Eberwein, A. E., Kulkarni, S. S., Rushton, E., Broadie, K. (2023). Glycosphingolipids are linked to elevated neurotransmission and neurodegeneration in a Drosophila model of Niemann Pick type C. Disease models & mechanisms, 16(10) PubMed ID: 37815467

BIOLOGICAL OVERVIEW

Classifying brainiac according to its role in development presents a problem. It has been classified as a neurogenic gene because of the neural hyperplasia (too many cells) induced in brainiac mutants. It is becoming clear, however, that brainiac besides being implicated in the Notch pathway, also interacts with Epidermal growth factor receptor (Egfr) signaling. The key to understanding the biological role of Brainiac comes from research into the role of Brn in oogenesis. Here Brainiac shows genetic interaction with Egf-r and gurken. However, the hallmark of the oocytic Egr-r-gurken interaction is zygotic dorsal-ventral polarity; with regard to this critical developmental cusp, Brn appears to be uninvolved, apparently having no effect. What then is the role of Brainiac in oogenesis, and how does a component of Egfr signaling come to be grouped with genes involved in Notch signaling?

brainiac mutants show a defect in the dorsal-ventral patterning of the ovarian follicle that results in defective dorsal appendages (anatomical features of Drosophila eggs). In mutants, dorsal appendages are shifted to the dorsal midline and become fused. A similar phenotype is apparent in mutants of gurken and Egf-r, the receptor ligand pair involved in determination of follicle cell identity. Gurken is made by the oocyte, and is involved in signaling to follicle cells, through the Egfr. Gurken signals are involved early in determining posterior follicle cell fate and later in determining dorsal follicle cell fate. In brainiac, Egf-r and gurken mutants, gaps appear in the follicular epithelium. It is believed that Gurken, acting through the Egfr, brings about the successful migration of prefollicular cells to surround each nurse cell-oocyte complex, forming a continuous follicle cell epithelium. Brainiac, a novel secreted protein produced by oocytes, interacts with this pathway either directly, through interference with the Gurken/Egfr interaction, or indirectly through an unknown receptor, thus modifying the results of Gurken signaling (Goode, 1996a).

Now ask the question above from a different perspective: how then does a protein classified as belonging in the Notch pathway become involved in Egfr signaling? One must look to the role of Notch in oogenesis to understand brainiac function. Like brainiac mutants, the animals mutant for Notch frequently develop egg chambers containing multiple oocyte-nurse cell complexes. In these mutants Notch is localized on the apical and lateral surface of all follicular epithelial cells. Stage 5 - 10 Notch mutant egg chambers have epithelial defects similar to those described for brainiac. The most striking phenotype observed for all three genes is a loss of apical-basal polarity and accumulation of follicular epithelial cells in multiple layers around the oocyte. Thus Notch is required in follicle cells for maintenance of the follicular epithelium. The observation that follicle cells show a propensity to detach from the oocyte rather than nurse cells an brainiac and Notch mutant egg chambers, starting at stage 4 and continuing through stage 9 of oogenesis, suggests that Brainiac and Notch are components of at least one oocyte-follicle cell adhesive system during stages 4 to 9 of oogenesis (Goode, 1996b)

Functional tests are consistent with the interdependence of the neurogenic and Egfr signaling process. brainiac and Notch mutations show dominant and synergistic interactions with mutations in components of the Egfr signaling process, for dorsal-ventral patterning and induction of cell proliferation and/or migration (Goode, 1992). These observations are consistent with the idea that cell adhesion, maintained by the Notch system, with the involvement of Brainiac, is required for the execution of inductive signals, carried out by the Egfr system. If Brainiac function in embryogenesis is the same as its function in oogenesis, then it is reasonable to assume that Brainiac cooperates with the Egfr in the development and maintenance of epithelial structure. Brainiac's involvement in lateral inhibition during early neurogenesis may be related to its involvement in maintaining epithelial structure within the neurogenic ectoderm during neuroblast segregation. Disruption of the continuity of the ectodermal epithelium may cause a neurogenic phenotype due to a compromised intercellular signaling capacity of neuroectodermal cells (Goode, 1996a).

brainiac encodes a glycosyltransferase putatively involved in glycosphingolipid synthesis

The Drosophila genes fringe and brainiac exhibit sequence similarities to glycosyltransferases. Drosophila and mammalian fringe homologs encode UDP-N-acetylglucosamine:fucose-O-Ser ß1,3-N-acetylglucosaminyltransferases that modulate the function of Notch family receptors. The biological function of brainiac is less well understood. Brainiac is a member of a large homologous mammalian ß3-glycosyltransferase family with diverse functions. Eleven distinct mammalian homologs have been demonstrated to encode functional enzymes forming ß1-3 glycosidic linkages with different UDP donor sugars and acceptor sugars. The putative mammalian homologs with highest sequence similarity to brainiac encode UDP-N-acetylglucosamine:ß1,3-N-acetylglucosaminyltransferases (ß3GlcNAc-transferases), and brainiac is shown to also encodes a ß3GlcNAc-transferase that uses ß-linked mannose as well as ß-linked galactose as acceptor sugars. The inner disaccharide core structures of glycosphingolipids in mammals (Galß1-4Glcß1-Cer) and insects (Manß1-4Glcß1-Cer) are different. Both disaccharide glycolipids served as substrates for Brainiac, but glycolipids of insect cells have so far only been found to be based on the GlcNAcß1-3Manß1-4Glcß1-Cer core structure. Infection of High Five(TM) cells with baculovirus containing full coding brainiac cDNA markedly increases the ratio of GlcNAcß1-3Manß1-4Glcß1-Cer glycolipids compared with Galß1-4Manß1-4Glcß1-Cer found in wild type cells. It is suggested that Brainiac exerts its biological functions by regulating biosynthesis of glycosphingolipids (Schwientek, 2002).

Thus brainiac encodes a ß3GlcNAc-transferase with broad acceptor substrate specificity having preference for ß-Man but also showing significant activity with ß-Gal terminating structures. Mannose-linked ß1-4 is found in the core structure of insect and nematode arthroseries glycolipids as mactosylceramide, and brainiac has activity with this glycolipid. Brainiac also shows activity with another disaccharide glycolipid, lactosylceramide, which represents the equivalent core structure upon which vertebrate glycosphingolipids are built. With the present knowledge of structures of Drosophila glycoconjugates mactosylceramide is the only likely natural substrate identified, indicating that Brainiac serves an important function in the biosynthesis of glycosphingolipids (Schwientek, 2002).

The acceptor substrate specificity of Brainiac with various mono- and di-saccharides and aglycon derivatives reveals clear preference for ß-linked mannose. Furthermore, Brainiac shows preference for dihexosides (Galß1-4Glc and Galß1-4Man), whereas disaccharides with penultimate N-acetylglucosamine represent poor substrates (Galß1-4GlcNAc and Manß1-4GlcNAc). In agreement with this, Brainiac used MacCer and LacCer glycolipid substrates in in vitro tests. Extended glycosphingolipids of Drosophila and other dipterans have been reported to contain two ß1-3 linked GlcNAc residues (e.g. Galß1-3GalNAcß1-4GlcNAcß1-3Galß1-3GalNAcalpha1-4GalNAcß1-4GlcNAcß1-3Manß1-4Glcß1-Cer). Since Brainiac shows poor activity with disaccharide structures containing internal n-acetylhexosamine and no activity with the disaccharide Galß1-3GalNAcalpha1-benzyl, it appears unlikely that Brainiac also catalyzes the addition of the outer GlcNAc residue (Schwientek, 2002).

Drosophila glycosphingolipids have all been reported so far to be based on the Ap₃Cer core structure. It is noteworthy though that relatively few studies have addressed Drosophila glycosphingolipids, as well as insect glycosphingolipids in general, compared with studies of vertebrate glycosphingolipids. In the biosynthesis of vertebrate glycosphingolipids built on LacCer, an important branch point occurs at the addition of the third monosaccharide residue. This is the determining step for synthesis of different classes of glycosphingolipids, designated (neo)lactoseries (GlcNAcß1-3Galß1-4Glcß1-Cer), (iso)globoseries Galalpha1-3/4Galß1-4Glcß1-Cer), and ganglioseries (GalNAcß1-4Galß1-4Glcß1-Cer). These classes of glycolipids are differentially expressed in cell types and during cell differentiation and have different properties and functions. There may be no analogous branch point in the biosynthesis of Drosophila glycosphingolipids, since only one CTH sequence, GlcNAcß1-3MacCer (Ap₃Cer, has been reported from this species. However, it is possible that additional structures and pathways exist. Although Galß1-4Manß1-4Glcß1-Cer is the major CTH component in High Five^TM cells, and this structure may represent an aberrant pathway confined to cultured insect cells, its appearance implies that a Galß1-4 transferase with substrate specificity for MacCer must be present in the insect repertoire. It is also possible that the alternative pathway relates to the lepidopteran, rather than dipteran, origin of the cell line. A stable brainiac transfectant of High Five^TM cells was not sucessfully established, but analysis of the glycosphingolipid profile of baculovirus infected cells shows that Brainiac functions and produces a significant shift in trihexoside ceramides to Ap₃Cer. Brainiac is homologous to vertebrate ß3GlcNAc-transferase enzymes that control the (neo)lactoseries pathway in vertebrates by forming GlcNAcß1-3Galß1-4Glcß1-Cer, and Brainiac uses LacCer similarly to the homologous mammalian ß3GlcNAc-transferases. This provides strong support for the proposed role for Brainiac in glycosphingolipid biosynthesis from a functional perspective (Schwientek, 2002).

The proposed function for Brainiac in Drosophila glycosphingolipid biosynthesis implies that brainiac mutants may lack extended glycosphingolipids. Drosophila does not appear to have close brainiac homologs, which would be predicted to have similar functions. More distant Drosophila homologs group independently or with vertebrate orthologs known to represent ß3-galactosyltransferases. It is therefore possible that this class of glycosphingolipids cannot be produced in brainiac mutant animals. Brainiac is required in the germ line during oogenesis and is also expressed zygotically. At present it is not technically feasible to isolate sufficient numbers of maternally and zygotically mutant animals to permit analysis of the glycosphingolipid composition. Failure to extend glycosphingolipids beyond MacCer could also lead to lack of acidic and zwitterionic glycosphingolipids in Drosophila that contain glucuronic acid linked to galactose residues and phosphoethanolamine linked to GlcNAc residues. Charged residues, glucuronic acid and sialic acids, of glycoconjugates are important for biological functions in vertebrates, and it is likely that glucuronic acid and phosphoethanolamine exert important functions in Drosophila as well (Schwientek, 2002).

The large vertebrate ß3-glycosyltransferase family homologous to Brainiac has been extensively characterized within the last few years. Functional subgroups with considerable apparent redundancies have been identified, and many of these have been assigned important roles in the biosynthesis of all glycosphingolipid classes in mammals. One group is represented by UDP-Gal:ßGlcNAc ß3-galactosyltransferases, ß3Gal-T1, -T2, and -T5, which are predicted to control synthesis of type 1 chain lactoseries structures on glycolipids and N- and O-linked glycoproteins. All three function in vitro with glycolipids, whereas only ß3Gal-T2 has activity with N-linked glycoproteins, and only ß3Gal-T5 functions with O-linked core 3 structures. Note that murine ß3Gal-T3 was originally erroneously proposed to function in lactoseries synthesis; however, ß3Gal-T3, renamed as ß3GalNAc-T1, is unique and functions in globoseries glycolipid biosynthesis forming GalNAcß1-3Galalpha1-4Galß1-4Glcß1-Cer. Surprisingly, a recent report indicated that this gene was essential in mice. However, the orthologous gene in man is inactivated in healthy individuals of the rare P^k blood group. ß3Gal-T4 is also unique and functions in ganglioseries glycolipid biosynthesis forming Galß1-3GalNAcß1-4Galß1-4Glcß1-Cer. Again, ß3Gal-T6 was originally erroneously reported as ß3GnT with a ß3GlcNAc-transferase activity similar to Brainiac; however, a recent report shows that this gene encodes the Gal-I enzyme involved in the proteoglycan core region synthesis (Galß1-3Galß1-4Xylß1-O-Ser). A single Drosophila ortholog (CG8734) is predicted to have similar enzymatic functions. The human core 1 ß3Gal-T (Galß1-3GalNAcalpha1-O-Ser/Thr) is only distantly related and groups in an independent clade with two Drosophila orthologs (Schwientek, 2002).

A dendrogram based on protein distance analyses of the putative catalytic units of the ß3-glycosyltransferase family depicts Brainiac in a subfamily with five mammalian orthologs, which are all known to function as ß3GlcNAc-transferases. ß3GnT2 functions in poly-N-acetyllactosamine synthesis (GlcNAcß1-3Galß1-4Glc[NAc]) of glycoproteins and glycolipids. ß3GnT3 was shown to function as a core 1 extension enzyme (GlcNAcß1-3Galß1-3GalNAcalpha1-O-Ser/Thr). The function of ß3GnT4 may be related to the function of ß3GnT2, although only low activity has been demonstrated thus far. ß3GnT5 also has similar functions, and it may have a primary function in glycosphingolipid biosynthesis (GlcNAcß1-3Galß1-4Glcß1-Cer). Finally, the most distant of the close ß3GlcNAc-T Brainiac orthologs, ß3GnT6, was recently shown to represent a core 3 enzyme (GlcNAcß1-3GalNAcalpha1-O-Ser/Thr). The mammalian ß3GnTs thus all use ßGal or alphaGalNAc as acceptor sugar, while Brainiac uses both terminal ßGal and ßMan. Human ß3GnT2 in contrast to Brainiac does not function with MacCer (Schwientek, 2002).

The phenotypes associated with brainiac mutations have led to the proposal that it might modulate the activities of several signaling pathways, including Delta/Notch and TGFalpha/EGF. Could the diversity of effects observed in these mutants be due to an influence of glycosphingolipids on signaling? In vertebrates, glycosphingolipids are known to modulate receptor function and signaling pathways through direct interaction with receptors and through formation of lipid rafts which provide structurally distinct membrane domains for localization of receptors and ligands. Drosophila lipid rafts have been identified and shown to be enriched with MacCer. It is therefore proposed that Brainiac exerts its biological functions in Drosophila by directing glycosphingolipid biosynthesis, rather than by directly modifying receptor molecules as established for Fringe. brainiac mutants may therefore provide a unique genetically tractable system to study the biological role of glycosphingolipids in vivo (Schwientek, 2002).

GENE STRUCTURE

Exons - 1

Bases in 3' UTR - 331

PROTEIN STRUCTURE

Amino Acids - 325

Structural Domains

Brainiac protein is novel. The hydrophilic cluster at the N-terminus appears to be a signal sequence, acting to facilitate secretion (Goode, 1996a).

EVOLUTIONARY HOMOLOGS

Fringe, a secreted protein involved in boundary formation, and Braniac, required for proper contact or adhesion between germline and follicle cells, may both be part of a large family of glycosyltransferases. Brn and FNG share several features: 1) they are developmentally regulated, secreted signaling molecules without known receptors, 2) they are required during epithelial patterning, 3) they interact genetically with the Notch and/or EGF receptor pathways, suggesting that they might modify the signaling mediated by these receptors, and 4) FNG and Brn both have at least two C. elegans homologs and several vertebrate homologs as well, suggesting the presence of multigene families. FNG and Brn show conserved sequence regions homologous with Haemophilus influenzae Lex1, essential for the biosynthesis of parasitic bacterium's extracellular lipooligosaccharides (LOS). Lex1 is a galactosyltransferase, adding galactose to glucose or N-acetylglucosamine residues of LOS. Secreted glycosyltransferases may use their ability to recognize specific carbohydrate moieties on cell surface molecules to trigger particular receptors; they might also play a crucial role in epithelial pattern formation by modifying these carbohydrate moieties at particular locations recognizable by various carbohydrate-binding domains of extracellular proteins. The carbohydrate status of the cell during development might even be a function of neighboring cells and not only of its own expression set of glycosyltransferases (Yuan, 1997).

A murine homolog of the Drosophila brainiac gene has been isolated and its highly specific expression pattern during development and adult life has been delineated. Particularly strong expression is found in the developing central nervous system, in the developing retina, and in the adult hippocampus. Targeted deletion of mouse Brainiac 1 expression leads to embryonic lethality prior to implantation. Null embryos can be recovered as blastocysts but do not appear to implant, indicating that mouse Brainiac 1, likely a glycosyltransferase, is crucial for very early development of the mouse embryo (Vollrath, 2001).

A murine cDNA coding for a beta1,3-N-acetylglucosaminyltransferase enzyme (beta3GnT) has been isolated. This enzyme is similar in sequence to Drosophila melanogaster Brainiac and to the murine and human beta1,3-galactosyltransferase family of proteins. The mouse beta 3GnT protein is 397 amino acids in length and contains 7 cysteine residues that are conserved in the human ortholog. beta 3GnT is a type II membrane protein localized to the Golgi apparatus. Enzyme assays with recombinant mouse beta 3GnT reveal that it has a preference for acceptors with Gal(beta1-4)Glc(NAc) at the non-reducing termini. Proton NMR analysis of product shows incorporation of GlcNAc in beta1,3 linkage to the terminal Gal of Gal(beta1-4)Glc(beta1-O-benzyl). Northern blot analysis reveals the presence of a single 3.0 kb transcript in all adult mouse and human organs tested, with highest levels in the kidney, liver, heart and placenta. The beta 3GnT gene is also expressed in a number of tumor cell lines. The human orthologue of beta 3GnT is located on chromosome 2pl5 (Egan, 2001).

Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, Caenorhabditis elegans was screened for mutants that resist Cry protein intoxication. Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. As in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, C. elegans bre-5 rescues the Drosophila brn mutant and BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans (Griffitts, 2003).

REGULATION

Characterization of Brainiac enzymatic function

Mutations at the brainiac locus lead to defective formation of the follicular epithelium during oogenesis and to neural hyperplasia. The brainiac gene encodes a type II transmembrane protein structurally similar to mammalian ß1,3-glycosyltransferases. The brainiac gene has been cloned from D. melanogaster genomic DNA and expressed as a FLAG-tagged recombinant protein in Sf9 insect cells. Glycosyltransferase assays show that Brainiac is capable of transferring N-acetylglucosamine (GlcNAc) to ß-linked mannose (Man), with a marked preference for the disaccharide Man(ß1,4)Glc, the core of arthro-series glycolipids. The activity of Brainiac toward arthro-series glycolipids was confirmed by showing that the enzyme efficiently utilizes glycolipids from insects as acceptors whereas it does not with glycolipids from mammalian cells. Methylation analysis of the Brainiac reaction product revealed a ß1,3 linkage between GlcNAc and Man, proving that Brainiac is a ß1,3GlcNAc-transferase. Human ß1,3GlcNAc-transferases structurally related to Brainiac are unable to transfer GlcNAc to Man(ß1,4)Glc-based acceptor substrates and fail to rescue a homozygous lethal brainiac allele, indicating that these proteins are paralogous and not orthologous to brainiac (Muller, 2002).

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:ß Man ß 1,3-N-acetylglucosaminyltransferase (ß 3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAc ß 1-3Man ß 1-4Glc ß 1-1Cer. This study demonstrates that egghead encodes a Golgi-located GDP-mannose:ß Glc ß 1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Man ß 1-4Glc ß 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 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 ß4-mannosyltransferase, and brainiac encodes a ß3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAcß1-3Manß1-4Glcß1-Cer, found widely in invertebrates but not vertebrates. This report provides direct in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, 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 (Galß1-4Glcß1-Cer) using a human ß4-galactosyltransferase (ß4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) results in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Manß1-4Glcß1-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).

DEVELOPMENTAL BIOLOGY

Embryonic

Highest expression of brainiac occurs during the 0 to 4 hour interval and 4 to 8 hour interval of embryogenesis (Goode, 1996a).

Adult

Early egg chambers in the vitellarium show uniform expression of brn in germ cells. brn does not appear to be expressed in follicle cells. brn transcription begins at the time that the cyst becomes surrounded by a monolayer of follicle cells. At stage 6, egg chambers shows uniform levels of expression in nurse cells as well as the oocyte. At stage 10 of oogenesis, Brn transcript becomes even more abundant in nurse cells, probably reflecting the maternal contribution of brn transcripts to early neurogenesis (Goode, 1996a).

Effects of mutation or deletion

brainiac mutant mothers produce embryos with epidermal hypoplasia and neural hyperplasia. This phenotype is consistent with the classification of brainiac as a neurogenic gene. Embryos from mutant mothers are zygotically rescuable. As for the well-characterized zygotic neurogenic loci, brainiac gene activity is thus required zygotically to prevent all cells of the neurogenic region from differentiating into neuroblasts (Goode, 1992).

The dorsal appendages are located at the dorso-lateral surface of the eggshell, and are derived from dorsal-lateral follicle cells. Homozygous brn females lay eggs with fused dorsal appendages, a phenotype associated with mutants of the Egf receptor (Egfr) and with gurken mutants. brn is required for determining the dorsal-ventral polarity of the ovarian follicle. In brn and grk mutants, dorsal appendages are fused along the dorsal midline, indicating that dorsal-lateral follicle cells are shifted to a more dorsal position. Surprisingly, although brn influences follicle fates, it has no effect on embryonic DV polarity. This is the first instance of a Drosophila gene required for determination of dorsal-ventral follicle cell fates that is not required for determination of embryonic dorsal-ventral cell fates. The temperature-sensitive period for brn effect on follicle cell fate begins at the inception of vitellogenesis (Goode, 1992).

brainiac and Egfr interact in the initial formation of the follicle cells in the germarium. brainiac/Egfr double mutants lay only a few or no eggs, in contrast to single mutants which lay hundreds of eggs. In double mutants most follicles have more than one oocyte and more than 15 nurse cells. These follicles are formed within the germarium and proceed to late stages of oogenesis. Occasionally such chambers will proceed with chorion synthesis and form an egg in the shape of a ball, lacking overt polarity. Prefollicular cells fail to migrate between each oocyte/nurse cell complex, resulting in follicles with multiple sets of oocytes and nurse cells. brn and Egfr function is also required for establishing and/or maintaining a continuous follicular epithelium around each oocyte/nurse cell complex (Goode, 1992).

Embryos from mature mutant brn eggs also develop a neurogenic phenotype that can be zygotically rescued if a wild-type sperm fertilizes the egg. The zygotic brn functions, as well as the brn requirement for determination of follicle cell fate, appear to be genetically separable functions of the brn locus. Genetic mosaic experiments show that brn is required in the germline for establishment of follicle cell fate whereas the Egfr is required in the follicle cells. Thus brn may be part of a germline signaling pathway differentially regulating successive Egfr-dependent follicle cell activities of migration, division and/or adhesion and determination during oogenesis. It appears that the functions of brn in oogenesis are distinct from those of Notch and Delta, two other neurogenic loci that are known to be required for follicular development (Goode, 1992).

gurken and brainiac exhibit dosage-sensitive interactions. Double heterozygotes of weak mutants lay eggs with partially fused dorsal appendages, while single mutants are completely wildtype. Animals heterozygous for strong alleles show slight defects in patterning of follicular epithelium, while double mutants lay completely ventralized eggs completely lacking dorsal appendages. Ovarioles from females homozygous for weak alleles of grk or brn resemble wild type in terms of follicular cell development. In contrast, egg chambers from animals homozygous for strong alleles consistently display both fused egg chambers and gaps in the follicular epithelium, frequently uncovering over half of the egg chamber. It is concluded that grk acts in concert with brn to achieve the migration of prefollicular cells to surround each nurse cell-oocyte complex and to form a continuous epithelium (Goode, 1996a).

The neurogenic gene brainiac is specifically required for epithelial development. egghead (egh), a gene with phenotypes identical to brn, encodes for a novel, putative secreted or transmembrane protein. By comparing the function of germline egh and brn to Notch during oogenesis, direct evidence has been found for the involvement of Notch in maintenance of the follicle cell epithelium, and the specificity of brn and egh in epithelial development during oogenesis. The most striking phenotype observed for all three genes is a loss of apical-basal polarity and accumulation of follicular epithelial cells in multiple layers around the oocyte. The spatiotemporal onset of this adenoma-like phenotype correlates with the differential accumulation of egh transcripts in the oocyte at stage 4 of oogenesis. In contrast to N, it is found that brn and egh are essential for the organization, but not specification, of stalk and polar cells (Goode, 1996b).

Both egh and brn are required for concerted border cell-main body epithelium migration. Polar cells at the anterior of the egg chamber are part of a larger group of cells, termed border cells, that break from the main body epithelium (referred to as MBE, that portion of the epithelium that maintains a cuboidal/columnar phenotype and ends up covering the oocyte). These cells become mesenchymal-like, and migrate through the center of the egg chamber at stage 9 of oogenesis. slow border cells (slbo) is specifically expressed in border cells starting at stage 8 of oogenesis. Although follicle cells at the posterior of brn and egh mutant egg chambers become mesenchymal-like, they do not express slbo, indicating that they have not adopted a border cell fate (Goode, 1996b).

Instead, analysis of slbo expression from stage 8 to stage 10 in these mutants reveals that the migration of the MBE and border cells is not always concerted, and is not normal. The MBE frequently moves ahead of border cells, and completes its migration well before their migration. Once the MBE has completed its migration, slbo expression initiates at the anterior and posterior of the oocyte, but expression appears weaker as compared to that in wild type. All of these defects are independent of whether multiple layers of follicle cells accumulate around the posterior of the oocyte, and are consistent with a requirement for brn, egh and Notch in regulating the relative epithelial state of the follicular epithelium during a dramatic morphogenetic reorganization (Goode, 1996b).

The expression patterns and functional requirements of brn, egh, and N lead to a proposal that these genes mediate follicular morphogenesis by regulating germline-follicle cell adhesion. This proposal offers explanations for (1) the involvement of egh and brn in N-mediated epithelial development, but not lateral specification; (2) why brn and egh embryonic neurogenic phenotypes are not as severe as N phenotypes, and (3) how egh and brn influence Egfr-mediated processes. The correlation between the differential expression of egh in the oocyte and the differential requirement for brn, egh, and N in maintaining the follicular epithelium around the oocyte, suggests that Egghead is a critical component of a differential oocyte-follicle cell adhesive system (Goode, 1996b).

A screen was performed for female sterile mutations on the X chromosome of Drosophila and new loci were identified that are required for developmental events in oogenesis: new alleles of previously described genes were identified as well. The screen has identified genes that are involved in cell cycle control, intracellular transport, cell migration, maintenance of cell membranes, epithelial monolayer integrity and cell survival or apoptosis. New roles are described for the genes dunce, brainiac and fs(1)Yb, and new alleles of Sex lethal, ovarian tumor, sans filles, fs(1)K10, singed, and defective chorion-1 have been identified (Swan, 2001).

Glycosphingolipids control the extracellular gradient of the Drosophila EGFR ligand Gurken

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. 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. In C. elegans, GSLs appear to be dispensable throughout life. 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. 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. 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. 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 ). 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. Since the expression of a GSL-dedicated human galactosyltransferase in Drosophila egh mutants rescues their viability and fertility in a brn-dependent fashion, 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 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. 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, 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. 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. 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, 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).

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date revised:   25 April 2024 

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