InteractiveFly: GeneBrief

egghead: Biological Overview | References

BIOLOGICAL OVERVIEW

Glycosphingolipids (GSLs) are present in all eukaryotic membranes and are implicated in neuropathologies and tumor progression in humans. Nevertheless, their in vivo functions remain poorly understood in vertebrates, partly owing to redundancy in the enzymes elongating their sugar chains. In Drosophila, a single GSL biosynthetic pathway is present that relies on the activity of the Egghead and Brainiac glycosyltransferases. Mutations in these two enzymes abolish GSL elongation and yield oogenesis defects, providing a unique model system in which to study GSL roles in signaling in vivo. This study used egghead and brainiac mutants to show that GSLs are necessary for full activation of the EGFR pathway during oogenesis in a time-dependent manner. In contrast to results from in vitro studies, it was found that GSLs are required in cells producing the TGFα-like ligand Gurken, but not in EGFR-expressing cells. Strikingly, it was found that GSLs are not essential for Gurken trafficking and secretion. However, this study characterized the extracellular Gurken gradient and showed that GSLs affect its formation by controlling Gurken planar transport in the extracellular space. This work presents the first in vivo evidence that GSLs act in trans to regulate the EGFR pathway and shows that extracellular EGFR ligand distribution is tightly controlled by GSLs. This study assigns a novel role for GSLs in morphogen diffusion, possibly through regulation of their conformation (Pizette, 2009).

Glycosphingolipids (GSLs) are ubiquitous components of eukaryotic cell membranes. They consist of a variable oligosaccharide chain attached to a ceramide lipid backbone (Cer) that tethers them to the lumenal leaflet of membranes. GSLs are mainly synthesized from Ceramide in the Golgi apparatus by a stepwise process in which unique glycosyltransferases add monosaccharides to a growing lipid-linked oligosaccharide chain. They are subsequently exported towards the plasma membrane, their principal location, where they are enriched together with cholesterol in membrane microdomains. The expression of a particular GSL is differentially regulated according to the developmental stage, the cell type and its differentiation state. The role of vertebrate GSLs has mostly been addressed in vitro (reviewed by Degroote, 2004; Sillence, 2004). These studies indicate that cell-surface GSLs participate in adhesion through the binding in trans of lectins or other GSLs. GSLs can also bind in cis to directly modulate the activity of receptor tyrosine kinases (RTKs) at the plasma membrane. GSLs are also thought to be involved in vesicular transport along the exocytic and endocytic pathways, sorting proteins into different compartments. Lastly, the presence of GSLs in membrane microdomains, which are considered as signaling platforms, may underlie many of their functions (Pizette, 2009).

There is, however, little in vivo evidence to support any of these presumed functions. In S. cerevisiae, mutants abolishing all GSL synthesis fail to show defects in intracellular trafficking (Lisman, 2004). In C. elegans, GSLs appear to be dispensable throughout life (Griffitts, 2005). In mammals, the vast majority of GSLs are built on glucosylceramide (GlcCer), and synthesis branches at the level of the third glycosyl residue to yield three classes (lacto-, globo- and ganglioseries). Knockout of the mouse GlcCer synthase gene (Ugcg) leads to early embryonic lethality, for unclear reasons; assessing the effects of knocking out downstream glycosyltransferases is complicated by redundancy in these genes and between different GSLs (reviewed by Sabourdy, 2008). Nonetheless, disrupting the ganglioseries pathway produces mice that display neurological abnormalities after birth. Interestingly, in humans, mutations affecting GSL synthesis and degradation trigger severe neuropathologies (reviewed by Kolter, 2006). Therefore, GSLs are at least required for proper function of the adult nervous system, but no firm link has yet been established between this requirement and their proposed cellular roles (Pizette, 2009).

Drosophila GSLs are simpler in structure than their vertebrate counterparts, with a single biosynthetic pathway described to date, giving rise to a family of differentially elongated molecules (Seppo, 2000). Egghead and Brainiac are glycosyltransferases responsible for GSL biosynthesis in the fly, catalyzing the addition of the second and third glycosyl residues of the GSL oligosaccharide chain (Schwientek, 2002; Wandall, 2003; Wandall, 2005). There is no redundancy in these enzyme functions and no alternate biosynthetic pathway. Hence, egh and brn mutants are devoid of elongated GSLs and provide a useful model system for studies of GSL functions in vivo. Importantly, mutations in each gene are lethal and cause identical phenotypes during oogenesis and embryogenesis that are reminiscent of loss-of-function in the Notch receptor and EGF RTK (EGFR) pathways (Goode, 1996a; Goode, 1996b). Since the expression of a GSL-dedicated human galactosyltransferase in Drosophila egh mutants rescues their viability and fertility in a brn-dependent fashion (Wandall, 2005), these data indicate that Drosophila GSLs are essential for development, perhaps by modulating signaling (Pizette, 2009).

During Drosophila oogenesis, activation of the EGFR pathway primarily depends on Gurken (Grk), an EGFR ligand similar to vertebrate TGFα, that is secreted by the oocyte. The EGFR-Grk couple acts twice to polarize the follicular epithelium as well as the future embryo along both anteroposterior (AP) and dorsoventral (DV) axes. Despite ubiquitous expression of EGFR in follicle cells, its activation is spatially restricted by asymmetric Grk localization. In early oogenesis, grk mRNA and protein are enriched at the posterior pole of the oocyte, and Grk activates EGFR in neighboring follicle cells, inducing them to adopt a posterior fate. At mid-oogenesis, these cells signal back to the oocyte, resulting in a reorganization of its cytoskeleton, a redistribution of oocyte maternal determinants along the AP axis, and the movement of the nucleus towards the anterior oocyte cortex. Since grk RNA remains associated with the oocyte nucleus, a new restricted source of Grk is created to limit the highest activation of EGFR to the adjacent follicle cells, instructing them to assume a dorsal identity. Respiratory appendages are eggshell structures derived from dorsolateral follicular cells and their examination is an excellent means to monitor EGFR signaling. Indeed, mild Grk or EGFR loss-of-function causes a fusion of the respiratory appendages owing to the absence of the dorsal-most cells (weak ventralization). By contrast, a more severe reduction in EGFR signaling abrogates the formation of these structures (complete ventralization) (Pizette, 2009 and references therein).

This study addresses the role of GSLs in EGFR signaling during oogenesis using egh and brn mutants. First, it was shown that GSLs exert a temporal control on the level of activation of EGFR. No evidence was found of a role for GSLs in the direct modulation of EGFR activity, but instead GSLs act at the level of the EGFR ligand Grk. Despite reports of GSL function in trafficking, the results indicate that GSLs are dispensable for Grk export to the plasma membrane and for its secretion. However, by observing the gradient of secreted Grk, this study shows that GSLs control Grk diffusion in the extracellular space (Pizette, 2009).

Once Grk is secreted, GSLs are necessary for its efficient diffusion in the extracellular space. The possibility cannot be dismissed that GSLs might have a slight influence on Grk secretion, a decrease in Grk secretion cannot explain the observed changes in the extracellular Grk gradient shape in the brn mutant or the higher levels of secreted Grk that accumulate above the source, as compared with the wild type. GSLs are thus crucial for the formation of a Grk gradient that is able to achieve maximal activation of EGFR (Pizette, 2009).

Surprisingly, GSLs were found to be involved only in the final step of Grk signaling during the establishment of the DV axis. Prior to DV patterning, Grk activates EGFR to set the AP axis of the egg. It was observed, however, that AP polarity is not compromised in egh and brn alleles. The analysis of the distribution of the intermediate product mactosylceramide (MacCer) during oogenesis also supports the idea that the GSL biosynthetic pathway is not active at the time AP patterning is established. Grk signaling therefore seems to be more sensitive to GSL function for the determination of DV fates (Pizette, 2009).

Even during this process, there appear to be differential requirements for GSL activity. DV patterning proceeds in two distinct temporal phases of EGFR signaling, but only the second is under the control of GSLs. In a first phase (between stages 8 and 10a), the EGFR pathway establishes embryonic DV polarity and dorsal follicle cell fates. This phase culminates in the induction of rho1 transcription in DA follicle cells and depends on paracrine signaling mediated by Grk. This initial phase is not overtly affected in brn mutants as evidenced by the observation that their embryos have a normal DV axis (Goode, 1992) and it was found that rho1 expression was still induced. By contrast, the second phase of EGFR signaling is triggered by rho1 expression and corresponds to an amplification of EGFR activity needed to split the RA. This phase is disrupted in the brn mutant because the expression of rho1 and aos is not upregulated, as exemplified by the fusion of the RA (Pizette, 2009).

According to Wasserman and Freeman, the amplification phase is independent of Grk and relies on autocrine EGFR signaling (Wasserman, 1998). However, this study shows that GSLs, unlike the other molecules implicated in this process, act in the germ line to regulate the distribution of extracellular Grk. This indicates that this phase is not solely autocrine and that there is still a need for Grk-mediated paracrine signaling (Pizette, 2009).

At stage 10a, the vitelline membrane is already being deposited as vitelline bodies in the extracellular space between the oocyte and the follicular epithelium. Morphological data show that these bodies have not yet fused, leaving space for a number of interdigitating microvilli emanating from the oocyte membrane and the apical side of the follicle cells. Since the brn mutation specifically affects Grk signaling at this stage, it is proposed that GSLs play a role in Grk accessibility to follicle cells and that this is likely to be mediated through the microvilli. In support of this, at stages at which elongated GSLs are not essential (AP patterning and the onset of DV patterning), the oocyte plasma membrane is closely apposed to the apical side of the follicle cells. This, therefore, supports the hypothesis that GSLs are only required when Grk is not easily accessible to its receptor (Pizette, 2009).

This study provides the first experimental description of the wild-type extracellular Grk gradient at stage 10a and uncovers an unexpected feature: at the dorsal midline, past the source, high and steady levels of Grk are maintained over about half of the AP axis length. This result is at odds with a mathematical modeling of the Grk gradient that predicted a shallow decrease from anterior to posterior. However, from a strong and constant level of Grk over half the dorsal midline, it is expected that the expression domains of the Grk primary target genes have an identical width along most of the AP axis. This is precisely what is observed for kekkon-1 and pipe, which are clearly EGFR primary target genes. It is thus suggested that a stripe-shaped source of extracellular Grk along the dorsal midline, rather than a point-like source of Grk above the oocyte nucleus, is more efficient in accommodating patterning across the entire epithelium (Pizette, 2009).

Besides contributing to the shape of the Grk gradient, high Grk levels along the dorsal midline might serve to upregulate rho1 expression, leading to higher EGFR activity, aos expression and splitting of the RA primordium. Indeed, in the absence of elongated GSLs, weak rho1 expression is retained but it is not upregulated or refined. Grk signaling is necessary for this step. Since, in the brn mutant, there is a reduction in the high levels of extracellular Grk along the dorsal midline, it is proposed that a low Grk threshold is sufficient to initiate and maintain rho1 transcription (as well as the spatial regulation of EGFR), whereas a higher Grk threshold increases rho1 expression levels (Pizette, 2009).

What could be the basis for the discrepancy between these results and the mathematical modeling of the Grk gradient? In the latter (Goentoro, 2006), EGFR expression was assumed to be uniform throughout the follicular epithelium. However, this study showed that at stage 10a, EGFR levels are lower along part of the dorsal midline in a region coincident with that of high Grk levels. Furthermore, it was found that decreasing Grk binding to EGFR increased Grk spreading. Therefore, at the dorsal midline, the reduction in EGFR levels might saturate receptor occupancy. This could allow a large quantity of Grk to remain unbound, facilitating its movement toward the posterior pole (Pizette, 2009).

The most striking result of this study is that GSLs shape the extracellular Grk gradient and play a role in Grk diffusion without apparently interfering with the regulation of Grk diffusion by EGFR. But what could that role be? Grk movement in the extracellular space between the oocyte and the follicular epithelium is complicated by the formation of the vitelline membrane. Grk could either be released into the extracellular space or it could remain associated with the oocyte plasma membrane and localize to its microvilli. These alternatives could not be distinguished, since immunofluorescent staining is of insufficient resolution and the extracellular space was not well preserved in the immunoelectron microscopy experiments. Others have nevertheless reported the presence of Grk on microvilli (Bokel, 2006). GSLs could therefore be important for Grk targeting to microvilli versus flat portions of the membrane. This, however, is unlikely because Grk still activates EGFR in the brn mutant, indicating that it can encounter its receptor. By contrast, what Grk fails to do in the mutant context is to concentrate along the dorsal midline at a distance from its point of secretion. This suggests that GSLs function in the planar transport of Grk along the AP axis, from one oocyte microvillus to the next, a hypothesis supported by the fact that the oocyte microvilli were found to be oriented parallel to the AP axis (Pizette, 2009).

An intriguing property of secreted Grk in the brn mutant context is that it is detected by extracellular staining and not conventional immunostaining. In an effort to understand the basis for this, it was found that secreted Grk is sensitive to the presence of detergent and to temperature, suggesting that its conformation relies on the presence of GSLs once it reaches the cell surface. Interestingly, GSLs induce a conformational change in the amyloid β-protein upon its release from the plasma membrane (reviewed by Ariga, 2008). It is thus possible that under the experimental conditions, Grk conformation is not fully restored, modifying its ability to diffuse (Pizette, 2009).

In this case, how could the two processes be linked? There is increasing evidence that the spreading of secreted molecules depends on elaborate events involving their multimerization and/or incorporation into higher-order structures such as lipoprotein particles. It is therefore tempting to speculate that a change in secreted Grk conformation that depends on plasma membrane GSLs reflects its packaging into special structures that are required for its efficient transport along microvilli. Since mammalian GSLs can be shed from the plasma membrane and are found circulating with secreted lipoprotein particles (see Lauc, 2006), GSLs could enhance Grk spreading by delivering it to these particles (Pizette, 2009).

Another, non-mutually exclusive means by which GSLs could affect Grk diffusion is linked to their enrichment in plasma membrane microdomains. Since GSLs can interact with proteins through their oligosaccharide chain, GSLs could bind Grk, or a Grk co-factor, sorting Grk into such domains. It has been reported that Grk is potentially palmitoylated, and palmitoylation is one of the signals that target proteins to membrane microdomains. Because Grk recruitment to these domains has not been addressed and because the detection of extracellular Grk by biochemical means in ovaries has so been so far elusive, understanding how GSLs regulate Grk diffusion will have to await the generation of better tools (Pizette, 2009).

Egghead and Brainiac are essential for glycosphingolipid biosynthesis in vivo

The Drosophila genes, brainiac and egghead, encode glycosyltransferases predicted to act sequentially in early steps of glycosphingolipid biosynthesis, and both genes are required for development in Drosophila. egghead encodes a β4-mannosyltransferase, and brainiac encodes a β3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAcβ1-3Manβ1-4Glcβ1-Cer, found widely in invertebrates but not vertebrates. This study reports in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, it was demonstrated that despite fundamental differences in the core structure of mammalian and Drosophila glycosphingolipids, the Drosophila egghead mutant can be rescued by introduction of the mammalian lactosylceramide glycosphingolipid biosynthetic pathway (Galβ1-4Glcβ1-Cer) using a human β4-galactosyltransferase (β4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) resulted in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Manβ1-4Glcβ1-Cer. The study demonstrates that glycosphingolipids are essential for development of complex organisms and suggests that the function of the Drosophila glycosphingolipids in development does not depend on the core structure (Wandall, 2005).

Invertebrates, C. elegans and Drosophila, have attracted considerable attention as model organisms for deciphering specific biological roles of complex carbohydrates. One elegant example of this was a number of studies leading to the identification of a series of glycosylation genes critical for vulval invagination in C. elegans, which were all shown to affect a common biosynthetic pathway for the assembly of the O-linked oligosaccharide linker region common for all proteoglycans (Selleck, 2000). Another example was the role of the O-linked fucose glycosylation pathway on the Notch receptor function (Haltiwanger, 2004). The Drosophila neurogenic genes brainiac and egghead encode glycosyltransferases essential for epithelial development during oogenesis and in the embryo. egghead and brainiac mutants display similar, non-additive defects, which has led to the proposal that they act in the same pathway. brainiac has been shown to encode a UDP-N-acetylglucosamine: βMan β1,3-N-acetylglucosaminyltransferase (β3GlcNAc-transferase), and egghead encodes a GDP-mannose:βGlc β1,4-mannosyltransferase, with putative functions in sequential steps in the biosynthesis of the core structure of arthro-series glycosphingolipids (GlcNAcβ1-3Manβ1-4Glcβ1-Cer) as predicted by in vitro analysis. Loss of either gene is predicted to abrogate glycosphingolipid biosynthesis at the di- or monosaccharide-ceramide step (Wandall, 2005).

Insect, nematode, and vertebrate glycosphingolipids share a common element consisting of Glcβ1-ceramide, after which they differ markedly in structure and complexity. Insect and nematode glycosphingolipids are built on Manβ1-4Glcβ1-ceramide (MacCer) predicted to be catalyzed by Egghead, while vertebrate glycosphingolipids are built on Galβ1-4Glcβ1-ceramide (LacCer) catalyzed by the β4-galactosyltransferases, β4Gal-T5 and -T6 (Nomura, 1998; Amado, 1999). Despite considerable differences in overall structures of glycosphingolipids among insects and vertebrates, it is clear that homologous glycosyltransferase genes conserved throughout evolution catalyze most biosynthetic steps (see egghead mutants lack mannosyltransferase activity and elongated glycosphingolipids). Egghead is perhaps the only exception suggesting that MacCer-based glycosphingolipids represent a specific functional basis for the diversification of the underlying biosynthetic pathways. Importantly, vertebrate glycosphingolipids based on the LacCer core diverge at the third biosynthetic step to form different classes of structures, which are differentially expressed in cells and are differentially expressed during development and differentiation. The vertebrate glycosphingolipid lacto-series is initiated by addition of β1,3GlcNAc to LacCer by brainiac orthologs designated β3GnTs. Interestingly, Drosophila brainiac functions both on the invertebrate and vertebrate precursor substrate MacCer and LacCer, while the vertebrate orthologs appear to only act on LacCer (Wandall, 2005).

This report presents direct evidence that Egghead and Brainiac do function in vivo in the glycosphingolipid pathway and are essential for glycosphingolipid biosynthesis in vivo. Furthermore, it was demonstrated that despite the fundamental difference in the structure of core glycosphingolipid, the Drosophila egghead mutant can be rescued by introduction of the corresponding enzyme from the human glycosphingolipid biosynthetic pathway. In contrast the fly glycosphingolipid biosynthetic pathway is not elongated in vertebrate cells. The results show that glycosphingolipids are essential for development of complex organisms and suggest that the function of Drosophila glycosphingolipids in development does not depend on the core structure (Wandall, 2005).

Drosophila lacking zygotic activity of the egh and brn genes die as pupae. Drosophila lacking maternal and zygotic activity of these enzymes are devoid of elongated glycosphingolipids and have a more severe defect, dying as embryos with a defect in correct specification of neural and epidermal cell types. Elongated glycosphingolipids also appear to be required for normal development of the mouse embryo. Mice mutant for the glucosylceramide synthase enzyme controlling the ultimate glycosphingolipid precursor die during gastrulation due to apoptosis in all germ layers but particularly in ectoderm (Yamashita, 1999). The mouse embryos lacking glucosylceramide synthase die at an earlier stage of embryogenesis than Drosophila egh and brn mutants. This may reflect a difference in the position at which truncation occurs. For example, elevated ceramide levels are known to be pro-apoptotic (Hannun, 2002). Accordingly, knockdown of the Glcβ1-Cer synthase in Drosophila by RNAi also leads to increased apoptosis, thought to be due to elevated ceramide levels (Kohyama-Koganeya, 2004). Whether ceramide levels are responsible for apoptosis in the mouse mutants in vivo remains to be determined (Wandall, 2005).

Egghead and Brainiac are expressed and required during oogenesis. In the absence of their function, development of the ovarian follicles is defective. Earlier reports have suggested that the activity of these genes was limited to the germ line, because phenotypes were not observed in somatic mutant clones in the follicular epithelia. Using the MacCer antibody on genetic mosaics this study has shown that Egghead and Brainiac are both present and active in the follicular epithelia (Wandall, 2005).

Interestingly, the orthologs of brn and egh do not appear to be essential for development of the nematode C. elegans (Griffitts, 2003). Instead, both genes are required for susceptibility to the crystal (5B) toxin from the bacterium Bacillus thuringiensis. It therefore appears that Drosophila has acquired functions for glycosphingolipids that are not shared among all invertebrates and that Drosophila presents an excellent model for studies of such functions in vivo (Wandall, 2005).

The phenotypes associated with brn and egh mutants initially suggested a role of these in Notch receptor modulation similar to but distinct from fringe. Given the demonstrated function of Brainiac and Egghead in glycosphingolipid biosynthesis it is tempting to suggest that extended glycosphingolipids in Drosophila might play a direct role in modulation of receptor functions in a manner similar to the effects of GM3 on the epidermal growth factor receptor (Miljan, 2002; Meuillet, 2000; Rebbaa, 1996). Alternatively, extended glycosphingolipids might play an indirect role on signaling by virtue of their contribution to the formation of lipid rafts and the recruitment of receptors to rafts. Another appealing possibility is that glycosphingolipids influence the cleavage of membrane-bound ligands, such as the activation of the epidermal growth factor receptor ligands Spitz, Gurken, and Keren by the Rhomboid family of secretases. Of special interest in this context is the possibility that glycosphingolipids could influence Rhomboid-2 cleavage of Gurken in oogenesis. Likewise in the case of Notch signaling, glycosphingolipid could affect the γ-secretase, which is organized in lipid rafts and cleaves the intracellular tail of Notch. The availability of Drosophila lacking elongated glycosphingolipids will provide an opportunity to investigate the functions of glycosphingolipids in cell signaling in vivo. In considering possible modes by which glycosphingolipids may act, it is intriguing that they can do so apparently normally when their core structure has been altered by replacing MacCer with LacCer. This observation provides a starting point for further humanization of the biosynthetic pathway by further replacement of Brainiac with enzymes responsible for the next steps in the mammalian lacto-, ganglio-, or globo-series biosynthetic pathways. Conversely, vertebrate cells with MacCer-based glycosphingolipids provide a unique genetic tool to address structure-function relationships for glycosphingolipids (Wandall, 2005).

Distinct contributions of beta 4GalNAcTA and beta 4GalNAcTB to Drosophila glycosphingolipid biosynthesis

Drosophila melanogaster has two beta4-N-acetylgalactosaminyltransferases, beta4GalNAcTA and beta4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcbeta,4GlcNAc). LacdiNAc is found as a structural element of Drosophila glycosphingolipids (GSLs) suggesting that beta4GalNAcTs contribute to the generation of GSL structures in vivo. Mutations in Egghead and Brainaic, enzymes that generate the beta4GalNAcT trisaccharide acceptor structure GlcNAcbeta,3Manbeta,4GlcbetaCer, are lethal. In contrast, flies doubly mutant for the beta4GalNAcTs are viable and fertile. This study describes the structural analysis of the GSLs in beta4GalNAcT mutants and finds that in double mutant flies no lacdiNAc structure is generated and the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer accumulates. It was also found that phosphoethanolamine transfer to GlcNAc in the trisaccharide does not occur, demonstrating that this step is dependent on prior or simultaneous transfer of GalNAc. By comparing GSL structures generated in the beta4GalNAcT single mutants, it was shown that beta4GalNAcTB is the major enzyme for the overall GSL biosynthesis in adult flies. In beta4GalNAcTA mutants, composition of GSL structures is indistinguishable from wild-type animals. However, in beta4GalNAcTB mutants precursor structures are accumulating in different steps of GSL biosynthesis, without the complete loss of lacdiNAc, indicating that beta4GalNAcTA plays a minor role in generating GSL structures. Together these results demonstrate that both beta4GalNAcTs are able to generate lacdiNAc structures in Drosophila GSL, although with different contributions in vivo, and that the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer is sufficient to avoid the major phenotypic consequences associated with the GSL biosynthetic defects in Brainiac or Egghead (Stolz, 2008).

Glycosphingolipids with extended sugar chain have specialized functions in development and behavior of Drosophila

Glycosphingolipids (GSL) are glycosylated polar lipids in cell membranes essential for development of vertebrates as well as Drosophila. Mutants that impair enzymes involved in biosynthesis of GSL sugar chains provide a means to assess the functions of the sugar chains in vivo. The Drosophila glycosyltransferases Egghead and Brainiac are responsible for the 2nd and 3rd steps of GSL sugar chain elongation. Mutants lacking these enzymes are lethal and the nature of the defects that occur has suggested that GSL might impact on signaling by the Notch and EGFR pathways. This study reports on characterization of enzymes involved in the 4th and 5th steps of GSL sugar chain elongation in vitro, and explores the biological consequences of removing the enzymes involved in step 4 in vivo. Two beta4-N-Acetylgalactosyltransferase enzymes can carry out step 4 (beta4GalNAcTA and beta4GalNAcTB), and while they may have overlapping activity, the mutants produce distinct phenotypes. The beta4GalNAcTA mutant displays behavioral defects, which are also observed in viable brainiac mutants, suggesting that proper locomotion and coordination primarily depend on GSL elongation. beta4GalNAcTB mutant animal shows ventralization of ovarian follicle cells, which is caused by defective EGFR signaling between the oocyte and the dorsal follicle cells to specify dorsal fate. GSL sequentially elongated by Egh, Brn and beta4GalNAcTB in the oocyte contribute to this signaling pathway. Despite the similar enzymatic activity, evidence is provided that the two enzymes are not functionally redundant in vivo, but direct distinct developmental functions of GSL (Chen, 2007; full text of article).

The egghead gene is required for compartmentalization in Drosophila optic lobe development

The correct targeting of photoreceptor neurons (R-cells) in the developing Drosophila visual system requires multiple guidance systems in the eye-brain complex as well as the precise organization of the target area. The egghead (egh) gene, encoding a glycosyltransferase, is required for a compartment boundary between lamina glia and lobula cortex, which is necessary for appropriate R1-R6 innervation of the lamina. In the absence of egh, R1-R6 axons form a disorganized lamina plexus and some R1-R6 axons project abnormally to the medulla instead of the lamina. Mosaic analysis demonstrates that this is not due to a loss of egh function in the eye or in the neurons and glia of the lamina. Rather, as indicated by clonal analysis and cell-specific genetic rescue experiments, egh is required in cells of the lobula complex primordium which transiently abuts the lamina and medulla in the developing larval brain. In the absence of egh, perturbation of sheath-like glial processes occurs at the boundary region delimiting lamina glia and lobula cortex, and inappropriate invasion of lobula cortex cells across this boundary region disrupts the pattern of lamina glia resulting in inappropriate R1-R6 innervation. This finding underscores the importance of the lamina/lobula compartment boundary in R1-R6 axon targeting (Fan, 2005).

Compartment boundaries play key roles in pattern formation during development, and the establishment of these boundaries is thought to be a general mechanism for creating the organization of different tissues in a multi-cellular organism. Multiple compartments have been identified in developing vertebrate and invertebrate central nervous systems, and a number of molecules including cell-cell signaling proteins and transcription factors have been implicated in their establishment. During brain development, different cellular compartments form a complex prepatterned environment which is required for the navigation of axons to their correct targets. For example, in the developing mammalian brain, the subplate, the ganglionic eminence and the thalamic reticular complex are involved in the patterning of connections between the thalamus and the cortex. Similarly, in the developing orthopteran brain, glial boundaries of compartment-like proliferative clusters are used by axons of pioneering neurons for the establishment of the primary axon scaffold that interconnects protocerebrum, deutocerebrum and tritocerebrum (Fan, 2005).

The fly visual system is an excellent model system for the study of cellular and molecular mechanisms of axon guidance. The adult Drosophila compound eye comprises some 750 ommatidia, each containing eight photoreceptor neurons (R-cells). During larval development, different classes of R-cells in the eye disc project through the optic stalk to a different synaptic layer in the brain. R1-R6 axons terminate in the lamina between rows of epithelial and marginal glial cells, forming the lamina plexus, while R7 and R8 axons pass through the lamina and terminate in the medulla. The formation of this R-cell projection pattern is known to involve complex bidirectional interactions between R-cell axons and different populations of cells in the target area. The molecular mechanisms that underlie these interactions have been studied intensively in the photoreceptor neurons of the developing eye, and to a lesser degree in the developing lamina. Thus, R-cells express a set of genes encoding cell surface receptors, signaling molecules and nuclear factors that have been shown to control target selection in lamina and medulla. In the lamina, glial cells appear to act as intermediate targets for R-cell axons and may be an important source of targeting information. When the organization of lamina glia is disrupted, large numbers of R1-R6 axons project through the lamina into the medulla (Fan, 2005).

Unlike the lamina and medulla, the mature lobula complex, composed of lobula and lobula plate, does not receive direct input from R-cells in the adult fly brain. However, during optic lobe development, morphogenetic movements of the optic lobe anlagen transiently bring the lobula complex primordium into close apposition to the developing lamina and medulla. Given this spatial proximity, correct targeting of R-cell axons and plexus formation in the developing lamina can be influenced by cells of the lobula complex primordium, especially if the formation of the boundary that separates the developing lamina from the lobula complex is disrupted. Evidence for a perturbation of the R-cell projection pattern due to invasion of the developing lamina by cells of the adjacent lobula complex has been obtained in slit or robo loss-of-function mutants, in which the lamina/lobula cortex boundary is disrupted resulting in cell mixing across the two optic lobe compartments (Fan, 2005).

This study investigated the role of egh, encoding a glycosyltransferase, in the formation of the R-cell projection pattern. The findings show that in egh loss-of-function mutants, R1-R6 axons form a disorganized projection pattern characterized by defects in the lamina plexus and aberrant projection of some R1-R6 axons through the lamina and into the medulla. Genetic analysis involving mosaics demonstrate that these defects are not due to a loss of egh function in the eye or in the neurons and glia of the lamina. Instead, clonal analysis and cell-specific genetic rescue experiments show that egh is required in cells of the lobula complex primordium. In the absence of egh, the lamina/lobula cortex boundary is disrupted as indicated by the disorganization of sheath-like glial processes at the interface between lamina glia and distal cells of the lobula cortex. Cell mixing across the lamina/lobula cortex boundary occurs, and neurons of the lobula cortex invade the developing lamina at the site of lamina plexus formation disrupting the pattern of lamina glia and resulting in inappropriate R1-R6 axonal projections. Further mutant analysis and genetic rescue experiments suggest that egh acts together with the glycosyltransferase gene brainiac (brn) in this process. These findings uncover a novel role of the egh gene in the developing Drosophila visual system and provide further support for the important role of the lamina/lobula compartment boundary in R1-R6 axon targeting (Fan, 2005).

The egh gene is essential for embryonic epithelial development and oogenesis (Goode, 1996a; Rubsam, 1998). Sequence analysis as well as enzymatic assays suggests that egh encodes a glycosyltransferase and functions in a glycosylation pathway (Wandall, 2003}. Given that some of the features of egh action are reminiscent of neurogenic gene action (Goode, 1996a}, the role of egh in nervous system development was investigated. No obvious zygotic phenotypes of egh loss-of-function were observed in the embryonic nervous system; however, clear defects of R-cell axonal connectivity were seen by using the marker mAb24B10 in third instar larval brains. Thus, in egh mutants, egh⁷ and egh³, the lamina plexus was discontinuous and of variable thickness. In addition, thicker axon bundles were found projecting to the medulla in comparison to the wild type situation (Fan, 2005).

Further analysis of egh mutants using Ro-τlacZ, a marker selectively expressed in R2-R5 axons, showed that some R2-R5 (and R1-R6) axon fascicles fail to stop in the lamina and, instead, projected to the medulla. Moreover, the R2-R5 axons that still terminated in the lamina were disorganized, showed perturbed fasciculation and formed abnormal lamina plexus patches when compared to wild type. The egh photoreceptor projection defects were fully rescued by placing a UAS-egh transgene under the control of the ubiquitously expressed tub-GAL4 driver in an egh mutant background. These results indicate that egh is required for correct R1-R6 axonal projections during larval development (Fan, 2005).

Lamina neurons are generated from a subpopulation of neuroblasts in the outer proliferation center (OPC). In a two-step process, neuroblasts give rise to lamina precursor cells (LPCs) and LPCs subsequently complete final divisions to produce mature lamina neurons. During this process, R-cell afferents release signals such as Hedgehog and Spitz to induce lamina neuron development. In turn, LPC progeny assemble into lamina columns which associate with older R-cell axon bundles. To assess lamina neuron differentiation in egh mutants, the early neuronal differentiation marker Dachshund (Dac} and the late neuronal differentiation marker ELAV were used. The expression pattern of Dac in the lamina was indistinguishable in wild type and egh mutants. Moreover, as in wild type, mature lamina neurons L1-L5, which form lamina columns, expressed ELAV in egh mutants. In egh mutants and in wild type, L1-L4 neurons formed a superficial layer, while L5 neurons resided in a medial layer which was just above the epithelial glia cells. These findings imply that egh is not required for the generation and differentiation of lamina neurons (Fan, 2005).

Lamina glial cells are generated by glial precursor cells located in two domains at the dorsal and ventral edges of the prospective lamina. Mature glia migrate into the lamina target field along scaffold axons which serve as migratory guides. Lamina glial cells have been identified as the intermediate targets of R1-R6 axons, and removal of glia disrupts R1-R6 axon targeting. In wild type, R1-R6 growth cones terminated between rows of epithelial and marginal glial cells, and the row of medulla glial cells lay beneath the marginal glial cells. In egh mutants, a layered assembly of glial cells was also found at the site of lamina plexus formation; however, these layers were clearly disorganized as compared to the wild type situation. Notably, defects in glial layer organization correlated with the gaps in the associated lamina plexus. This suggests that egh is not required for the initial generation and migration of glial cells into the target area, but that the final pattern of glial cells in the developing lamina is perturbed in egh mutants (Fan, 2005).

Cells of the lobula complex are derived from the inner proliferation center (IPC). During optic lobe development, the lobula complex primordium transiently moves into close apposition to the developing lamina. Recently, the existence of a boundary region between the developing lamina and lobula cortex has been demonstrated, and evidence for a perturbation of the R-cell projection pattern due to the invasion of the developing lamina by cells of the lobula cortex has been obtained in slit and robo loss-of-function experiments. A comparable phenotype was observed in egh mutants. Thus, in wild type, lobula distal cell neurons, which form the anterior edge of the lobula cortex, were separated from the adjacent posterior face of the developing lamina by a precise boundary region. In contrast, in egh mutants, this boundary region between lobula and lamina was no longer apparent, and streams of lobula distal cell neurons crossed into the base of the developing lamina. Moreover, these sites of lobula distal cell neuron invasion correlated with sites of structural defects in the developing lamina plexus (Fan, 2005).

Thus, in the absence of egh, a number of mutant phenotypes occur in the optic lobe: (1) disruption of R1-R6 axon targeting in the lamina, (2) perturbation of lamina glial organization, (3) invasion of lobula cortex distal cells into the lamina and (4) disruption of the glial sheath at the lamina/lobula cortex boundary region. It is hypothesized that these phenotypes are causally related in egh mutants, in that disruption of the lamina/lobula boundary and invasion of lobula cortex distal cells into the adjacent lamina cause a displacement of lamina glial cells, resulting in aberrant photoreceptor projection patterns (Fan, 2005).

The generation of the R-cell projection pattern involves complex bidirectional interactions between R-cell axons and different populations of cells in the target region. R-cell axons provide signals for induction of proliferation and differentiation of lamina neurons and for differentiation and migration of glial cells. In turn, lamina glial cells act as intermediate targets for R1-R6 growth cones. When these glial cells are missing or reduced, as occurs in nonstop and jab1/csn5 mutants, large numbers of R1-R6 axons project aberrantly through the lamina into the medulla. Given this crucial role of lamina glia for correct R1-R6 axonal projections, the disorganization of lamina glia in egh mutants is likely to result in aberrant R1-R6 projection patterns. Indeed, in egh mutants, defects in lamina glial layer organization correlate spatially with defects in the associated lamina plexus (Fan, 2005).

It is conceivable that the aberrant R-cell projection in egh mutants might be due, at least in part, to defects in lamina neurons, which are the final targets of R1-R6 axons. However, in egh mutants, generation and differentiation of lamina neurons appear normal, and animals with MARCM mutant clones in lamina neurons have normal R-cell projection patterns. Defects in R-cells themselves also unlikely contribute substantially to the projection defect since egh mutant R1-R6 photoreceptors project normally into wild type optic lobes, and R-cell fate and determination appear normal in egh mutant clones in the eye. Thus, the most reasonable explanation for the disrupted R-cell axonal projections in egh mutants is that they are a consequence of the perturbation of lamina glia (Fan, 2005).

Lamina glia cells migrate to the lamina target field from their progenitor zones. In egh mutants, the initial generation and migration of glial cells to the lamina appear unaffected. Epithelial and marginal glia in large egh mutant clones which contain the glia and their precursors, are arranged normally in appropriate layers at the site of formation of a normal lamina plexus. Moreover, expression of Egh protein in the lamina glia in egh mutants does not rescue the phenotype. Therefore, it seems unlikely that the mispositioning of glial cells in egh mutants is due to defects in the glial cells, their precursors or their migratory behavior (Fan, 2005).

These observations imply that the mispositioning of lamina glia in egh mutants is a secondary consequence of other disruptions in optic lobe development. MARCM mutant clonal analysis indicates that the characteristic defects in the lamina plexus are associated with cells of the lobula complex primordium. Moreover, the egh mutant phenotype is rescued in experiments in which Egh protein is expressed in the lobula cortex. A good candidate for the lobula-associated disruption in optic lobe development in egh mutants is the observed invasion of lobula cells into the base of the developing lamina. In egh mutants, the distal cell neurons invade and intermingle with lamina glial cells, and this cell intermixing correlates spatially with the displacement of the lamina glia at the base of the developing lamina. The possibility cannot be ruled out that the displacement of the lamina glia is caused primarily by unidentified signals from the lobula cortex, with distal cell invasion into the disrupted glial layers occurring secondarily. However, the most reasonable explanation for the observed glial cell mispositioning phenotype is that it is due to the invasion and intermingling of lobula cells into the lamina (Fan, 2005).

Glial cells are thought to play a major role in the formation and maintenance of many compartments in the central nervous system, and some of the most prominent compartments in the insect brain, including the optic ganglia, are delimited by sheath-like glial septa. In the developing visual system of Drosophila, the boundary area that separates the cells of the developing lamina from the transiently adjacent cells of the developing lobula complex is delimited by sheath-like glial cell processes which extend from the lateral surface of the brain to the posterior face of the developing lamina plexus. In wild type, no intermixing of the two cell populations across this boundary area occurs. In egh mutants, the glial sheath interface at the boundary is disrupted and this disruption correlates with the invasion of lobula distal cells into the lamina. The perturbation of the glial sheath interface at the lamina/lobula cortex boundary could be the secondary consequence of the invasion of lobula distal cells into the developing lamina that occurs in egh mutants. Alternatively, the glial sheath perturbation may contribute directly to the compartmentalization defect observed in egh mutants (Fan, 2005).

In vitro and in vivo analysis suggests that egh encodes a glycosyltransferase and functions in a common signaling pathway with another glycosyltransferase encoded by brn (Goode, 1996a; Wandall, 2003). These two glycosyltransferases are capable of catalyzing sequential elongation steps in glycosphingolipid biosynthesis (Wandall, 2005}. This report found that egh brn double mutants have R-cell projection defects that are comparable to those observed in egh or brn single mutants. Furthermore, expression of the human β4-galactosyltransferase β4GalT6, which restores glycosphingolipid biosynthesis in egh mutant animals (Wandall, 2005}, can rescue the R-cell projection phenotype in egh mutants but not in egh brn double mutants. Taken together, these findings suggest that brn functions downstream of egh in a glycosphingolipid biosynthetic pathway that is required for correct compartmentalization during optic lobe development. This, in turn, suggests that egh might be involved in regulating the organization of lipid composition in the plasma membrane by controlling the biosynthesis of glycosphingolipids. In vertebrates, glycosphingolipids are known to have functions in cell adhesion, growth, regulation, differentiation, cell interaction, recognition and signaling (Watts, 2003; full text of article}, and all of these processes may contribute to compartment formation (Fan, 2005).

Many studies in mammalian cells suggest that particularly ordered lipid environments are enriched with signaling molecules including transmembrane and glycosylphosphatidylinositol (GPI)-anchored receptors as well as intracellular signaling intermediates. Despite differences in the chemical structure of their lipids, Drosophila membranes contain microdomains with a similar protein and lipid composition as their mammalian counterparts which are believed to provide suitable microenvironments to enable selective protein-protein interactions as well as local initiation of signal transduction. This is also supported by the fact that mammalian lactosylceramide glycosphingolipid biosynthetic pathway can functionally replace the Drosophila mactosylceramide glycosphingolipid biosynthetic pathway (Wandall, 2005}. Although the existence of cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) remains a controversial issue, lipid microdomains have been shown to play a direct role in organizing spatial signaling during cell chemotaxis and axon guidance by concentrating the gradient-sensing machinery at the leading cell edge. Hence, egh might be important for the organization of lipid microdomains which in turn are required for selective signal transduction in compartmentalization (Fan, 2005).

Slit/Robo signaling has been shown to be critical for the formation of the lamina/lobula cortex boundary and for the prevention of invasion and cell intermingling across this boundary. Given the similarity of the slit/robo and egh mutant phenotypes in the developing optic lobe, it is conceivable that Egh might interact with Slit/Robo in the compartmentalization process. The expression of Slit and Robo proteins in egh mutants is largely wild type-like. Moreover, in a series of genetic studies, no genetic interactions were uncovered between egh and slit or robo in the developing optic lobes. While these findings are in accordance with the notion that the Slit/Robo signaling pathway and the Egh glycosyltransferase function independently in optic lobe development, it is still not clear whether the Slit/Robo signaling pathway is functionally affected in egh mutants. Further detailed analyses such as the cellular localization of Robo proteins in egh mutant lobula cells may help answer this question (Fan, 2005).

Recent studies on heparan sulfate proteoglycans (HSPGs) suggest that a Slit/Robo signaling-related transmembrane HSPG, Syndecan (Sdc), and a GPI-linked glypican, Dally-like protein (Dlp), are expressed in the lobula cortex and are involved in visual system assembly. Thus, it will be interesting to investigate the possible roles of HPSGs, such as Sdc and Dlp, in mediating egh action on compartmentalization of Drosophila visual system. Other interesting candidates that may contribute to the egh action in visual system development include Notch and EGFR signaling. These candidates have been reported to play a role in compartmentalization and have been shown to interact with the egh and brn pathway during oogenesis (Fan, 2005).

Drosophila egghead encodes a beta 1,4-mannosyltransferase predicted to form the immediate precursor glycosphingolipid substrate for brainiac

The neurogenic Drosophila genes brainiac and egghead are essential for epithelial development in the embryo and in oogenesis. Analysis of egghead and brainiac mutants has led to the suggestion that the two genes function in a common signaling pathway. Recently, brainiac was shown to encode a UDP-N-acetylglucosamine:beta Man beta 1,3-N-acetylglucosaminyltransferase (beta 3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAc beta 1-3Man beta 1-4Glc beta 1-1Cer. This study demonstrate that egghead encodes a Golgi-located GDP-mannose:beta Glc beta 1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Man beta 1-4Glc beta 1-1Cer. Egghead is unique among eukaryotic glycosyltransferase genes in that homologous genes are limited to invertebrates, which correlates with the exclusive existence of arthroseries glycolipids in invertebrates. It is proposed that brainiac and egghead function in a common biosynthetic pathway and that inactivating mutations in either lead to sufficiently early termination of glycolipid biosynthesis to inactivate essential functions mediated by glycosphingolipids (Wandall, 2003).

The egghead gene product influences oocyte differentiation by follicle cell-germ cell interactions in Drosophila melanogaster

Oogenesis in Drosophila is a useful model for studying cell differentiation. This study analyzed the role of the egh gene in these processes with the aid of a newly isolated viable but female sterile allele. This mutation results in diverse variable defects in oogenesis. The most frequent defect being follicles that have either more or less than the normal number of 16 germ cells. This is caused by erroneous splitting and/or fusion of correct clusters of 16 cystocytes. The entire follicle has a rather flexible structure in this allele, most obvious by a highly variable position of the oocyte within the follicle. Moreover, a second oocyte can also develop in egh clusters. This is exclusively observed in aberrant follicles that are generated by the aforementioned splitting/fusion process. Surprisingly, even a germ cell which is distinct from the two pro-oocytes can differentiate into an oocyte under these circumstances. Hence, determination of the oocyte is definitely not fixed when germ cell clusters are enveloped by prefollicular cells, and interactions between follicle cells and germ cells must play an important role in oocyte specification. Molecular analysis proves that the oocyte-specific transcript of the egh gene is drastically reduced in this viable allele (Rubsam, 1998).

Search PubMed for articles about Drosophila Egghead

Amado, M., Almeida, R., Schwientek, T. and Clausen, H. (1999). Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions. Biochim. Biophys. Acta 1473: 35-53. PubMed ID: 10580128

Ariga, T., McDonald, M. P. and Yu, R. K. (2008). Role of ganglioside metabolism in the pathogenesis of Alzheimer's disease - a review. J. Lipid Res. 49: 1157-1175. PubMed ID: 18334715

Bokel, C., Dass, S., Wilsch-Brauninger, M. and Roth, S. (2006). Drosophila Cornichon acts as cargo receptor for ER export of the TGFalpha-like growth factor Gurken. Development 133: 459-470. PubMed ID: 16396907

Chen, Y. W., et al. (2007). Glycosphingolipids with extended sugar chain have specialized functions in development and behavior of Drosophila. Dev. Biol. 306(2): 736-49. PubMed ID: 17498683

Degroote, S., Wolthoorn, J. and van Meer, G. (2004). The cell biology of glycosphingolipids. Semin. Cell Dev. Biol. 15: 375-387. PubMed ID: 15207828

Fan, Y., et al. (2005). The egghead gene is required for compartmentalization in Drosophila optic lobe development. Dev. Biol. 287(1): 61-73. PubMed ID: 16182276

Goentoro, L. A., Reeves, G. T., Kowal, C. P., Martinelli, L., Schupbach, T. and Shvartsman, S. Y. (2006). Quantifying the Gurken morphogen gradient in Drosophila oogenesis. Dev. Cell 11: 263-272. PubMed ID: 16890165

Goode, S., Wright, D. and Mahowald, A. P. (1992). The neurogenic locus brainiac cooperates with the Drosophila EGF receptor to establish the ovarian follicle and to determine its dorsal-ventral polarity. Development 116: 177-192. PubMed ID: 1483386

Goode, S., Morgan, M., Liang, Y. P. and Mahowald, A. P. (1996a). Brainiac encodes a novel, putative secreted protein that cooperates with Grk TGF alpha in the genesis of the follicular epithelium. Dev. Biol. 178: 35-50. PubMed ID: 8812107

Goode, S., Melnick, M., Chou, T. B. and Perrimon, N. (1996b). The neurogenic genes egghead and brainiac define a novel signaling pathway essential for epithelial morphogenesis during Drosophila oogenesis. Development 122: 3863-3879. PubMed ID: 9012507

Griffitts, J. S., Huffman, D. L., Whitacre, J. L., Barrows, B. D., Marroquin, L. D., Muller, R., Brown, J. R., Hennet, T., Esko, J. D. and Aroian, R. V. (2003). Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions. J. Biol. Chem. 278: 45594-45602. PubMed ID: 12944392

Griffitts, J. S., Haslam, S. M., Yang, T., Garczynski, S. F., Mulloy, B., Morris, H., Cremer, P. S., Dell, A., Adang, M. J. and Aroian, R. V. (2005). Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science 307: 922-925. PubMed ID: 15705852

Haltiwanger, R. S., and Lowe, J. B. (2004). Role of glycosylation in development. Annu. Rev. Biochem. 73: 491-537. PubMed ID: 15189151

Hannun, Y. A. and Obeid, L. M. (2002). The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J. Biol. Chem. 277: 25847-25850. PubMed ID: 12011103

Kohyama-Koganeya A, Sasamura, T., Oshima, E., Suzuki, E., Nishihara, S., Ueda, R. and Hirabayashi, Y. (2004). Drosophila glucosylceramide synthase: a negative regulator of cell death mediated by proapoptotic factors. J. Biol. Chem. 279, 35995-36002. PubMed ID: 15210713

Kolter, T. and Sandhoff, K. (2006). Sphingolipid metabolism diseases. Biochim. Biophys. Acta 1758: 2057-2079. PubMed ID: 16854371

Lauc, G. and Heffer-Lauc, M. (2006). Shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins. Biochim. Biophys. Acta 1760: 584-602. PubMed ID: 16388904

Lisman, Q., Pomorski, T., Vogelzangs, C., Urli-Stam, D., de Cocq van Delwijnen, W. and Holthuis, J. C. (2004). Protein sorting in the late Golgi of Saccharomyces cerevisiae does not require mannosylated sphingolipids. J. Biol. Chem. 279: 1020-1029. PubMed ID: 14583628

Meuillet, E. J., Mania-Farnell, B., George, D., Inokuchi, J. I. and Bremer, E. G. (2000). Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431. Exp. Cell Res. 256: 74-82. PubMed ID: 10739654

Miljan, E. A., Meuillet, E. J., Mania-Farnell, B., George, D., Yamamoto, H., Simon, H. G. and Bremer, E. G. (2002). J. Biol. Chem. 277: 10108-10113. PubMed ID: 11796728

Nomura, T., Takizawa, M., Aoki, J., Arai, H., Inoue, K., Wakisaka, E., Yoshizuka, N., Imokawa, G., Dohmae, N., Takio, K., Hattori, M. and Matsuo, N. (1998). Purification, cDNA cloning, and expression of UDP-Gal: glucosylceramide beta-1,4-galactosyltransferase from rat brain. J. Biol. Chem. 273: 13570-13577. PubMed ID: 9593693

Pizette, S., Rabouille, C., Cohen, S. M. and Thérond, P. (2009). Glycosphingolipids control the extracellular gradient of the Drosophila EGFR ligand Gurken. Development 136(4): 551-61. PubMed ID: 19144719

Rebbaa, A., Hurh, J., Yamamoto, H., Kersey, D. S. and Bremer, E. G. (1996). Ganglioside GM3 inhibition of EGF receptor mediated signal transduction. Glycobiology 6: 399-406. PubMed ID: 8842703

Rubsam, R., et al. (1998). The egghead gene product influences oocyte differentiation by follicle cell-germ cell interactions in Drosophila melanogaster. Mech. Dev. 72: 131-140. PubMed ID: 9533964

Sabourdy, F., Kedjouar, B., Sorli, S. C., Colie, S., Milhas, D., Salma, Y. and Levade, T. (2008). Functions of sphingolipid metabolism in mammals-lessons from genetic defects. Biochim. Biophys. Acta 1781: 145-183. PubMed ID: 18294974

Schwientek, T., Keck, B., Levery, S. B., Jensen, M. A., Pedersen, J. W., Wandall, H. H., Stroud, M., Cohen, S. M., Amado, M. and Clausen, H. (2002). The Drosophila gene brainiac encodes a glycosyltransferase putatively involved in glycosphingolipid synthesis. J. Biol. Chem. 277: 32421-32429. PubMed ID: 12130651

Selleck, S. B. (2000). Proteoglycans and pattern formation: sugar biochemistry meets developmental genetics. Trends Genet. 16: 206-212. PubMed ID: 10782114

Seppo, A., Moreland, M., Schweingruber, H. and Tiemeyer, M. (2000). Zwitterionic and acidic glycosphingolipids of the Drosophila melanogaster embryo. Eur. J. Biochem. 267: 3549-3558. PubMed ID: 10848971

Sillence, D. J. and Platt, F. M. (2004). Glycosphingolipids in endocytic membrane transport. Semin. Cell Dev. Biol. 15: 409-416. PubMed ID: 15207831

Stolz, A., et al. (2008). Distinct contributions of beta 4GalNAcTA and beta 4GalNAcTB to Drosophila glycosphingolipid biosynthesis. Glycoconj. J. 25(2): 167-75. PubMed ID: 17876704

Wandall, H. H., Pedersen, J. W., Park, C., Levery, S. B., Pizette, S., Cohen, S. M., Schwientek, T. and Clausen, H. (2003). Drosophila egghead encodes a beta 1,4-mannosyltransferase predicted to form the immediate precursor glycosphingolipid substrate for brainiac. J. Biol. Chem. 278: 1411-1414. PubMed ID: 12454022

Wandall, H. H., Pizette, S., Pedersen, J. W., Eichert, H., Levery, S. B., Mandel, U., Cohen, S. M. and Clausen, H. (2005). Egghead and brainiac are essential for glycosphingolipid biosynthesis in vivo. J. Biol. Chem. 280: 4858-4863. PubMed ID: 15611100

Wasserman, J. D. and Freeman, M. (1998). An autoregulatory cascade of EGF receptor signaling patterns the Drosophila egg. Cell 95: 355-364. PubMed ID: 9814706

Watts, R.W. (2003) A historical perspective of the glycosphingolipids and sphingolipidoses. Philos. Trans. R. Soc. Lond., B Biol. Sci. 358: 975-983. PubMed ID: 12803932

Yamashita, T., Wada, R., Sasaki, T., Deng, C., Bierfreund, U., Sandhoff, K. and Proia, R. L. (1999). A vital role for glycosphingolipid synthesis during development and differentiation. Proc. Natl. Acad. Sci. 96: 9142-9147. PubMed ID: 10430909

date revised: 2 May 2009

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.