PMC

Ultrastructural characteristics of larval photoreceptor terminals (lpt). A: 3D digital model of larval optic neuropil (LON, gray) and outer optic anlage (OOA, brown), dorso-posterior view. One photoreceptor terminal is shown (green). B: Model of LON and the same photoreceptor terminal as in A. Note varicose and bouton-shaped (bt) thickenings of photoreceptor terminal. Output synapses (sy) of this terminal are shown in magenta, bolwig nerve (bn). C: Statistics of the twelve lpt analyzed. D: TEM image of two adjacent bt of photoreceptor terminals, with synapse (sy; note characteristic T-bar and synaptic density, arrowhead). Other profiles in this image include optic lobe pioneer (OLP) and (uncharacterized) target neuron (tn). E, F: TEM images of photoreceptor terminal (shaded green), showing thin connector (cn) between adjacent boutons (bt). Arrowhead in F (magnified view of connector) points at array of microtubules. Note also profile of electron-dense glial cell (gl) adjacent to photoreceptor terminal and the epithelial cell (ep). Bars: Scale bars: 0.5μm (D), 1.5 μm (E), 0.2 μm (F).

Projection neurons of the larval optic neuropil (LON). A–C: 3D digital models of LON, showing optic lobe pioneers (OLP) in shades of blue, and PDF-dendritic branches (PDF) in shades of orange, yellow and red; cellbodies of the outer optic anlage epithelium (OOAep) are translucent grey. The same colors are used in panels E–I, which show TEM sections of LON at different levels. Cell bodies of OLPs are located laterally adjacent to OOAep, flanking the entering Bolwig’s nerve (bn) (A, F, G). Note multiple short, dendritic branches of OLP fibers, preferentially in distal LON (LONdist; arrowheads in A–C). PDF neuritis are found preferentiall in the proximal LON (LONprox) and LONdist. Cell bodies of PDF neurons, shown schematically in (D), are located dorsally adjacent to the OOA. Cell body fibers form a bundle (PATpdf) that penetrates through the OOA epithelium (green arrowhead in D and E; represents a section of the OOA epithelium near its outer, apical boundary; note sub-apical adherens junctions shown by white arrowheads). Cortex neuron cell body (co) flanking the OOAep (in E). PDF-neuronal fibers form bundle in proximal LON (arrowhead in I) and extend branches throughout this compartment (B, C, F, G, H), glia cells (gl), PAT primary lineage related axon tract (I, H). Scale bars: 2 μm (F, G), 1μm (H, I, E).

Topology of larval optic neuropil (LON) as part of series of contiguous transmission electron microscopy (TEM) images. A: Montage of TEM images of one L1 larval brain hemisphere (co cortex; np neuropil; OOA outer optic anlage). B: High magnification of section of larval visual system, boxed in (A). Shown are epithelial outer optic anlage (OOAep; rendered brown), distal LON (LONd; purple), defined by presence of large diameter photoreceptor terminals (arrows), proximal LON (LONp; red), axon tracts of primary lineages flanking optic anlage (primary axon tract; PAT; yellow). C: 3D digital model of L1 larval brain hemisphere, lateral view. Neuropil compartments (dark gray; CPL centro-posterior lateral compartment) and larval visual system (rendered in same colors as in ) are shown as landmarks. Parallel lines demarcate the brain “slice” included in the TEM stack that was used for the analysis of the larval optic neuropil in this paper. D: 3D digital model of LON and OOA, reconstructed from TEM stack. The model is shown at an orientation that is used most frequently in the following figures of this paper: the view is from dorso-posterior (indicated by red arrow in (C), exposing the LONd and LONp, enclosed by the hemicylindrical outer optic anlage. E: TEM image of proximal LON (boundaries indicated by arrowheads) at higher magnification. Scale bars: 5μm (A), 2 μm (B, E).

Glial element of the larval optic neuropil (LON). A: TEM section showing outer optic anlage epithelium (OOAep) and LON (LONd: distal LON; LONp: proximal LON). OOA epithelium is shaded brown; glial processes in green; optic lobe pioneers (OLP) in blue; primary axon tracts (PAT) in yellow. Arrow points at interface between apical membranes of OOA epithelial cells and neurons (ne), which lacks any glial layer. Surface glia (sg) surrounds brain surface, including OOA and optic lobe pioneers (OLP). Note cortex glial cell (cg) directly adjacent to OOA and LON, cortex neuron cell bodies (ne) (A, C). B: Single confocal section of first instar larval brain hemisphere, labeled with Nrv2-Gal4 driven GFP (green; glial cell bodies and processes) and anti-Repo (glial nuclei, magenta).. sg have repo-positive nuclei, but do not express the Nrv2 driver. Neuropil glia (ng) surrounds central brain neuropil (np), cortex (co). C: TEM section showing lateral part of the OOAep and brain cortex. Electron-dense cortex cg emits processes in between neurons of cortex (arrowheads). Note also Bolwig’s nerve (bn), surrounded by peripheral glial sheath (pg), which contacts both surface glia (sg) and cortex glia. D: 3D digital model of LON (gray) and glial processes present in LON (green). Note relative scarcity of glial lamellae in distal LON, compared to proximal LON. Arrowhead in D (and A) points at straight glial process that extends from the entry point of the bn to the center of the LON. E–H: TEM images showing details of glial structure at high magnification. E: septate junctions (arrowheads) between pg (surrounding larval photoreceptor axons, lpa) and sg. F: Auto-septate junction (arrowhead) formed by mesaxonal peripheral glia around photoreceptor axons of bn. G: Glial lamella (ng) between OOAep and neurites of LONp. H: Interface between LONd, containing boutons of larval photoreceptor terminals (lpt), and OOA epithelium. Note absence of glial sheath (arrow).

Topographically ordered projection of larval photoreceptor terminals in larval optic neuropil (LON). A–C: TEM sections of larval photoreceptor terminals and LON at different anterior-posterior levels, indicated by lines in panel H. The twelve individual photoreceptor axons are rendered in different colors and arbitrarily annotated by numbers, shown in (A′). A, A′: Bolwig’s nerve (bn) as it contacts the outer optic anlage (OOAep). Presumed Rh5-positive axons (shades of blue and purple) occupy a centro-medial position within nerve; Rh6-positive axons (shades of green to orange) are peripherally. B: Section of central part of LON. C: Section of posterior part of LON. D–F: Single sagittal confocal sections of early larval visual system (OOAep labeled by anti-DE-cadherin antibody). Planes of sections are indicated in panel G. Rh5-positive and Rh6-positive photoreceptor axons are differentially labeled by rh5-GFP and rh6-GFP. Note that Rh5-positive terminal axons (blue) occupy a posterior location in LON (arrowhead in E), and terminate mainly in proximal LON (F), which does not receive terminal axons of Rh6-positive axons (green). G–N: 3D digital models of LON (gray) and individual photoreceptor axon terminals, rendered in same colors as used in TEM images in A–C). Models of left column (G, I, K, M) present dorsal view; right column offers lateral views. G, H: three presumed Rh5-positive photoreceptor terminal axons, recognizable by their posterior position in LON (arrowhead in H) and termination in proximal (i.e., medial) LON (arrow in G). I, J: Rh6-positive receptor axons 1, 4, 11, traveling medially in Bolwig’s nerve (A′), have terminal boutons in anterior LON. K, L: Rh6-positive axons 2, 5, 12, located at intermediate levels in nerve, terminate preferentially in central domain of LON. M, N: Lateral Rh6-positive axons 3, 6, 10 terminate in posterior LON. Scale bars: 0.5μm (A′), 1 μm (A), 2 μm (B, C, D), 3 μm (D, E, F).

Elements of the larval optic neuropil (LON). All panels show schematic or confocal images representing frontal section of a first instar larval brain hemisphere; lateral to the left, dorsal up. A: Labeling with anti-Neuroglian (Ngl), showing neuronal cell bodies in cortex (co) and nerve processes, forming central neuropil (np). Z-projection of a confocal stack (3 μm). Added to the section is a 3D digital model (anterior view) of outer optic anlage (OOA, beige), inner optic anlage (IOA, gray), LON, purple, and Bolwig’s nerve (bn, magenta), primary axon tract (PAT). B: 3D digital model of brain hemisphere (posterior view), showing brain surface (light gray), neuropil compartments (dark gray: BPL, baso-posterior lateral compartment; CPL centro-posterior lateral compartment; BPM baso-posterior medial compartment; CX calyx), and visual system (coloring as in A). C: Labeling with anti-Bruchpilot (Brp), a marker for synapses, outlines neuropil compartments, including LON. Z-projection of a confocal stack (3 μm). CPLd dorsal subdivision of CPL; CPI centro-poterior intermediate compartment; CPM; central-posterior medial compartment). D: Schematic representation of neuropil compartments of central brain and of larval visual system, including main neuronal elements contributing to LON: larval photoreceptors (lp, red), bn (red); optic lobe pioneers (OLP, blue), PDF neurons (PDF; green), serotonergic neurons (5-HT; yellow), OOA (brown). E: Z-projection of a confocal stack (17 μm) showing larval photoreceptor projections. Rh5 and Rh6 are differentially labeled by rh5-GFP (blue) and rh6-LacZ (red). Anti-Drosophila E-cadherin (DEcad, green) labels the OOA surrounding LON. Note that terminals (lpt) of Rh5-positive axons are located more medially in LON than terminals of Rh6 terminals. F: Labeling with antibody 22C10 (red) and anti-Ngl (green), Z-projection of a confocal stack (22 μm). 22C10 marks (among many other, central brain neurons) the OLP, whose cell bodies are located laterally in the OOA. OLP fibers project through LON towards central brain. G: GFP driven by pdf-Gal4 labels the PDF neurons (red; cell bodies rendered in purple to set them apart from dendrites), anti-Ngl (green). Z-projection of a confocal stack (20 μm). PDF cell bodies are located in brain cortex adjacent to OOA; profuse dendritic branches are formed in the LON, and axons project out of the LON towards the CPLd compartment of the central brain. H: Labeling with anti-Serotonin (5-HT; red), showing pair of serotonergic neuronal cell bodies with neurites projecting towards LON. Z-projection of a confocal stack (20 μm). GFP driven by Nervana2-Gal4 (Nrv2; green) labels glial cells. I: Z-projection of a confocal stack (29 μm) showing a third instar larval brain labeled with anti-PDF (Green), Rh6-projections with anti-Rh6 (blue) and GFP driven by Tdc2-Gal4 labels (Oct; red). Subset of octopaminergic neurons projects into LON. Scale bars: 10μm (A, C, E, F, G, I), 15 μm (H).

Topology of larval optic neuropil (LON) throughout development. A, B: 3D digital model of mid third instar larval brain hemisphere, postero-lateral view. Optic anlagen (outer optic anlage: OOA; inner optic anlage: IOA) have grown and form crescent-shaped epithelia at the ventro-lateral brain surface (rendered in gray). Space in between OOA and IOA is filled by neurons of medulla (cell bodies not shown) whose fibers have started to form the neuropil of the adult medulla (proximal medulla, Mp, purple; distal medulla, Md, light blue); bolwig nerve (bn, brown). The LON is accreted to the medial edge of the medulla neuropil. C, D: Frontal single confocal section of late third instar larval (C) and adult (D) brain hemisphere (lateral to the left). Different optic neuropils are rendered in different colors (Md, blue; Mp, purple; Lo, lobula faint pink; Lp, lobula plate, red). In C, Neurons are labeled by anti-DN-cadherin (DNcad; faint gray). The IOA and OOA are DNcad negative and stand out as dark spaces. Labeling of synapses with anti-Brp is added to global neuronal staining with anti-DNcad,; this results in bright signal demarcating larval neuropil compartments containing mature synapses, including the larval optic neuropil (red arrowhead). Neuropil compartments stained with anti-DN-cad (BPL, baso-posterior lateral compartment; CPL centro-posterior lateral compartment; CPLd centro-posterior lateral dorsal compartment; VLPp, ventrolateral protocerebrum, posterior subdivision). E: frontal single confocal section of adult brain at a more anterior level than section shown in (D). Shown in this panel is anterior surface and medial rim of the distal medulla, to which the accessory medulla (Ma), the descendant of the LON, is attached (red arrowhead; enlarged view shown in inset). Neuropil compartment stained with anti-DN-cad (VLPa ventrolateral protocerebrum, anterior subdivision). F: Z-projection of a confocal stack (39 μm) of late third instar larval brain hemisphere. Neurons are labeled by 22C10 antibody (green). Larval photoreceptor terminals are differentially labeled by rh5-GFP (blue), rh6-LacZ (red) and demarcate the larval optic neuropil. Note that 22C10-positive cell bodies of optic lobe pioneers (OLPs) have been “pulled away” from their original location (which was right next to the LON; see ) by the expanding optic lobe. G, H: Frontal single confocal section of late larval (G) and adult (H) brain hemisphere labeled with anti-DN-cad. As explained for panel (C), anti-Brp was added in G to enhance staining of larval compartments containing mature synapses, including the LON. Labeling of PDF neurons (pdf-Gal4 > UAS-GFP; green) illustrates the topological relationship between larval and adult visual system. PDF-neuronal dendrites branch in larval LON and adult accessory medulla (red arrowhead). Axons project to larval CPLd compartment (G), which grows during metamorphosis to become the adult superior lateral protocerebrum, posterior subdivision (SLPp in H). PDF neurons add additional dendrites branching profusely throughout adult medulla (orange arrowhead); axons added during metamorphosis project contralaterally through the great commissure (CCX central complex; p peduncle; LH, lateral horn).