Glycobiology Advance Access originally published online on May 23, 2007
Glycobiology 2007 17(8):820-827; doi:10.1093/glycob/cwm056
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Published by Oxford University Press 2007
O-linked glycan expression during Drosophila development
2 Developmental Glycobiology Unit, NIDCR, National Institutes of Health, Building 30, Room 426, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370
1 To whom correspondence should be addressed; Fax: +1 301 4020897; E-mail: kelly.tenhagen{at}nih.gov
Received on October 23, 2006; revised on April 18, 2007; accepted on May 17, 2007
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentReferences |
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Mucin-type O-linked glycosylation is an evolutionarily conserved protein modification that is essential for viability in Drosophila melanogaster. However, the exact role of O-glycans and the identity of the crucial apoproteins modified with O-linked N-acetylgalactosamine (O-GalNAc) remain unknown. In an effort to elucidate the O-linked glycans expressed during Drosophila development, we have employed fluorescent confocal microscopy using a battery of lectins and an antibody specific for the GalNAc
–Ser/Thr structure (Tn antigen). Confocal microscopy provides high-resolution images of the diversity of glycans expressed in many developing organ systems. In particular, O-glycans are highly expressed on a number of ectodermally derived tissues such as the salivary glands, developing gut, and the tracheal system, suggesting a role for O-glycans in cell polarity and tube formation common to these organs. Additionally, O-glycans are found in the developing nervous system and within subregions of developing tissues known to be active in cell signaling events. This study provides us with temporal and spatial information regarding O-glycan expression as well as a set of reagents for the isolation of glycoproteins from specific developmental stages and organ systems. This information will aid us in identifying the in vivo substrates of the UDP–GalNAc: polypeptide N-acetylgalactosaminyltranferases, in a continuing effort to define the biological role of O-linked glycoproteins during development. Key words: development / Drosophila / glycosylation / lectins / pgant
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentReferences |
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Mucin-type O-linked glycosylation is an evolutionarily conserved protein modification found in diverse species including mammals, fish, insects, worms, and some types of fungi (Hagen et al. 1999
; Schwientek et al. 2002; Ten Hagen and Tran 2002; Ten Hagen, Fritz et al. 2003; Ten Hagen, Tran et al. 2003). The initiation of this form of O-glycosylation is performed by a family of enzymes known as the UDP–GalNAc: polypeptide N-acetylgalactosaminyltranferases (ppGaNTases or ppGalNAcTs in mammals and PGANTs in Drosophila), which catalyze the transfer of N-acetylgalactosamine (GalNAc) from the nucleotide sugar, UDP–GalNAc, to the hydroxyl group of serines and threonines on protein substrates (Ten Hagen, Fritz et al. 2003; Hang and Bertozzi 2005). Members of this family have characteristic substrate preferences as well as patterns of expression during development (Kingsley et al. 2001; Ten Hagen and Tran 2002; Ten Hagen, Fritz et al. 2003; Ten Hagen, Tran et al. 2003; Tian and Ten Hagen 2006). Previous work has demonstrated that certain isoforms have been conserved over the course of evolution, with unique orthologous pairs existing in mammals and in the fruit fly, Drosophila melanogaster (Schwientek et al. 2002; Ten Hagen and Tran 2002; Ten Hagen, Tran et al. 2003). Biochemical analyses revealed that these orthologs share functional conservation, as they have similar substrate preferences in vitro as well as sites of addition within those substrates (Ten Hagen, Tran et al. 2003). These data suggest a key biological role for these enzymes that is required for both insect and vertebrate development.
Recent studies have begun to identify crucial biological roles for members of this enzyme family. Familial tumoral calcinosis in humans, which is characterized by the development of subdermal calcified tumors, hyperphosphatemia, and increased bone density, results from mutations in the ppGaNTase-T3 isoform in certain families (Topaz et al. 2004). Additionally, studies in Drosophila have demonstrated that one isoform (pgant35A) is essential for viability and development (Ten Hagen and Tran 2002; Schwientek et al. 2002). A recent study from our laboratory has found that this isoform is involved in regulating epithelial tube formation during development of the embryonic respiratory system in Drosophila (Tian and Ten Hagen 2007). While these recent studies have begun to elucidate the biological importance of members of this family, the repertoire of in vivo substrates upon which the enzymes act remains a major gap in our knowledge. In an effort to define the temporal and spatial expression of O-glycans during Drosophila development as well as to identify reagents to isolate O-linked glycoproteins, we have performed high-resolution confocal microscopy using lectins that detect a wide array of O-linked structures. The information provided here corroborates previous developmental expression patterns seen for the pgant gene family (Tian and Ten Hagen 2006) and provides evidence for the widespread presence of O-glycans in many developing organ systems. The reagents employed can now be used in an informed proteomics-based approach to isolate the in vivo substrates of the PGANT enzymes.
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Results |
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Lectin staining during early-mid Drosophila embryogenesis (stages 3–10)
Drosophila embryos were stained with fluorescently labeled lectins (Table I) to detect various O-linked glycans present during development. Additionally, lectins that detect N-glycans were also employed for comparison. During the early stages of Drosophila embryogenesis, from initial cellularization to the multilayered embryo formed after gastrulation (stages 3–10), the glycans detected are predominantly those recognized by Jacalin (O-linked glycans) (Tachibana et al. 2006
) and concanavalin A (ConA; N-linked glycans) (Figure 1). Jacalin and ConA both stain the outer ectodermal layer of cells during cellular blastoderm (stages 3–5). Later, at germ band elongation stages (stages 9–10), ConA continues to stain the ectodermal layer. Jacalin detects the ectoderm, the amnioserosa (a structure involved in cell adhesion and migration during dorsal closure), and the developing tracheal system (tracheal placodes).
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Lectin staining during mid-embryogenesis (stages 11–14)
During later stages of embryonic development, glycans detected by other lectins are now observed in addition to those seen during early embryogenesis. O-glycans detected by Jacalin are found on the epidermis and the remainder of the amnioserosa (Figure 2). Additionally, O-glycans detected by peanut agglutinin (PNA) are now seen in the epidermis and developing central nervous system (CNS) (Figure 2), similar to what was seen previously by D'Amico and Jacobs (1995)
. Soybean agglutinin (SBA) staining is now seen intensely on ectodermally derived structures, such as the foregut, hindgut, salivary gland, and portions of the tracheal system (respiratory system), similar to previous studies (D'Amico and Jacobs 1995; Fredieu and Mahowald 1994). Vicia villasa agglutinin (VVA) (Manimala et al. 2005) is also found in the foregut, hindgut, salivary gland, and tracheal system. ConA staining of N-glycans is predominantly seen on the epidermis. Wheat germ agglutinin (WGA), which detects sialic acid and chitin, the N-acetylglucosamine (GlcNAc) polymer, is now present and found in the epidermis as well as in ectodermally derived structures such as the hindgut, foregut, salivary glands, and tracheal system that had not been detected in previous studies (D'Amico and Jacobs 1995; Fredieu and Mahowald 1994).
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Lectin staining during late embryogenesis (stages 15–17)
Figure 3 shows the staining pattern of seven lectins in late-stage embryos. Jacalin is again found in the epidermal layer. Additionally, confocal sectioning through the embryo reveals very weak Jacalin staining in the expanding tracheal system. PNA is again found predominantly in the CNS at this stage. Dolichus biflorus agglutinin (DBA) is detected for the first time in the dorsal longitudinal trachea (dlt) and epidermis; intense staining is also seen in the pharynx. In addition to epidermal staining, confocal sectioning of SBA-stained embryos reveals signal in the foregut and tracheal system at this stage as well (Figure 3). VVA staining is found in a number of tissues, including the epidermis, pharynx, esophagus, salivary gland, and hindgut and tracheal system. Confocal sections through the ConA-stained embryo show staining in the pharynx, esophagus, and portions of the trachea that had not previously been seen using standard immunohistochemistry. WGA is again found in the salivary gland, esophagus, and tracheal system. Embryos that are homozygous for a mutant pgant35A gene (Tian and Ten Hagen 2007
) were also stained with the aforementioned lectins. While ConA staining is largely unaffected in these mutants, the staining patterns of lectins detecting O-glycans in certain tissues are greatly reduced (Jacalin staining along the epidermis; DBA staining in the tracheal system; and VVA staining in the foregut, hindgut, salivary glands, and tracheal system). However, not all O-glycan staining in these mutants is abrogated (PNA staining in the CNS, DBA staining in the pharynx, and SBA and VVA staining in the epidermis), indicating a role for other members of the pgant family in the glycosylation of protein substrates during embryonic development.
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Tn antibody staining during Drosophila embryogenesis
We also employed an antibody (Tn Ab) known to be highly specific for the unmodified Tn antigen (Tn Ag; GalNAc
–Ser/Thr) formed by the pgant family to visualize O-glycans expressed during Drosophila embryogenesis. The Tn Ab recognizes the central yolk component at early stages of development (Figure 4A and B). As embryogenesis proceeds, staining can be seen in many tissues of ectodermal origin, including the developing tracheal placodes, the invaginating hindgut and salivary glands (Figure 4C–G) (stages 10–12). Confocal sectioning through stage 13 embryos (Figure 4H and I) reveals intense staining in many tissues, including the expanding tracheal system, foregut, hindgut, salivary glands, the malpighian tubules (functional equivalent of the kidney), and posterior spiracles (part of the respiratory system). Tn Ab staining is seen in these tissues at stage 17 as well (Figure 4K–M) using confocal sectioning, with notable staining throughout the ramified tracheal system. Under higher magnification, Tn Ab staining can be seen along the apical surfaces and luminal space of the developing malpighian tubules (Figure 4O), tracheal system (Figure 4P–R), salivary glands (Figure 4S and T), foregut (Figure 4U), and hindgut (Figure 4V). Additional punctate staining can be seen within the cytoplasm of the cells that comprise these organs, which probably represents vesicles transporting these O-linked proteins to the apical/luminal surfaces. Tn Ab staining is shown at the bottom for homozygous null mutants in one member of the pgant family in the fly (pgant35A). There is a marked decrease in Tn Ab staining seen in certain tissues within the mutants as well as phenotypic abnormalities in these embryos, suggesting that the GalNAc–Ser/Thr structure on proteins plays an important role during embryonic development. Indeed, recent work from our laboratory has demonstrated that pgant35A plays a role in the proper development of the embryonic tracheal system (Tian and Ten Hagen 2007). Residual Tn Ab staining seen in certain tissues is probably the result of the activity of the additional members of this enzyme family that initiate O-linked glycosylation.
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Lectin and Tn Ab staining of third instar larval imaginal discs
During the larval stages of development, epithelial sacs are formed within the larvae that will grow and differentiate to become the adult structures of the fly. These sacs or infoldings are known as imaginal discs. We next examined the imaginal discs of larvae after staining with lectins and the Tn Ab (Figure 5). In the eye-antennal discs, which are destined to become the adult eye, antenna and head, staining was seen with Tn Ab, VVA, Jacalin, and WGA in a distinct line along the morphogenic furrow of the presumptive eye. This region of the developing eye is an area of intense cell signaling as cells transition from a rapidly dividing state to a differentiated state, thus implicating glycans in these processes. Diffuse staining across the surface of the eye disc was seen for SBA. ConA stained only the peripheral regions of this disc.
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The wing disc, leg disc, and haltere disc were stained as a group. Notable staining was seen for SBA and Jacalin, which detected the ventral region of the wing disc slightly more than the dorsal region. Additionally, Jacalin and VVA were seen in a band of cells along the border of the dorsal and ventral regions of the wing disc, another region characterized by cell signaling events. VVA, SBA, Jacalin, WGA, and the Tn Ab stained the circumference of the ridges of the wing, leg, and haltere discs. ConA was only found around the peripheral regions of each disc.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentReferences |
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The results presented here shed new light on the spatial and temporal expression of specific O-linked glycans during Drosophila development. Through the use of fluorescent confocal microscopy, we are able to visualize the patterns of O-linked glycan expression throughout embryogenesis and in imaginal discs. We show specific developmental expression of a variety of mucin-type O-linked glycans using the lectins Jacalin, PNA, DBA, SBA, and VVA. Additionally, we show striking expression of the Tn Ag (GalNAc
–Ser/Thr) using an antibody specific for this structure. By examining serial Z sections through the three-dimensional samples, we are able to see O-glycans present in sub-regions of internal structures and developing organs within the embryo.
Previous studies examining lectin-staining patterns during Drosophila development employed immunohistochemical methods using standard microscopy (Fredieu and Mahowald 1994; D'Amico and Jacobs 1995). While these studies provided a great deal of information on glycoconjugate expression, confocal microscopy allows visualization of a plane of focus within the embryo, allowing structures not previously seen to be stained. In our studies, we detected additional staining on internal structures and developing organs not previously seen with DBA, ConA, and WGA. Our studies also employed additional lectins (Jacalin and VVA) that detect a number of other O-linked glycans. We also used an antibody specific to the unmodified Tn Ag to obtain striking images of O-glycan expression throughout the development of many organ systems. Finally, we provide the first examination of glycans present in imaginal discs, the larval-derived epithelial sacs that will eventually develop into the adult structures of the fly. Our results provide high-resolution imaging of the diversity of O-glycans present during many stages of Drosophila development.
Our results highlight the diverse organ systems and developmental processes potentially influenced by O-glycans, as organ- and tissue-specific patterns were observed for a number of lectins. For example, the lectins Jacalin and PNA, which detect various elaborations of the Tn Ab structure, are found specifically in the amnioserosa and CNS, respectively. Additionally, Jacalin and SBA stained the ventral portion of the wing imaginal disc more than the dorsal region, suggesting a role for these glycans in that specific wing compartment. Of further note is the staining seen for Jacalin and VVA along a band of cells separating the dorsal and ventral portions of the wing disc. This is a region where critical cell signaling events take place in the developing disc, potentially implicating Jacalin- and VVA-specific glycans in these processes.
Additional expression patterns of interest were those seen with the Tn Ab, VVA, Jacalin, and WGA in the morphogenic furrow of the developing eye. The morphogenic furrow is an epithelial indentation of cells which moves from posterior to anterior as the eye develops, leaving differentiated ommatidial clusters posterior to the position of the furrow. Signaling events occur anterior and posterior to the furrow that restrict the boundaries and movement of the furrow (Pignoni and Zipursky 1997; Cho et al. 1998). Previous work identified a glycoprotein expressed in the furrow that regulates Notch–Delta signaling events during the differentiation of the eye (Fetchko et al. 2002). The lectin staining seen in this region may reflect the presence of glycoproteins such as this one that are acting to regulate signaling pathways around the region of the furrow. Future mutational work in the fly will be necessary to understand the role of each glycan in these developmental processes.
It is particularly interesting to note that the most intense and widespread staining seen during embryogenesis was that of the Tn Ab, which detects the unmodified GalNAc–Ser/Thr structure. Those lectins that detect more extended O-glycan structures (Jacalin and PNA) were more restricted in their tissue distribution. These results suggest that the primary O-glycans present in many tissues within the developing fly embryo are the unmodified Tn Ag. It also suggests a unique developmental role for the glycans detected by Jacalin in the amnioserosa and by PNA in the developing nervous system.
Many of the tissues stained by the Tn Ab, such as the salivary glands, developing gut and tracheal system, are of ectodermal origin and are comprised of polarized epithelial cells actively involved in secretion. Higher magnification images reveal apical and luminal staining for the Tn Ab in most of these tissues, suggesting a role for Tn Ag-containing proteins along the apical surface in regulating epithelial development and tube formation common to these tissues (Andrew et al. 2000; Weinkove and Leevers 2000). Indeed, we have recently found that pgant35A is required for the proper development of epithelial tubes in the embryonic tracheal system (Tian and Ten Hagen 2007). The loss of this enzyme results in loss of tracheal tube integrity, concomitant with a decrease in apical Tn Ab staining within the tracheal system (Tian and Ten Hagen 2007). Here, we show that the tracheal staining with a number of O-glycan-specific lectins was also diminished in the pgant35A mutants. Other types of glycans, such as those detected by WGA and ConA, were not appreciably affected in pgant35A mutants. Thus, the Tn Ab is probably detecting key Tn Ag-decorated glycoproteins involved in tracheal tube integrity and may therefore be a useful reagent for the isolation of these crucial molecules.
The lectin and Tn Ab staining data reported in this study are in good agreement with gene expression patterns seen previously for the pgant family members (Tian and Ten Hagen 2006). pgant gene expression was found in the embryonic fore-, mid-, and hindgut, salivary glands, amnioserosa, trachea, pharynx, esophagus, proventriculus, and epidermis, as is seen when staining with lectins and antibodies that are specific for O-glycans. pgant expression was also detected in the same areas of the imaginal discs that stained with various O-glycan-specific lectins, such as the morphogenic furrow of the eye disc and the ventral region of the wing discs. Additionally, one member of the core 1 ß1,3-galactosyltransferase family [which adds galactose (Gal) in a ß1,3-linkage to GalNAc–Ser/Thr, forming the core 1 structure] was found to be highly expressed in the amnioserosa (Muller et al. 2005), a tissue intensely stained, in our study, by Jacalin. Few expression data are currently available for other genes contributing to glycan biosynthesis. It will be of interest to see when and where other genes involved in both N- and O-glycan biosynthesis are present and expressed in Drosophila.
The data presented here provide the community with a new set of markers and tools for interrogating the development of various organ systems during embryogenesis. In the case of the Tn Ab, vivid images of the developing tracheal system are obtained beginning at stages very early in development and continuing until the end of embryogenesis. In contrast to other markers, which are first seen late or are present for a limited time, the Tn Ab will be useful for discerning aberrations in tracheal development throughout embryogenesis as well as visualizing development of the foregut, hindgut, salivary gland, and malpighian tubules. Additionally, knowing the patterns of lectin- and glycan-specific antibody staining during development can provide us with tools for the isolation of glycoproteins expressed in specific places at specific times. Identification of the substrates for the polypeptide GalNAc transferases remains a continuing challenge, as there is no reliable way to predict sites of glycosylation in all instances. In contrast to N-linked glycosylation, there is no consensus sequence for O-GalNAc addition, no single reagent that will detect all O-linked glycoforms and no endoglycosidase that will remove all O-linked chains regardless of their length or structure. The Tn Ab or lectins of interest can be used as affinity reagents to isolate O-glycosylated substrates present during normal embryonic development. The identification of O-linked glycoproteins will be a crucial step toward understanding the role O-glycans play in diverse developmental processes.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentReferences |
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Preparation of Drosophila embryos and larval tissues
Wild-type Drosophila embryos (Oregon R) were collected and fixed as previously described (Tian and Ten Hagen 2006
). pgant35A maternal/zygotic (m/z) mutant embryos were collected as described previously (Tian and Ten Hagen 2007). The third instar wandering larvae were dissected and inverted to expose imaginal discs and other organs, and then treated as described previously (Tian and Ten Hagen 2006).
Whole-mount lectin and antibody staining
Immunostaining with lectins and the Tn Ab was performed according to standard procedures. The fluorescent-conjugated lectins, Canavalia ensiformis (ConA), Dolichos biflorus (DBA), Artocarpus integrifolia (Jacalin), Vicia villosa (VVA), and Triticum vulgare (WGA) were purchased from EY Laboratories (San Mateo, CA). Alexa fluorescent-conjugated Arachis hypogea (PNA) and Glycine maximus (SBA) were purchased from Molecular Probes (Eugene, OR). All lectins were used at 10 µg/mL. Mouse monoclonal anti-Tn Ab (Ca3638; 1:50) was the kind gift of Dr Richard Cummings who had acquired the stocks of antibodies and hybridomas from the late Dr Georg F. Springer (Avichezer et al. 1997). Fluorescein isothiocyanate conjugated secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and used at the concentration of 1:100. Carbohydrate inhibition controls were performed by pre-incubation of lectins with mannose (Man; for ConA), Gal (for Jacalin, PNA, and SBA), GlcNAc (for WGA), or GalNAc (for VVA, DBA, and Tn Ab) for 30–60 min at room temperature. All sugar inhibitors were purchased from Sigma (St. Louis, MO) and used at 0.2 M.
Mounting and imaging
Stained third instar larvae were dissected in 2% 1,4-diazabicyclo-[2.2.2.]octane (Sigma) in 70% glycerol/phosphate-buffered saline to separate imaginal discs and other organs. Embryos and larval tissues were mounted in the aforementioned solution containing Hoechst 33342 (Molecular Probes; 1:20000) to counterstain nuclei and then stored at –20°C. Zeiss LSM 510 confocal laser scanning microscope was used for sample analysis. Optical thicknesses of confocal images of embryos were between 1 and 3 µm. Imaginal discs are shown as confocal projection images using the LSM browser software (Zeiss, Thornwood, NY). Embryos were staged according to Hartenstein (1993). Images were processed by the LSM Image Browser and assembled in Photoshop.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentReferences |
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None declared.
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Acknowledgment |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentReferences |
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We would like to thank Dr Lawrence Tabak for helpful discussions and Dr Richard Cummings for the Tn antibody. This research was supported by the Intramural Research Program of the NIDCR, NIH.
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Abbreviations |
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CNS, central nervous system; ConA, concanavalin A; DBA, Dolichus biflorus agglutinin; dlt, dorsal longitudinal trachea; Gal, galatose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Man, mannose; m/z, maternal/zygotic; PNA, peanut agglutinin; ppGaNTase or ppGalNAcT or pgant, UDP–GalNAc:polypeptide N-acetylgalactosaminyltransferase; SBA, Soybean agglutinin; VVA, Vicia villosa; WGA wheat germ agglutinin
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References |
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