Eph receptor tyrosine kinase-mediated formation of a topographic map in the Drosophila visual system

Abstract

Roles for Eph receptor tyrosine kinase signaling in the formation of topographic patterns of axonal connectivity have been well established in vertebrate visual systems. Here we describe a role for a Drosophila Eph receptor tyrosine kinase (EPH) in the control of photoreceptor axon and cortical axon topography in the developing visual system. Although uniform across the developing eye, EPH is expressed in a concentration gradient appropriate for conveying positional information during cortical axon guidance in the second-order optic ganglion, the medulla. Disruption of this graded pattern of EPH activity by double-stranded RNA interference or by ectopic expression of wild-type or dominant-negative transgenes perturbed the establishment of medulla cortical axon topography. In addition, abnormal midline fasciculation of photoreceptor axons resulted from the eye-specific expression of the dominant-negative EPH transgene. These observations reveal a conserved role for Eph kinases as determinants of topographic map formation in vertebrates and invertebrates.

Fig. 1.

EPH domain structure and distribution in the late third larval instar stage. a, Comparison of EPH (nomenclature adopted for consistency with the Flybase Database of theDrosophila Genome) to mouse (EphA3) and C. elegans (VAB-1) Eph receptors. The degree of identity to EPH for each domain of EphA3 and VAB-1 is shown: glob, globular; CR, cysteine-rich; FNIII, fibronectin type III repeat; JXM, juxtamembrane region;TK, tyrosine kinase; PDZ, PDZ domain;SAM, sterile α motif. The dominant-negative EPH transgene used in these studies is shown to the right of the sequence similarity comparisons; amino acids 675–1035 are deleted in this construct. b, Diagram of the developing eye (ed)–antennal (ad) disc from the lateral perspective image shown in c and f; axons posterior to the morphogenetic furrow (mf) from dorsal ommatidia project to dorsal optic lobe positionsd, whereas those from ventral ommatidia project to ventral target sites v in the lamina or more medial medulla regions (shaded crescent). The centripetal projections of cortical cell axons (red) to the underlying medulla are also shown. The midline position is indicated by the black arrow, and the location of the lobula complex (lob) is indicated. c, Photoreceptors in the eye disc (ed) and their axons in the optic stalk (os) and lamina (lam) are strongly stained by anti-EPH antibody (blue) in the late third-instar stage (∼125 hr AEL). Glia cells in the brain and optic stalk are stained by anti-OMB (red). d, Same picture as c but showing EPH staining only.e, A bright-field in situ hybridization of a third larval instar eye (ed)–antennal (ad) disc (left side) probed with digoxigenin-labeled eph mRNA. eph is expressed in cells anterior to the morphogenetic furrow (mf), consistent with immunostaining patterns. The anteroposterior, dorsoventral orientation of the eye disc is indicated in the top left corner. A Western blot of third larval instar CNS tissue is shown on the right.Lane 2 shows extracts of CNS stained with anti-EPH sera in which a ∼110 kDa band, the predicted size for EPH based on its open reading frame, is recognized. This band is not recognized by the preimmune sera (Lane 1), indicating specificity of this antibody for EPH. f, A more medial focal plane than (d) showing the medulla neuropil that lies directly beneath the lamina. EPH (blue) is strongly expressed by cortical cells (med. cortex) at the prospective dorsoventral midline (indicated by m) and virtually absent from cells at the most dorsal (d) and ventral (v) regions forming a dorsoventral gradient of expression in the medulla neuropil (med. n'pil). Some of the cortical axons that occupy dorsal and ventral neuropil positions are contributed by OMB-positive neurons (red). g, h, Same image asf, showing EPH staining only (g) or anti-HRP only (h). i, A view of the optic ganglia from the horizontal perspective at the third-instar larval stage. Photoreceptor axons enter the lateral portion of the brain hemisphere through the optic stalk (os; lateral is to the left). The R1–R6 axons terminate (R1–6 ter) after passing through the lamina cortex (lam), whereas R7–R8 continue medially beyond the R1–R6 termination point into the medulla neuropil (med. n'pil), in which HRP antigen is concentrated (green in i). As can be seen by comparing j (anti-EPH alone) and k(anti-HRP alone), EPH is concentrated in the older axons the lie at the prospective posterior (P) of the medulla neuropil and lamina (see also Fig. 2). Axons at anterior retinotopic positions (A in j) display less EPH antigen. Axons from the medulla cortex that project into the lobula (lob) also display a position-specific concentration of EPH antigen at their growth cone termini.

Fig. 2.

EPH expression during the formation of synaptic circuitry in the visual ganglia. The visual ganglia were isolated at three pupal stage time points (a–c; 24 hr APF;d–f, 45 hr APF; g–i, 65 hr APF) and stained with anti-EPH antibody (blue in a, d, g; alone in b, e, h), anti-HRP (green in a, d, g; alone inc, f, i) and Mab24B10, which specifically stains photoreceptor cells and their axons (red in a, d, g). As first noted in the late third-instar stage, EPH antigen continues to display a graded concentration on the anterior (A), posterior (P) axis at the early pupal time point (a–c). EPH is most strongly concentrated in the R1–R6 growth cone termini (R1–6 ter) at the prospective posterior of the lamina and cortical cell axonal termini at the prospective posterior of the medulla neuropil (med. n'pil; compare b, c). By 45 hr APF (d–f), EPH antigen is downregulated in the eye and concentrated in lamina synaptic cartridges (lam cart), which begin to form by this time. EPH antigen begins to display a columnar distribution in the medulla neuropil and is localized to a specific layer in the lobula (lob). By 65 hr APF (g–i), EPH antigen is absent from the eye and concentrated in the synaptic neuropils of the lamina, medulla, and lobula (h). An X denotes the position of the first optic chiasm.

Fig. 3.

RNAi inhibition of ephexpression causes defects in medulla architecture. Animals injected with either buffer alone (a–c) or 2 mg/ml dsephRNA (d–f) at the syncytial blastoderm embryonic stage were dissected at the late third instar.a–c, Lateral view of the medulla neuropil (med. n'pil) of an animal injected with buffer alone showing normal development. The R7–R8 axons (anti-Mab24B10; redin a) project normally into the neuropil to form a crescent. The larger crescent formed by the centripetal projections of the medulla cortical cell axons (med. cortex) is revealed by anti-HRP staining (b; greenin a, d). The location of the lobula complex (lob) is indicated. Anti-EPH staining (c; blue in a) reveals a wild-type dorsoventral gradient of expression. In animals injected with dsephRNA (d–f), dramatic disorganization is evident in the R7–R8 axon projections (red in d) and medulla cortical cell axon projections (e; green ind) to the neuropil. These defects are associated with a complete loss of EPH immunoreactivity in the brain (f).

Fig. 4.

Retinal and cortical cell axons project abnormally when the graded distribution of EPH is disrupted through expression ofeph^DN andeph⁺ transgenes. The ey-GAL4 driver alone does not affect optic ganglia development (a–c). Both cortical cell (med. cortex) axon projections to form the medulla neuropil (med. n'pil; anti-HRP stain;green in a; alone in b) and retinal axon projections (anti-Mab24B10 stain; bluein a; alone in c; R7–R8) are patterned normally. Medulla glial cells stained with anti-Repo antibodies (red in a) distribute themselves along the anterior [outer medulla glia (OMG)] and posterior [inner medulla glia (IMG)] faces of the medulla normally. The location of the lobula complex (lob) is indicated. The presence of a single copy ofP[UAS-eph^DN ] under control of the ey-GAL4 driver (d–f) disrupted optic ganglia structures specifically at the midline (region bracketed by arrowheads in d). The absence of strong anti-HRP staining in the central medulla of these animals is evident in d (green) ande (anti-HRP staining only), in which ectopic anti-HRP staining is detected (arrowhead in e). Retinal axons project to more anterior positions at the midline (region bracketed by arrowheads in f) and exhibit abnormal fasciculation (anti-24B10 staining;blue in d; shown alone inf; R7–R8). Ectopic expression ofeph⁺ in the pattern ofomb (g–i) also disrupted formation of the medulla neuropil. Medulla cortical cell axons (anti-HRP stain; green color in g; alone in h), especially those from dorsal (d) and ventral (v) regions (midline indicated by m), failed to project in direct centripetal manner into the neuropil, resulting in considerable disorganization (compare region indicated by arrow ing and h with b). Retinal axon projections (red in g; alone ini; R7–R8) show some disorganization, albeit less dramatic than what was observed using theeph^DN transgene.

Fig. 5.

Specific expression ofeph^DN in cortical cell populations using the ap-GAL4 driver disrupts the medulla neuropil architecture. Cortical cells bearing ap-GAL4 were labeled with GFP in recombinant animals (see Results); when crossed intoeph^DN lines, all GFP-positive cells also express the transgene. In animals harboring the ap-GAL4driver only (a–c), cortical cell (med. cortex) axons project centripetally [dorsal (D); ventral (V)] into the medulla neuropil (med. n'pil), forming a lattice-like meshwork (green ina; alone in b). Retinal axons project topographically into the medulla neuropil, forming a crescent (blue in a; alone in c; R7–R8). The pattern of axon projections into the medulla neuropil is disrupted in animals harboring both ap-GAL4 andeph^DN (d–f). Cortical cell axons project aberrantly (arrows ine), creating gaps in the neuropil (arrowheads in e), which has a fuzzy, undefined quality. In addition, the cortical cells themselves appear to be disorganized, most notably at the midline. Photoreceptor projections (f; R7–R8) exhibit some abnormal midline fasciculation in these animals. The fine structure of the neuropil is also disrupted when ap-GAL4 is used to driveeph⁺ in these same cells (g–i). In these animals, the effects are primarily associated with dorsoventral structures (arrows in h) rather than at the midline. Photoreceptor projections (anti-24B10 stain; blue ing; alone in i; R7–R8) were primarily normal in these animals.

Fig. 6.

Somatic clones expressingeph^DN indicate that EPH-mediated topographic guidance is important for both photoreceptor and cortical cells. Animals harboring hsFLP₁₂₂, P{UAS-CD8-GFP}, P{tubα₁>y⁺, CD2>GAL4}, andUAS-eph^DN were subjected to a brief heat shock to induce FLP expression. Recombination between the repeated FRT sites (indicated by >) yields GAL4⁺ clones marked by GFP expression; these clones also expresseph^DN . Retinal axons (R7–R8) abnormally fasciculated (region bracketed by arrowheads inc; anti-24B10 staining; blue ina; alone in c) when the eye tissue was composed of large clones (eye disc not shown). Expression ofeph^DN throughout the cortex (a, d) resulted in defects primarily localized to the midline. The higher-magnification view in d of the region indicated in b demonstrates the position-dependent effects of EPH signaling. Cortical cell axon projection defects (abnormal fasciculation and/or topographic projection) were enhanced at borders between wild-type tissue and tissue expressingeph^DN (arrows ind). Axon projections dorsoventral to the clone boundaries (arrowhead in d) were often wild type in appearance. EPH-mediated topographic mapping in the developing visual system is modeled in e; antennal disc (ad), eye disc (ed), morphogenetic furrow (mf), medulla cortex (med. cortex), medulla neuropil (med. n'pil), dorsal (d), and ventral (v) orientations are indicated. The distribution of EPH in the medulla is indicated by the grayscale shading. Medial axons (redcells) of both photoreceptors and cortical cells exhibit the greatest requirement for EPH signaling. Intermediate dorsoventral positions (yellow cells) require less EPH function, whereas extreme dorsoventrally located cells (blue) require the least degree of EPH signaling.