QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 14 January 2009
doi: 10.1242/dev.031104

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pizette, S. Articles by Thérond, P. Search for Related Content PubMed PubMed Citation Articles by Pizette, S. Articles by Thérond, P. Social Bookmarking                         What's this?

Glycosphingolipids control the extracellular gradient of the Drosophila EGFR ligand Gurken

¹ Institute of Developmental Biology and Cancer, CNRS UMR 6543, Centre de Biochimie, Université de Nice, Parc Valrose, 06108 Nice Cedex 02, France.
² The Cell Microscopy Centre, Department of Cell Biology, Institute of Biomembranes, University Medical Centre Utrecht, AZU Room H02.313, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
³ Temasek Life Sciences Laboratory, 1 Research Link, Singapore 117604, Republic of Singapore.

View larger version (34K): [in this window] [in a new window]   Fig. 1. GSL biosynthetic pathway and establishment of DV polarity during oogenesis. (A,B) Glycosphingolipid (GSL) biosynthetic pathway in mammals (A) and Drosophila (B). Glycan chain biosynthesis proceeds from right to left. (A) Mammalian GSLs are classified into three groups, ganglio-, lacto- and globoseries, depending on which glycosyl residue is added to the invariant diglycosylceramide core. These different triglycosylceramides can be further elongated by glycosyl residues and/or modified by sialic acid (not represented here). (B) Simplified version of the longest Drosophila GSL species isolated to date. All other characterized species correspond to shorter forms of this GSL. Egh catalyzes the addition of the second glycosyl residue (a mannose), whereas Brn catalyzes the addition of the third (an N-acetylglucosamine), providing the biosynthetic intermediates for further elongated GSLs. (C) Schematics highlighting the events taking place during DV patterning in Drosophila ovaries. See text for details. (D-G) Dorsal view of Drosophila eggs; anterior is to the left. In wild-type (wt) eggs (D), two separate respiratory appendages (RAs) of dorsolateral origin are seen at the anterior side of the egg (arrows). In mutants affecting DV polarity, these structures are either fused, such as in a hypomorphic Egfr allele and in a brn allele (arrow in E and G, respectively), or missing, such as in a strong hypomorphic combination of cni alleles that severely compromises Grk secretion (F, arrow points to a knob of residual appendage material).

View larger version (92K): [in this window] [in a new window]   Fig. 2. GSLs are needed for full activation of the EGFR pathway. (A-D') Stacks of confocal images showing dorsal views (anterior is left) of stage 11 Drosophila egg chambers processed for RNA in situ hybridization with an argos probe (aos, red in A,B, shown alone in A',B') or a rhomboid1 probe (rho1, red in C,D, shown alone in C',D'). In wild-type samples (A,C), aos is expressed in dorso-anterior (DA) follicle cells in a triangle (arrow in A') and rho1 transcripts are enriched in two L-shaped stripes (C'). In brn mutant samples (B,D), aos expression is almost extinguished (arrow in B'), whereas low-level, rather uniform rho1 expression remains (bracket in D').

View larger version (62K): [in this window] [in a new window]   Fig. 3. The GSL biosynthetic pathway is active in both germ line and follicle cells. (A) Upon loss of brn function, the intermediate product mactosylceramide (MacCer), synthesized by Egh, cannot be extended and accumulates. This product is specifically recognized by our antibody. (B) Confocal images of brn homozygous mutant Drosophila egg chambers of the indicated stages stained with anti-MacCer antibody (blue). The asterisk marks an egg chamber that could not be staged because it is compound. MacCer staining (shown alone in the bottom row of panels) is detected in the germarium (germ) and in newly budded egg chambers. Expression disappears between stages 4 and 7. From stage 8 onward, MacCer staining is present at the membrane of follicle cells. It is also observed in nurse cells at their membranes (arrows), but mostly in intracellular aggregates. In oocytes, MacCer staining is uniform at stage 8 and shifts towards the anterior cortex between stages 9 and 10 to remain strong in the vicinity of the nucleus. Wild-type egg chambers are negative for MacCer staining (not shown). Anterior is to the left and dorsal is up.

View larger version (93K): [in this window] [in a new window]   Fig. 4. A stage-specific requirement for GSLs in Grk distribution. (A,B) Confocal images of stage 10a Drosophila egg chambers processed for RNA in situ hybridization with a grk probe (red). grk mRNA is tightly localized around the nucleus in both wild-type (A) and brn mutant (B) oocytes. (C-I') Confocal images of egg chambers labeled by immunofluorescence for Grk protein. (C-D') Stage 8 egg chambers. In both wild-type (C) and brn mutant (D) egg chambers, Grk (red, shown alone in C',D') is detected in the cytoplasm and at the cortex of the oocyte (labeled for F-actin, green), in the close vicinity of the nucleus, as well as in adjacent follicle cells (arrows in insets in C',D'). (E-E'') Stage 10b wild-type egg chamber showing an enlargement of the DA region of the oocyte and follicular epithelium. (F-I') Stage 10a egg chambers. G,I show enlargements of the DA region of the oocyte and follicular epithelium from the chambers shown in F,H, respectively. In wild-type specimens (E-G'), Grk (red, shown alone in E'',F',G') is present in the oocyte cytoplasm close to the nucleus, and in neighboring follicle cells (arrows). The bulk of Grk, however, lies in the extracellular space between the cortices of the oocyte and the follicular epithelium (identified by F-actin staining, green, as shown alone in E'). brn mutant oocytes (H,I') exhibit levels of Grk (shown alone in H',I') in their cytoplasm that are comparable to those in the wild type. Grk, however, is absent from the extracellular space and is only observed in the anterior-most follicle cells (arrow in I'; compare with the wild type, arrows in G') and in border cells (arrowheads in I'; the border cell cluster in not in the plane of the section in the wild type). Anterior is to the left, dorsal is up. N, oocyte nucleus.

View larger version (82K): [in this window] [in a new window]   Fig. 5. GSLs are dispensable for Grk trafficking to the plasma membrane. (A-D') Confocal images of stage 10a Drosophila egg chambers labeled by immunofluorescence for Grk (red). Enlargements of the DA region of the oocyte and follicular epithelium are shown in A'-D'. Anterior is to the left and dorsal is up. In cniAA12/cniAR55 mutant samples (C,C'), Grk is not secreted and localizes neither to the extracellular space separating the oocyte from the follicular epithelium, nor to follicle cells (compare with wild-type sample, A,A'). Instead, it diffuses into the ER lumen of the oocyte, where it is trapped (reticular staining surrounding the nucleus in C'). In samples from the grkDC allele (D,D'), which expresses an uncleavable Grk, Grk is also absent from the extracellular space and from follicle cells, but it reaches the oocyte cortex and it is dispersed throughout the oocyte cytoplasm (yellow staining in D' corresponds to Grk overlapping with the cortex stained for F-actin in green). In brn mutant samples (B,B'), despite the lack of Grk in the extracellular space and in most follicle cells, Grk does not mislocalize or accumulate in the oocyte cytoplasm. (E) Western blot analysis of Grk cleavage. Extracts from wild-type ovaries, and from wild-type or brn mutant ovaries expressing an intracellularly Myc-tagged grk construct in their germ line (driven by nosGAL4). Tubulin (Tub) was used as a loading control. In both wild-type and brn mutant backgrounds, the anti-Myc antibody detects a comparable amount of the cleaved intracellular (IC) portion of Grk, revealing similar cleavage efficiencies. (F,G) Transmission electron micrographs of the DA region of stage 10a egg chambers. Ultrathin cryosections were immunolabeled for Grk (10 nm, gold). A tER-Golgi unit close to the oocyte nucleus is shown (brackets). The most abundant labeling for Grk in a wild-type (F) or brn mutant (G) oocyte is at the tER-Golgi units. Note that the density of this labeling is similar in the two backgrounds. Scale bars: 200 nm.

View larger version (83K): [in this window] [in a new window]   Fig. 6. GSLs are not required for Grk to maintain the spatial regulation of EGFR. Confocal images of stage 10a Drosophila egg chambers from the wild type (A-A''), and from cni (B-B'') and brn (C-C'') mutants, labeled by immunofluorescence for EGFR. Enlargements of the anterodorsal (A'-C') and the anteroventral (A''-C'') parts of the follicular epithelium are shown. EGFR distribution is asymmetric in wild-type egg chambers (A), the protein being barely detectable (A') in most of the dorsal midline (region delimited by arrowheads in A), while it is enriched at the apical and lateral membranes of ventral follicles cells (A''). cni mutant egg chambers (B), however, display EGFR staining characteristic of ventral follicle cells throughout their epithelium (compare B' with B''). In brn mutant egg chambers (C), EGFR distribution remains asymmetric along the DV axis, with lower protein levels in DA follicle cells (region delimited by arrowheads in C; compare C' with C''). In all samples, F-actin staining is uniform throughout the follicular epithelium (not shown). Anterior is to the left, dorsal is up. N, oocyte nucleus.

View larger version (60K): [in this window] [in a new window]   Fig. 7. GSLs shape the extracellular Grk gradient. (A,B) Stacks of confocal images showing dorsal views (anterior is left) of wild-type (A) and brn (B) stage 10a Drosophila egg chambers, labeled by immunofluorescence for extracellular Grk. The oocyte nucleus is beneath the asterisk. Under such conditions, Grk is exclusively visualized in the extracellular space. (C-E) Plots of average Grk staining intensity along the planes indicated by the arrows in A, from three to five wild-type and brn mutant specimens. A value of 0 on the x-axis corresponds to the dorsal-most point of planes C,D and to the anterior-most point of plane E. In wild-type egg chambers (A), extracellular Grk (red in C-E) forms a gradient along the DV axis (C,D). Along the AP axis (E), Grk levels are low at the source (asterisk marks the position of the oocyte nucleus, see also C) and rapidly rise to reach a plateau. In brn mutant egg chambers (B, yellow in C-E), the shape of the gradient is different in that more Grk accumulates at the source than in the wild type (C), but the length of the plateau is shortened along the AP axis (E). (F-K') Confocal images of stage 10a egg chambers labeled by immunofluorescence for extracellular Grk (red). Enlargements of the DA region of the oocyte and follicular epithelium in G,I,K show the presence of Grk (shown alone in G',I',K') in the extracellular space. In wild-type (F,F') and brn mutant (H,H') samples, the extent of extracellular Grk diffusion towards the posterior pole is similar, whereas in Egfr mutant samples (J,J'), Grk diffuses further (arrows in F',H',J' delimitate extracellular Grk staining, see text for quantifications). Anterior is to the left, dorsal is up.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter