with PBS, embryos were incubated in 20% sucrose/PBS at 4°C overnight before embedding in OCT compound in iso-pentane/dry ice bath. Frozen blocks were stored at –80°C until sectioning. Frozen sections (8 µm) were used for both regular hematoxylin and eosin staining and immunolocalization of HS and fibronectin. The distribution of HS was detected by monoclonal antibody against HS 10E4 epitope (David et al., 1992Go) (Seikagaku) followed by fluorescein isothiocyanate–conjugated rabbit anti-mouse IgM (Sigma). Fibronectin was detected by monoclonal antibody against chick fibronectin (B3/D6, Developmental Studies Hybridoma Bank at the University of Iowa), followed by fluorescein isothiocyanate–conjugated goat anti-mouse IgG (Santa Cruz). Immunostained sections were examined using a Zeiss LSM510 confocal microscope.


    Acknowledgements
 
We are grateful to Dr. Zhengliang Wu and Andre Love for help on characterizing the native and modified heparins and to Miroslaw Lech for his help with the mass spectrometer. We thank Drs. Clifford J. Tabin and YiPing Chen for providing chick Nodal and Pitx2 probes. The monoclonal antibody B3/D6 against chick fibronectin was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biological Sciences at the University of Iowa. This work was supported by HL59479 and HL65231 from the NIH (to R.D.R.).


    Footnotes
 
2 To whom correspondence should be addressed; e-mail: rdrrosen{at}mit.edu

1 Present address: Department of Pathology and Laboratory Medicine, University of Wisconsin–Madison, Madison, WI 53706 Back


    Abbreviations
 
CDSNAc, completely desulfated and N-acetylated heparin; CS, chondroitin sulfate; ECM, extracellular matrix; FGF, fibroblast growth factor; GAG, glycosaminoglycan; HPLC, high-performance liquid chromatography; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; L–R, left–right; NDSNAc, N-desulfated and N-acetylated heparin; ODS, O-desulfated heparin; PBS, phosphate buffered saline; PCR, polymerase chain reaction


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 Materials and methods
 References
 
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