A Retinal Axon Fascicle Uses Spitz, an EGF Receptor Ligand, to Construct a Synaptic Cartridge in the Brain of Drosophila  Zhen Huang, Ben-Zion Shilo, Sam Kunes  Cell  Volume 95, Issue 5, Pages 693-703 (November 1998) DOI: 10.1016/S0092-8674(00)81639-6

Figure 1 The Developing Eye and Brain at the Third Instar Larval Stage (A and B) The visual system from the lateral perspective. Photoreceptor cells in the eye disc commence neural differentiation at the morphogenetic furrow (mf), which moves in a posterior-to-anterior direction at this stage. The eight axons of an ommatidial R cell cluster navigate as a fascicle through the optic stalk (os) into the brain. Shown schematically in (A) are three of the ∼500 R cell clusters present at this time. The R1-R6 axons terminate at retinotopic destinations in the lamina target field (blue color in [A]). At the crescent-shaped lamina furrow (lf), recently arriving retinal axons trigger lamina precursor cells (LPCs) to undergo a terminal division and express early differentiation markers (such as Dachshund; blue color). A subset of postmitotic LPCs undergo neuronal differentiation, giving rise to cartridge ensembles at the posterior of the lamina (red circles). The confocal micrograph in (B) shows postmitotic LPCs in the lamina labeled to detect DAC (blue color), lamina cartridge neurons (αELAV staining; red color), and photoreceptor cells, their axons, and other neuronal cell membranes (labeled by αHRP; green color). The cartridge neurons, expressing both DAC and ELAV, are the pink cells among the retinal axons at the posterior of the lamina target field. Anterior is to the left, dorsal up. lob, lobula complex. Scale bar in (B) is 24 μm. (C and D) The visual system from the horizontal perspective. The eye and lamina display a temporal gradient of development on their anteroposterior axes. In (C), three ommatidia represented at different anteroposterior positions in the eye disc send their axons through the os to corresponding anteroposterior positions in the lamina, where the R1-R6 axons terminate. LPCs undergo their terminal division at the posterior margin of the lf and initiate their expression of early markers such as DAC (blue color). Within the lamina, postmitotic LPCs are organized into columns in precise register with the axons that regulate their differentiation. At the anterior, the differentiation of the first L-neuron (probably L1) is indicated by the onset of ELAV expression (red color). In more posterior columns, additional L-neurons join the fascicle-associated column. At the posterior of the lamina, complete cartridge ensembles form two layers, with L1-L4 localized superficially and L5 in a deep layer near the R1-R6 growth cone termini. The confocal micrograph in (D) shows DAC-positive postmitotic LPCs (blue color) and ELAV-positive cartridge neurons (red staining; coexpression of ELAV and DAC yields a pink color). The DAC-positive LPCs form vertical columns sandwiched between retinal axon fascicles (labeled by αHRP; green color). An arrow marks the position of the lamina furrow. An arrowhead marks the most recently arriving retinal axon fascicle at the anterior of the lamina target field. Anterior is to the left, lateral up. Scale bar in (D) is 12 μm. Cell 1998 95, 693-703DOI: (10.1016/S0092-8674(00)81639-6)

Figure 2 Retinal Axons Provide a Local Cue for Cartridge Neuron Differentiation and EGF Receptor Activation (A–C) A lateral view of an so1 specimen in which ommatidial R cell clusters have formed only in the ventral portion of the eye disc. The specimen shown in (B) and (C) is depicted schematically in (A). The photoreceptor cells (red color in [A]; stained in [B] and [C] by αELAV [red color], αHRP [green color], and αDAC [blue color]) send their axons through the optic stalk (os) into the lamina, where they project to appropriate ventral locations (the dorsoventral midline is marked by a black bar in [B] and [C]). Lamina differentiation, indicated by the expression of DAC and ELAV, is restricted to the ventral region that receives innervation. The high magnification view in (C) reveals that cartridge neuron differentiation (pink cells) is restricted to the immediate vicinity of the retinal axon fascicles (between the arrowheads). (D–F) hh− retinal axons induce cartridge neuron differentiation locally in animals in which hh+ has been supplied ectopically in the brain. The inactivation of hh+ at 84 hr AEL in a hhts2 animal results in an arrest of morphogenetic furrow (mf) progression at the posterior of the eye disc, as shown schematically in (D). In (E), a small group of hh− retinal axons (αHRP; green color) has innervated the dorsal portion of a large DAC-positive cell population induced by a flp-out P[Tubα1>y+,CD2>hh+] clone (blue color; shown schematically in [D]). Cartridge neuron differentiation (indicated by ELAV expression [red color]) is restricted to the immediate vicinity of retinal axon fascicles (between the arrowheads). In (F), a similar specimen is stained to reveal AOS expression (blue color). AOS expression (shown alone in the inset) is seen as punctate extracellular staining associated with the ELAV-positive cells. lf, lamina furrow; lon, larval optic nerve; ed, eye disc; lob, lobula. Anterior is to the left, dorsal up. Scale bar in (B) is 30 μm. Scale bar in (C), for (C), (E), and (F), is 12 μm. Cell 1998 95, 693-703DOI: (10.1016/S0092-8674(00)81639-6)

Figure 3 EGF Receptor Expression in the Lamina Is Induced by HH; EGFR Activity Is Both Necessary and Sufficient for Cartridge Neuron Differentiation (A–C) EGF receptor expression in the visual systems of wild-type (A), hhts2 (B), and “eyeless” animals with ectopic hh+ expression (C). In the wild type, EGFR expression (αEGFR staining in red color in [A–C], shown exclusively in [A′–C′]) is observed in the eye disc (ed) and at high levels in the lamina (pink color in [A] due to labeling with both αEGFR and αDAC [blue color]). In a hhts2 animal (B and B′) shifted to the nonpermissive at 94 hr AEL to generate hh− retinal axons (revealed by αHRP staining, green color), neither DAC nor EGFR expression is detected in the lamina target field. EGFR expression remains detectable in the eye disc. In (C), ommatidial differentiation was prevented by shifting a hhts2 animal to the nonpermissive temperature in the first instar stage (27 hr AEL; eye disc not shown). Lamina differentiation (indicated by DAC expression, blue color) was induced by the late flp-out induction of hh+ expression from the P[Tubα1>y+,CD2>hh+] transgene. EGFR expression (shown exclusively in [C′]) is stronger among the DAC-positive LPCs at the posterior of the lamina (lf, lamina furrow). lob, lobula. Scale bar in (A), for (A–C), is 24 μm. (D–F) Cartridge neuron differentiation in the wild type (D) and with the expression of transgenes encoding a dominant-negative (DN-) (E) and activated (λ-) (F) EGF receptor. In all three panels, postmitotic LPCs are shown in blue (DAC staining), cartridge neurons in red (αELAV staining), and retinal axons and other neuronal membranes in green (αHRP staining). In the wild type (D), cartridge neurons (pink color) are found at the posterior of the lamina. In an animal (E) in which strong late flp-out activation of P[Tub>CD2>GAL4] drives the expression of the P[UAS-DN-EGFR] transgene, cartridge neuron differentiation fails to occur, as indicated by the absence of ELAV-positive cells. However, retinal innervation and DAC expression appear normal. In an animal (F) in which the late flp- out activation of P[Tub>CD2>GAL4] drives the activated EGFR construct P[UAS-λ-EGFR], cartridge neurons are observed throughout a large portion of the lamina target field. In all panels, anterior is to the left, dorsal is up. Scale bar in (D), for (D–F), is 18 μm. Cell 1998 95, 693-703DOI: (10.1016/S0092-8674(00)81639-6)

Figure 6 The Expression of an Active SPI Fragment, sSPI, Induces Ectopic Neuronal Differentiation in the Lamina (A and B) Cartridge neuron differentiation in the lamina of the wildtype (A, A′) and animals expressing a truncated “active” SPI fragment, sSPI (B, B′). Expression of sSPI was induced in animals harboring the P[UAS-sSpi] and P[hsGAL4] transgenes by moderate heat shock conditions (34°C for 9 hr) immediately prior to dissection. In the wild type (A, A′), ELAV-positive cells (red color; shown alone in A′) are found in two layers (L1-L4 and L5) only in the posterior two-thirds of the lamina at this stage. In animals dissected immediately after heat shock treatment (B, B′), nearly all of the DAC-positive cells (blue color) in each fascicle-associated column are ELAV-positive (shown alone in B′), including cells at the very anterior of the lamina. (C and D) Expression of sSPI induces ectopic Argos expression in the lamina. In the wild type (C), AOS (blue color) is detected as punctate staining surrounding the ELAV-positive cartridge neurons (red color). The expression of aos in cartridge neurons was confirmed by examining nuclear localized β-galactosidase expression in an aos enhancer trap line (not shown). In a specimen in which ectopic neuronal differentiation has been induced by sSPI, AOS antigen is detected throughout the lamina target field, including LPCs at the anterior adjacent to the lamina furrow. (E and F) Expression of sSPI induces ectopic L5 neurons. The differentiation of L5 neurons was monitored by their expression of brain-specific homeobox (BSH; blue color). In the wild type (E), L5 neurons differentiate in a single medial layer (pink staining due to the expression of both BSH and ELAV [red color]). Ectopic sSPI expression (F) under the conditions described above induces ectopic neuronal differentiation throughout the lamina target field. BSH-positive (pink) cells occupy a 3–4 cell thick medial layer adjacent to the R1-R6 axon termini. os, optic stalk. In all panels, anterior is to the left, lateral is up. Scale bar in (A), for (A–F), is 12 μm. Cell 1998 95, 693-703DOI: (10.1016/S0092-8674(00)81639-6)

Figure 4 Spitz, Synthesized in the Eye, Is Transported along Retinal Axons into the Brain The visual systems of wild-type (A) and spiSCP2/spi0E92 animals (B) stained with a rat monoclonal αSPI antibody, which recognizes the extracellular portion of SPI. In the wild type, SPI antigen is detected in R cell clusters posterior of the morphogenetic furrow (indicated by a vertical white bar) in the eye disc (ed). SPI antigen is detected on retinal axons in the optic stalk (os) and in the lamina. SPI antigen is also detected on the larval optic nerve (lon). In the spiSCP2/spi0E92 specimen (B), SPI expression is greatly diminished. Cell 1998 95, 693-703DOI: (10.1016/S0092-8674(00)81639-6)

Figure 5 spi+ Is Required in the Eye for Cartridge Neuron Differentiation in the Lamina (A and B) Analysis of spiT25 mosaic animals. A spi clone in the ventral portion of the eye disc (outlined schematically in [A]) is associated with the absence of cartridge neurons in the retinotopically corresponding portion of the lamina (indicated by the arrowhead in the blue lamina target field in [A]). In (B) (left panel), the spi clone in the eye is marked by the absence of the P[arm-lacZ] marker (α-β-galactosidase staining in blue; clonal boundary indicated by white outline). The area within the clone harbors primarily R8 cells (αELAV staining, red color). In the brain ([B], right panel), cartridge neurons (αELAV staining, red color) are present only in the dorsal lamina target field, which is innervated by spi+ axons. The ventral portion of the lamina (indicated by an arrowhead) lacks cartridge neurons. In (A) and (B), a black horizontal bar marks the brain’s dorsoventral midline. Scale bar in (B), left panel, for ([B–F], left panels), is 24 μm. Scale bar in (B), right panel, for ([B–F], right panels), is 20 μm. (C–F) Ommatidial assembly in the eye and cartridge neuron differentiation in the lamina were monitored in the wild-type (C), spiSCP2/spiT25 (D), spiSCP2/spiA14 (E), and spiSCP2/spiT25 animals with specific spi+ expression in R cells (F). The wild-type eye disc ([C], left panel) displays a regular array of R cell clusters (αELAV staining, red color) posterior of the morphogenetic furrow (indicated by a vertical bar in [C–F]). Their axons induce ELAV expression in the DAC-positive (blue color) lamina target field ([C], right panel). In the spi hypomorphs (D and E), which were rescued to the early third instar stage by ectopic spi+ expression from the P[hsGAL4] and P[UAS-spi+] transgenes (see Experimental Procedures), some irregularity is observed in the eye ([D and E], left panels), particularly in anterior columns, but most posterior columns contain a normal R cell complement. In a significant proportion of these animals (see the text), cartridge neuron differentiation (indicated by the expression of ELAV) fails to occur ([D and E], right panels). The early marker DAC (blue color) is induced normally. In (F), the presence of the P[GMR-GAL4] eye-specific driver rescues cartridge neuron differentiation in the spi background. lf, lamina furrow. lob, lobula. In all panels, anterior is to the left, dorsal is up. Cell 1998 95, 693-703DOI: (10.1016/S0092-8674(00)81639-6)

Figure 7 Signaling Pathway for Lamina Cartridge Assembly On their arrival in the brain, retinal axons deliver HH, whose activity is required for several events, including the onset of EGF receptor expression, that render LPCs competent for cartridge neuron differentiation. Retinal axons provide the EGF receptor ligand Spitz, which triggers the differentiation of cartridge neurons in association with each ommatidial fascicle. This local activity of each fascicle results in a precise match of ommatidial and cartridge units. Cell 1998 95, 693-703DOI: (10.1016/S0092-8674(00)81639-6)