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First published online 14 December 2005
doi: 10.1242/dev.02197

Development 133, 307-317 (2006)
Published by The Company of Biologists 2006
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The endocytic pathway and formation of the Wingless morphogen gradient

Eric Marois, Ali Mahmoud and Suzanne Eaton*

Max-Planck Institute for Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.



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  		Fig. 1. Wingless is found in Rab5 and Rab7 endosomes. Wing imaginal discs ubiquitously expressing moderate levels of CFP-Rab5 and YFP-Rab7 stained with antibodies against Wg. Panels depict the different channels from a single confocal section 4 µm below the apical surface (including a small part of the Wg expression domain, at bottom of panel). Yellow arrowheads indicate co-localization between Wg and CFP-Rab5, blue arrowheads co-localization between Wg and YFP-Rab7. Scale bar: 20 µm.

 


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  		Fig. 2. Wingless spreads further when Rab5-dependent internalization is blocked. (A-C) Wg staining in single confocal sections of discs that expressed Rab5SN for 4 hours (A) or 5 hours 30 minutes (B,C) in the dorsal compartment. Apical, middle and basal sections are shown. Discs in A,B were stained using a protocol detecting both intra- and extracellular Wg; C shows extracellular Wg. (D) Single xz section perpendicular to the dorsal/ventral boundary of a disc 5 hours 30 minutes after the onset of Rab5SN expression. Dorsal Rab5SN-expressing compartment is to the right. The dorsal-ventral compartment boundary is indicated by a white line. Cadherin (green) marks apical junctions. Elevated Wg (red) accumulates apically at the level of and above junctions. Green arrows indicate junctions within the overlying peripodial membrane. (E) Single confocal section through the middle (8 µm below apical surface) of a wing disc that overexpressed GFP-Dlp for 24 hours in the dorsal compartment (indicated by brackets). The section passes through lateral cell membranes. Top: endogenous GFP-Dlp fluorescence. Bottom: extracellular Wg. GFP-Dlp overexpression elevates Wg on the lateral surface. Scale bar: 20 µm.

 


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  		Fig. 3. Inhibiting the degradative or recycling pathways does not affect extracellular Wingless distribution. (A,B) Wg conventional staining of wing discs expressing (A) Rab7TN, (B) Rab4SN + Rab11SN (inset shows nuclei positive for activated Caspase). Compare dorsal expressing and ventral non-expressing compartments. (A',B') Wg extracellular staining of the same genotypes. The dorsal-ventral boundary is indicated by a red line. Wg extracellular distribution is not altered by any of these treatments, but note intracellular Wg accumulation in A. Scale bars 20 µm.

 


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  		Fig. 4. Arrow knockdown causes intracellular Wg accumulation and loss of Wg signaling. (A) Anti-Arrow staining of wing disc with arrow RNAi induced in the dorsal compartment. Dorsal cells lack Arrow, except in four small clones that failed to excise the HcRed cassette and initiate RNAi (imaged using higher gain than Fig. 5C to maximize residual Arrow detection). (B) Top panel shows a control wing. Middle panel shows a wing from a fly that emerged 6 days after arrow RNAi induction in the posterior compartment. Lower panel shows a wing from a fly that emerged 6 days after induction of GFP-Dlp overexpression in the posterior compartment. (C) Projection of 12 confocal sections 1 µm apart from a disc that expressed arrow RNAi in the dorsal compartment for 48 hours, stained for Wg. (D) Single confocal section of the disc shown in (C) showing Wg, CFPRab5 and YFPRab7. Yellow arrowheads show examples of co-localization between Wg and endosomes. Scale bar: 20 µm.

 


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  		Fig. 5. Rab5SN expression alters subcellular localization and mRNA levels of Fz2 and Arrow. (A) In-situ hybridization to Fz2 mRNA in a disc after 4 hours 30 minutes Rab5SN expression in the posterior compartment. Inset shows Rab5SN expression domain (green). (B) In-situ hybridizations detecting arrow mRNA in discs of different genotypes. Left: wild type. Middle: disc expressing GFP-Wg in the posterior compartment for 6 hours. Right: disc expressing Rab5SN in the posterior compartment for 4 hours 30 minutes. (C) Discs expressing Rab5SN in the dorsal compartment for 5 hours 30 minutes stained for Arrow, Fz2 and Armadillo to mark apical junctions. Apical, middle sections and the basal region are shown. Note the accumulation of Fz2 and Arrow above the junctions. Large arrows in middle sections indicate the apical cell surface. In the basal region, the wing pouch curves so that the apical-basal axes of the epithelial cells at the edges of the wing pouch lie parallel to the focal plane. Thus, a complete longitudinal section of these cells is visible. Small arrows point to their basal sides. (D) Larger magnification of the areas boxed in C. Armadillo staining reveals the apical junctions of two rows of epithelial cells facing each other. Fz2 and Arrow accumulate above cellular junctions in Rab5SN-expressing tissue. (E) Single confocal section of a wing imaginal disc expressing YFP-Rab7, CFP-Rab5 and stained for Arrow. Arrowheads indicate co-localization between Arrow and Rab5/7 endosomes. Quantifying ten confocal sections corresponding to the apical-most 10 µm of the disc shown indicates that 49% of the brightest Arrow spots co-localize with either YFP-Rab7 or CFP-Rab5. Scale bar: 20 µm.

 


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  		Fig. 6. Rab5SN expression alters subcellular localization and mRNA levels of Dlp. (A,B) Wing disc after 5 hours 30 minutes Rab5SN expression in dorsal compartment, stained for Dlp. Dlp accumulates more strongly apically (A) than basal-laterally (B) in response to Rab5SN expression. (C) In-situ hybridization to dlp mRNA in a wild-type disc (top) and a disc after 4 hours 30 minutes Rab5SN expression in the posterior compartment (to the right). (D-G) Single confocal section of a disc stained for endogenous Dlp and expressing the indicated FP-Rab proteins. Quantifying ten confocal sections corresponding to the apical-most 10 µm of this disc indicates that 19% of the brightest Dlp spots co-localize with either YFP-Rab7 or CFP-Rab5. D, dorsal compartment; V, ventral compartment.

 


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  		Fig. 7. Removal of Dlp by PI-PLC treatment or by RNAi does not affect Wg accumulation on Rab5SN-expressing cells. (A-D) Imaginal discs after 5 hours 30 minutes Rab5SN expression in the dorsal compartment (dorsal Rab5SN-expressing tissue indicated by brackets). Images are projections of 1-µm-spaced confocal sections over 20 µm. (A) Disc mock-incubated for 1 hour, stained for Dlp. (B) Disc mock-incubated for 1 hour, stained for Wg. (C) Disc incubated for 1 hour with PI-PLC, stained for Dlp. (D) Disc incubated for 1 hour with PI-PLC, stained for Wg. Wg is not released with Dlp. (E,F) Discs subjected to Dlp RNAi for 40 hours in the dorsal compartment (indicated by brackets) stained for Dlp (E) or extracellular Wg (F). Dlp protein is undetectable and extracellular Wg fails to spread in dorsal cells. (G,H) Apical extracellular Wg staining of discs in which Rab5SN has been expressed for 5 hours in the dorsal compartment (indicated by brackets), either in the presence (G) or absence (H) of Dlp protein. (H') shows depletion of Dlp by RNAi in the dorsal compartment of a disc also expressing Rab5SN. (I-L) Disc ubiquitously expressing YFP-Rab7, overexpressing Dlp in the dorsal compartment (indicated by bracket) and stained for Wg. A projection of three sections 3 µm below the apical membrane is shown. (I) Wg imaged with high gain, emphasizing plasma membrane recruitment. (J) Wg imaged with low gain, emphasizing punctate endosomal staining. (K) YFPRab7-labeled endosomes. (L) Overlay of Wg and YFPRab7.

 


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  		Fig. 8. A model for Wingless gradient formation.

Cartoon of Wg trafficking through wild-type tissue (upper panel) and Dlp overexpressing tissue (lower panel). Wg is released from producing cells (red) and moves into receiving tissue, where it is endocytosed from apical and basal surfaces. Dlp overexpression (green in lower panel) biases Wg localization to the lateral surface, where it can diffuse without being endocytosed. This causes both elevation of Wg protein levels and extension of its spreading range (illustrated in flow charts). This model presupposes that Wg moves freely between apical, lateral and basal surfaces. If apical junctions establish a fence preventing diffusion of membrane proteins between apical and basal-lateral domains, Wg movement would have to occur via apical/basal-lateral transcytosis.

 




© The Company of Biologists Ltd 2006