Central projections of photoreceptor axons originating from ectopic eyes in Drosophila

Abstract

Ectopic expression of the retinal determination gene eyeless (ey) induces the formation of supernumerary eyes on antennae, legs, wings, and halteres. These ectopic eyes form ommatidia that contain photoreceptors and accessory cells and respond to light. Here, we demonstrate that ectopic eyes on antennae and legs extend axonal projections to the central nervous system. Furthermore, electroretinograms and morphological evidence indicate that the photoreceptor axons of at least the antennal ectopic eyes can form completely constituted ectopic synapses with foreign postsynaptic elements and suggest that transmission at these sites may be functional. However, the ectopic axons do not connect to their correct optic lobe targets and do not project deeply into the neuropile, but rather form synapses at superficial positions in the neuropils. By means of confocal and electron microscopy we show that these ectopic synapses resemble normal synapses, albeit with some distinct morphological differences. Our data strongly suggest that the developmental programs controlling photoreceptor synaptogenesis and visual map formation depend to a considerable extent on presynaptic and thus photoreceptor-autonomous steps. Our data also suggest that photoreceptor axon projections and the establishment of the highly stereotypical neural circuitry in the optic lobe, the normal target neuropil, may depend on target-specific cues that appear to be absent from the antennal lobe and thoracic ganglion.

Fig. 1.

Electroretinograms of normal and ectopic eyes (A–D) and confocal images of ectopic photoreceptor axon projections (E–O). (A) ERG of a WT eye consists of a positive on transient (left arrowhead), a negative sustained potential of photoreceptor origin, and a negative off transient (right arrowhead). The transients coincide with the onset and offset of the light. (B) ERG of an ectopic eye on the leg of a w/w;dpp^blinkGAL4/UE10 fly. Only the photoreceptor potential can be detected. (C) ERG of an ectopic eye on the wing of a MS1096/w;UE10/+ fly. A small photoreceptor potential is recorded. Also note the slow repolarization after lights off. (D) ERG of an ectopic eye on the antenna of a w/w;dpp^blinkGAL4/UE10 fly. A small photoreceptor potential is observed. In addition, on and off transients were recorded. The bar beneath the ERG recording indicates the duration of the light stimulus. (E) Schematic overview of ectopic photoreceptor axons (marked with asterisk and arrow) projecting to the antennal lobe (AL) glomeruli. (F) Confocal images corresponding to the overview in E. The photoreceptor axons immunolabeled with monoclonal anti-DLG antibody 4F3 (magenta) enter the brain and project to the antennal lobe immunolabeled with anti-Bruchpilot antibody nc82 (green). (G–I) Higher magnification of boxed area in F, with H and I representing single confocal channels. (J–0) Schematic overview (J) of the ventral ganglion shown in K–N. T1-T3, thoracic neuromeres 1–3; A, abdominal neuromere. (K) Confocal image corresponding to boxed area in J. Thoracic ganglion immunolabeled with anti-HRP antibody (magenta) and nc82 (green). (L) Higher magnification of the boxed area in K. (M and N) Single confocal channels of L. (O) Boutons of ectopic photoreceptors in the thoracic ganglion.

Fig. 2.

Ectopic photoreceptor terminals form synaptic contacts in foreign territories. (A) A stained photoreceptor axon bundle from an antennal eye off the left hand side of the section can be seen extending through the scape (scp), along the antennal nerve (AN; right rectangle) and into the antennal lobe (AL; left rectangle) in a light micrograph of an unstained 2-μm semithin section before re-embedding. (B–D) Electron micrographs from the same section as in A. (B) Bundle of axon profiles in the antennal nerve (right rectangle in A) with HRP-labeled plasma membranes. (C) Profiles of axons with labeled membranes in the superficial layers of the antennal lobe, beneath the cortex, enlarged from left rectangle in A. Three labeled axon profiles enclosed in the rectangle are further enlarged in D. (D) Three profiles of synaptic terminals, containing mitochondria (m), have a presynaptic profile (rectangle, enlarged in E). (E) Presynaptic T-bar ribbon (arrowhead) in terminal, opposite profiles of two of the postsynaptic elements (asterisks). The presynaptic element has many profiles of synaptic vesicles (arrow), one of which appears to be labeled with HRP reaction product possibly endocytosed from the plasma membrane (short arrow), but lacks the characteristic glial invaginations at capitate projections.