Polarized secretion of Drosophila EGFR ligand from photoreceptor neurons is controlled by ER localization of the ligand-processing machinery

Abstract

The release of signaling molecules from neurons must be regulated, to accommodate their highly polarized structure. In the developing Drosophila visual system, photoreceptor neurons secrete the epidermal growth factor receptor ligand Spitz (Spi) from their cell bodies, as well as from their axonal termini. Here we show that subcellular localization of Rhomboid proteases, which process Spi, determines the site of Spi release from neurons. Endoplasmic reticulum (ER) localization of Rhomboid 3 is essential for its ability to promote Spi secretion from axons, but not from cell bodies. We demonstrate that the ER extends throughout photoreceptor axons, and show that this feature facilitates the trafficking of the Spi precursor, the ligand chaperone Star, and Rhomboid 3 to axonal termini. Following this trafficking step, secretion from the axons is regulated in a manner similar to secretion from cell bodies. These findings uncover a role for the ER in trafficking proteins from the neuronal cell body to axon terminus.

Figure 1. Rho-3 exclusively mediates Spi secretion from photoreceptor axons.

(A–D) Lateral views of developing eye disc and lamina from late third-instar larvae. Photoreceptor cell bodies in the eye disc (e.d.) express the pan-neuronal marker ElaV (red, shown separately in single-primed panels). Photoreceptor axons, marked by HRP (blue), extend from the eye disc through the optic stalk (o.s.) and terminate at the lamina. The posterior lamina, in which ElaV is expressed, is marked by an arrowhead and outlined, and magnified in insets. Dac (green, shown separately in double-primed panels) and ElaV (red) expression in the lamina reflects Hh and Spi secretion from photoreceptor axons, respectively, and the triggering of the signaling pathways in the future lamina cartridge neurons. Scale bar: 40 µm. (A) In wild-type (wt) late third-instar larva, ElaV is expressed in the eye disc and lamina. (B) Schematic of (A). Note the retinotopic projections of photoreceptor axons in the lamina. At this developmental stage, not all photoreceptors have differentiated yet, hence only the posterior part of the lamina is invaded by retinal axons, and ElaV expression (yellow) is detected only there. (C) In eyes bearing large rho-1 clones, ElaV and Dac are normally expressed in the lamina (inset), despite some morphological abnormalities. (D) Large rho-3 clones eliminate EGFR activation in the lamina. ElaV expression is missing from the lamina (inset). Note that ElaV is still expressed in the eye disc, indicating that Rho-1 and Rho-3 redundantly mediate Spi secretion from cell bodies. Dac is normally expressed in the lamina, demonstrating that rho-3 mutants do not suffer from general secretion defects. Anti-HRP staining (blue) shows that rho-3 axons are correctly targeted to the lamina. (E) Anti-EGFR staining (red) in wild-type lamina shows many endocytic puncta (inset in E′, arrows) at the posterior of the lamina, associated with the ElaV-expressing cells (green). Scale bar: 20 µm. l.f., lamina furrow. (F) Spi–GFP (green) expressed in the eye by GMR–Gal4 is secreted from photoreceptor axons, and co-localizes with EGFR (red) in endocytic puncta (arrows) in lamina cells. Scale bar: 10 µm. (G) Spi–GFP (green) in which the Rhomboid cleavage site was mutated fails to localize with EGFR (red) in the lamina cells. Scale bar: 10 µm. (H) In a lamina from rho-3 mutants, EGFR distribution (red) shows a reduced number of endocytic puncta (inset in H′), suggesting that the receptor is not engaged by the ligand on the surface of lamina cells. ElaV expression (green) is specifically missing from the lamina. Scale bar: 20 µm. (I) Spi–GFP (green), expressed in the eye of rho-3 mutants is not secreted from the axons, and does not co-localize with EGFR in lamina cells. Scale bar: 10 µm. (J and K) Schemes of Spi secretion from axons. In wild-type larvae (J), Spi (green ovals) is secreted from axons and co-localizes with EGFR (red) in endocytic puncta in lamina cells. In the absence of cleavage by Rho-3 (K), Spi fails to be secreted from photoreceptor axons, and does not co-localize with EGFR in the lamina, which, in turn, is not internalized.

Figure 2. Subcellular localization of Rhomboids is mediated by their cytoplasmic N-termini and first luminal loop.

(A) A gRho-1 construct, YFP tagged at the C-terminus (green), localizes to discrete punctate structures (arrows in [A′]). ElaV (red) shows photoreceptor nuclei. The morphogenetic furrow is to the left. Scale bar: 5 µm. (B) Rho-3, expressed from a genomic construct and tagged with GFP at the C-terminus (green) shows both ER (arrowheads in [B′]) and punctate (arrows in [B′]) localization. Like the Rho-1 puncta, Rho-3 puncta are more abundant in apical optical sections (not shown). ElaV (red) shows photoreceptor nuclei. Primed panels show single channels for YFP or GFP. Scale bar: 5 µm. (C–F) Subcellular localization of GFP-tagged Rho-1, Rho-3, R1L1-R3, and R3L1-R1 (green), expressed in the eye disc by GMR–Gal4. ElaV (blue) marks photoreceptor nuclei, and FasIII (red) stains membranes. Primed panels show a single channel for GFP. Scale bar: 5 µm. The schemes at the top of each panel show the topology of the proteases (N-termini are in the cytoplasm; C-termini are luminal; not to scale). Also shown are the positions of the catalytic serine (S) and histidine (H), embedded in the fourth and sixth transmembrane helices, respectively. Rho-1 is orange; Rho-3 is green. (C) GFP–Rho-1 localizes to apical punctate structures (arrows in [C′]). (D) GFP–Rho-3 localizes to the apical structures (arrows in [D′]) and the peri-nuclear ER (arrowheads in [D′]). (E) The N terminus and first luminal loop of Rho-3 were replaced with that of Rho-1. These sequences are sufficient to confer a Rho-1-like localization to GFP–R1L1-R3 (arrows in [E′]). (F) Rho-1 in which these sequences are derived from Rho-3 (GFP–R3L1-R1) is localized to the ER (arrowheads in [F′]) and the apical puncta (arrows in [F′]).

Figure 3. ER localization of Rho-3 facilitates Spi secretion from axons.

(A–E) All constructs are GFP tagged at the N-termini, inserted into the same genomic location, and expressed in R2, R5, and R8 by MT14–Gal4. ElaV is red; Dac (green) and HRP (blue) mark photoreceptor axons. (A) Lateral view of a wild-type (wt) lamina from a late third-instar larva, with the typical ElaV triangle at the posterior. (B) GFP–Rho-1, expressed in R2, R5, and R8 fails to rescue the rho-3 phenotype, as indicated by the lack of ElaV-positive cells within the population of Dac-positive precursors. (C) A GFP–Rho-3 transgene restores ElaV expression to the lamina of a rho-3 mutant. (D) When Rho-3 is not localized to the ER, as is the case of the GFP–R1L1-R3 chimera, it fails to promote Spi secretion from the axons and induce EGFR activation in the lamina. (E) An ER-enriched Rho-1 (GFP–R3L1-R1) rescues the rho-3 phenotype. (A′–E′) Single channel for ElaV staining. (F) Quantification of the results from (A–E). ElaV-positive cells in the lamina were counted in 8–10 specimens per genotype. The difference between ER-resident and non-ER-resident proteases is statistically significant (ANOVA). (G) Rho-1–HA (red, shown separately in [G′]) expressed in wild-type MARCM clones (marked by GFP, green) is localized to the typical apical puncta. (H) ER-to-Golgi trafficking is blocked in sed5 MARCM clones, marked by GFP. Rho-1–HA (red, shown separately in [H′]) expressed in the mutant photoreceptors is retained in the peri-nuclear ER. (I) Horizontal view of a rho-3 mutant lamina. HRP (green) marks retinal axons and outlines the lamina; Dac is blue. No ElaV-positive cells are seen in rho-3 mutant lamina (red, shown separately in [I′]). (J) ElaV expression (red, shown separately in [J′]) is restored to a small population of cells at the posterior of the lamina of rho-3 mutants after elimination of one copy of sed5. Scale bars: 10 µm.

Figure 4. Spi secreted from axons is not processed in the ER.

(A–D) Lateral views of late third-instar larva laminae. ElaV (red, shown separately in primed panels), Dac (green), and HRP (blue). The posterior of the lamina, where EGFR activation is evident by ElaV expression, is marked with an arrowhead and is outlined. (A) Wild-type (wt) lamina showing the typical ElaV staining at its posterior. (B) A GFP–Rho-3 transgene, expressed in R2, R5, and R8 by MT14–Gal4 restores ElaV expression in rho-3 mutants (compare with Figure 3B). (C) When Rho-3 is localized exclusively to the ER by a KDEL tag (GFP–Rho-3–KDEL) it fails to rescue the rho-3 mutant phenotype. (D) An ER-retained form of Rho-1 (GFP–Rho-1–KDEL) fails to rescue the rho-3 mutant phenotype. (E and F) Spi does not require cleavage for translocating in the axons. Spi–GFP was expressed in the eye disc by GMR–Gal4, and its distribution in axons in the optic stalk (o.s.) was monitored. (E) Wild-type Spi–GFP expressed in a wild-type genetic background is detected throughout the axons. (F) Mutating the Rhomboid cleavage site in Spi–GFP does not alter its distribution in axons. (G) Cleavage of Spi in the ER does not occur in rho-3 mutants, yet the distribution of the ligand in axons is similar to wild-type. (H) Quantification of Spi distribution in axons. Mean pixel intensities were determined at the entry point of the optic stalk into the brain, and at the eye disc. A ratio of mean pixel intensity in the eye to mean pixel intensity in the optic stalk was calculated per specimen; 7–10 specimens were used for each quantification. The differences observed are not significant (ANOVA). Scale bars: 10 µm.

Figure 5. The ER facilitates Rho-3 trafficking to axons.

(A–C) Endogenous ER markers are detected throughout the axons of photoreceptor neurons. (A) A GFP gene trap in the endogenous PDI. GFP immunoreactivity is detected along the axons (not shown) and at their termini, as they invade the lamina. (B) ER-retained proteins are revealed by anti-KDEL immunostaining along the length of the axon. The inset shows a magnification of the axonal termini in the lamina. (C) ER exit sites, marked by dSec16, showing a smooth staining and some brighter puncta in the axons and in lamina cells. The inset shows dSec16 puncta (arrowheads) in axons which have reached the lamina. (D and E) Expression of GFP–KDEL in the eye disc by mδ0.5–Gal4. (D) RNA in situ hybridization with a GFP probe, showing that GFP–KDEL mRNA is restricted to cell bodies in the eye disc. No signal is detected in the optic stalk (o.s.) or the lamina (outlined). (E) GFP–KDEL protein can reach the axon through the ER, and is detected along the entire length of the axon. (F) ManII–GFP (green), expressed in wild-type MARCM clones, is present throughout the axon. The outlined area (asterisk) is a clone in the lamina cells. (F′) shows the ManII–GFP separately, with an enlargement of one fascicle. The Golgi is detected as discrete units (arrowhead), with a “beads on a string” appearance. (G) rho-3 mRNA is confined to cell bodies in the eye disc, and is not detected in the axonal projections into the lamina (outlined). (H) The ER localized gRho-3–GFP (green, and in [H′]) is localized to the eye disc, and is also enriched in axons. Arrowhead in (H′) marks the larval optic nerve (l.o.n.) where nonspecific staining occurs. (I) gRho-1–YFP (green, and in [I′]) is localized specifically to the eye disc, and does not reach the axons. Arrowhead in (I′) marks the larval optic nerve. (J) When gRho-1–YFP is targeted to the ER (gRho-1–YFP–KDEL, green and in [J′]), it is translocated along the axon bundle. Scale bars: 10 µm.

Figure 6. Co-trafficking of Spi, S, and Rho-3 sensitizes EGFR activation in the lamina to S gene dosage.

(A–C) The localization of Spi, S, and Rho-3 was examined in the optic stalks of specimens expressing HA- (red) or GFP-tagged (green) versions of the proteins in the eye disc by GMR–Gal4. (A) Spi–HA co-localizes with Rho-3–GFP in the axons. (B) Spi–GFP co-localizes with S–HA. (C) S–HA co-localizes with Rho-3–GFP. (D–F) S stabilizes Spi during their joint axonal trafficking. (D) The levels of Spi–GFP (green), expressed on its own in the eye disc, decay along the axons. (E) Co-expression of S–HA with Spi–GFP stabilizes the ligand. l.o.n., larval optic nerve; o.s., optic stalk. (F) Quantification of the effect of S expression on Spi. Mean pixel intensities of Spi–GFP were determined every 10 µm along the optic stalk, from the point where the optic stalk leaves the eye disc (distance  = 0). GFP intensity was normalized to 100 at point 0. Seven specimens were examined per genotype. Student′s t-test shows that the difference at the most distal point is statistically significant. (G and H) EGFR signaling is more sensitive to S levels in the lamina than in the eye. EGFR activation in both tissues is assayed by ElaV expression (red), Dac (green), and HRP (blue). (G) S heterozygous eye disc. EGFR phenotypes associated with S^+/− (misrotated ommatidia and missing photoreceptors) lead to the slightly abnormal appearance of ElaV staining, but the phenotype is not severe. The inset shows that photoreceptor axons extend normally to the brain. (H) The lamina of the same specimen as in (G) shows a severe reduction in EGFR activation. Only a small number of cells (arrows) at the posterior of the lamina express ElaV, although Dac expression is unperturbed. Scale bars: 5 µm.

Figure 7. Regulation of Spi secretion by endosomal trafficking.

(A and B) The apical Rho-1 puncta are Rab4/14 endosomes. Rho-1–HA (red), expressed in the eye disc, co-localizes with YFP-tagged Rab4 or Rab14 (green). (A) Co-localization of Rho-1–HA and YFP–Rab4. (B) Co-localization of Rho-1–HA and YFP–Rab14. (C) Rho-1–GFP shows the typical punctate staining of Rab4/14 endosomes when expressed in the eye disc. (D) Upon co-expression of Rab11DN, Rho-1–GFP is mislocalized. Some weak punctate staining is still detectable, but most GFP immunolabeling appears sub-membranal. (E) Expression of Rab11 RNAi in the eye disc by GMR–Gal4 yields EGFR phenotypes such as missing photoreceptors (arrows in [E′]), misrotated ommatidia (arrowhead), and defective ommatidial spacing. Importantly, although ElaV staining (red, and in [E′]) reveals these defects, R8 differentiation (Senseless, blue, and in [E″]) is unaffected. (F) R8-specific expression of Rab11DN by Sca–Gal4 disrupts EGFR signaling in adjacent cells, indicating that Rab11 is involved in ligand secretion. For example, two ommatidia with only four outer photoreceptors (*) and an R8 cell (#) are marked. (G) Adult flies expressing Rab11DN by GMR–Gal4 have small eyes. (H) Co-expression of Rho-1 or Rho-3 (not shown) rescues the defects associated with Rab11DN, suggesting that these defects are partly due to a failure to properly target Rhomboids and secrete Spi. (I) Expression of Rab11DN in the eye also leads to a defect in EGFR signaling in the lamina. ElaV staining in the lamina (red, and in [I″]) is strongly reduced (arrowhead in [I″] shows residual staining), while Dac expression (green, and in [I′]) is not affected. (J) Model. ER-facilitated trafficking of the Spi-processing machinery to axon termini promotes Spi secretion from the axons to the lamina. Scale bars: 5 µm.