Development of the anterior visual input pathway to the Drosophila central complex

Abstract

The anterior visual pathway (AVP) conducts visual information from the medulla of the optic lobe via the anterior optic tubercle (AOTU) and bulb (BU) to the ellipsoid body (EB) of the central complex. The anatomically defined neuron classes connecting the AOTU, BU, and EB represent discrete lineages, genetically and developmentally specified sets of cells derived from common progenitors (Omoto et al., Current Biology, 27, 1098-1110, 2017). In this article, we have analyzed the formation of the AVP from early larval to adult stages. The immature fiber tracts of the AVP, formed by secondary neurons of lineages DALcl1/2 and DALv2, assemble into structurally distinct primordia of the AOTU, BU, and EB within the late larval brain. During the early pupal period (P6-P48) these primordia grow in size and differentiate into the definitive subcompartments of the AOTU, BU, and EB. The primordium of the EB has a complex composition. DALv2 neurons form the anterior EB primordium, which starts out as a bilateral structure, then crosses the midline between P6 and P12, and subsequently bends to adopt the ring shape of the mature EB. Columnar neurons of the central complex, generated by the type II lineages DM1-4, form the posterior EB primordium. Starting out as an integral part of the fan-shaped body primordium, the posterior EB primordium moves forward and merges with the anterior EB primordium. We document the extension of neuropil glia around the nascent EB and BU, and analyze the relationship of primary and secondary neurons of the AVP lineages.

Keywords: RRID:BDSC_33065; RRID:BDSC_38821; RRID:BDSC_38865; RRID:BDSC_40372; RRID:BDSC_44409; RRID:BDSC_48625; RRID:BDSC_48860; RRID:BDSC_50101; RRID:BDSC_50349; RRID:BDSC_5137; RRID:BDSC_66685; RRID:BDSC_6799; RRID:BDSC_7127; RRID:BDSC_8751; RRID:DGGR_105240; brain; central complex; development; drosophila; lineage; visual pathway.

Figure 1

Anterior visual pathway of the Drosophila brain. Schematic frontal view of brain hemisphere depicting the neuronal elements forming the anterior visual pathway, as described in Omoto et al. (2017). Medullo-tubercular (MeTu) neurons project via the anterior optic tract to the lateral and intermediate domain of the anterior optic tubercle; AOTU). Two hemilineages, DALcl1d and DALcl2d, form the tuberculo-bulbar (TuBu) neurons that connect the tubercle to the bulb (BU). Lineage DALv2, formed by one surviving hemilineage (Kumar et al., 2009), forms multiple classes of ring (R) neurons with dendrites in the bulb, and axons in the ellipsoid body (EB). These classes (symbolized by dark red and light red shading) comprise sublineages born at different developmental intervals (J.L. and V.H., unpublished). Box at bottom of diagram presents linear arrangement of the elements of the AVP.

Figure 2

Larval primordia of the central complex and anterior visual pathway. (A) Schematic view of larval brain hemisphere (top: anterior view, dorsal up; bottom: dorsal view, posterior up). Secondary lineages contributing to the primordia of anterior visual pathway and central complex are rendered in different colors; mushroom body (gray) is shown as reference. (B, B′, C, C′) Z-projections of frontal confocal sections of late larval brain hemisphere labeled with anti-Bruchpilot (Nc82; green), anti-DN-cadherin (DNcad; white in B, C; blue in B′, C′) and anti-Neurotactin (BP106; white in B′, C′). Note domains which are negative for Nc82, indicating absence of synapses; these domains are positive for DNcad and BP106, showing presence of (undifferentiated) secondary fibers and filopodia which make up primordia of adult-specific compartments. Labeled are primordia of anterior optic tubercle (prAOTU), anterior ellipsoid body (prEBa), bulb (prBU), fan-shaped body (prFB), noduli (prNO. (D–K): Z-projections of confocal sections of late larval brain hemispheres in which individual cell classes forming part of anterior visual pathway and central complex are labeled by specific Gal4 drivers (green). In (D–L), DNcad antibody labels neuropil (white); (D′–G′) show only GFP expression. (D–G′) and (I, J) show frontal sections, (H) and (K, L) are digitally tilted dorsal views. (D, D′) Tuberculo-bulbar (TuBu) neurons of DALcl1/2 lineages, labeled by R52B11-Gal4. Shown to the left of hatched line is a more anterior plane, containing tufts of proximal filopodia (marked by arrow in D′) that constitute the primordium of the anterior optic tubercle. To the right of the hatched line is a slightly more posterior plane, featuring endings and distal filopodia of TuBu neurons, which form the primordia of the superior and inferior bulb (prBUs, prBUi), respectively. (E, E′) Axonal endings of medullo-tubercular (MeTu) neurons in the primordium of the anterior optic tubercle, labeled by R53B08-Gal4. (F–H) Ring (R) neurons of DALv2 lineage, labeled by Period (Per)-Gal4 (F, F′, H) or as MARCM clone (G, G′). DALv2 neurons form a straight, medially-directed bundle, the anterior lateral ellipsoid body tract (LEa), which extends posterior to the medial lobe of mushroom body (ML in panel A) to end close to the midline. Filopodial tufts of these axons form the primordia of the anterior ellipsoid body (prEBa; arrowheads in F′, G′) and, further laterally, the bulb (prBU; arrows in F′, G′). (I–L) Subset of columnar neurons of the central complex, labeled by R19G02-Gal4. These neurons form part of four type II lineages, DM1/DPMm1, DM2/DPMpm1, DM3/DPMpm2, DM4/CM4, as shown in panel (A). Axons project forward and proximal filopodia form the primordium of the protocerebral bridge (arrowheads in K; prPB in A). Axons then branch and turn medially, forming the posterior plexus of the fan-shaped body (FBppl in A, I, K). Filopodial tufts branch in the primordium of the fan-shaped body (A, I, K) and the posterior ellipsoid body (prFB/EBp in I, K). Axons continue forward, branching towards medially and laterally, forming the anterior plexus of the fan-shaped body (FBapl; A, J). Distal tips of this projection defines the primordium of the gall of the LAL (prLALg; A, J, K). For other abbreviations, see List of Abbreviations. Bar: 25μm

Figure 3

Morphogenesis of the ellipsoid body and bulb. (A–D′) and (G–N′) Z-projections of frontal confocal sections of pupal brain hemispheres at different stages, indicated in upper left corner of panels (P3 3hrs after puparium formation (APF); P16 16hrs APF; P24 24hrs APF; P48 48hrs APF). In panels (A–D) and (G–N), specific neuron populations are labeled by Gal4-drivers indicated at lower left corner of panels; antibody against DN-cadherin marks neuropil (white). (A–D′) R40G10-Gal4 labels preferentially inner ring (Ric) neurons throughout development. (E, F) Schematic anterior view of pupal brain hemisphere at 6hrs (E) and 24hrs (F) after puparium formation (APF). Secondary lineages contributing to the primordia of the anterior visual pathway are rendered in different colors; mushroom body (gray) is shown as reference. (G–H″) Per-Gal4 and Poxn-Gal4 are expressed in all R-neurons, but allow one to distinguish the inner from outer ring during early pupal stages. (I–J′) EB1-Gal labels R2 class of outer ring neurons. (K–N): R83H08-Gal4 (magenta) is expressed in tuberculo-bulbar neurons and labels the developing bulb primordium (prBUs, prBUi, prBUa) of left brain hemisphere. Panels (K′–N′) show opposite hemisphere, labeled only with anti-DN-cadherin (white). For other abbreviations, see List of Abbreviations. Bar: 25μm

Figure 4

Morphogenesis of the anterior optic tubercle. Panels show Z-projections of frontal confocal sections (left column; A, C, E, G) and corresponding, digitally-tilted horizontal sections (right column; B, D, F, H) of brain hemisphere at different stages of development, from 6hrs APF (P6) to Adult (stages indicated in upper right corner of panels). In panels of right column, the area in upper left boxed by gray line represents a dorsal level, including the prAOTU; remainder of the panels show horizontal sections at a more ventral level, including the prBU. Proximal arborizations of TuBu neurons, labeled by R83H08-Gal4 (green), mark the developing primordium of the anterior optic tubercle (prAOTU); distal arborizations outline the primordium of the bulb (prBU). Neuropil is labeled by anti-DN-cadherin (white). In early pupa (P6; A–B′) the prAOTU forms an undifferentiated, ovoid, DN-cadherin-rich domain at the anterior-lateral surface of the superior lateral protocerebrum (SLP). GFP-positive fibers of TuBu neurons are largely restricted to the lateral half of the prAOTU (A), which will give rise to the intermediate and lateral subdomains of this compartment (in/l in A′). The medial part of the prAOTU (m in A′) does not receive GFP-positive fibers. At P24 (C–D′), a sharp boundary separates the medial from the intermediate/lateral subdomain (arrowhead in C′, D′). At P48 (E–F′), the GFP-positive domain has differentiated into a lateral (l) and intermediate (i) subdomain. During late pupal development, these become further subdivided into smaller subdomains, including the intermediate medial (im), intermediate lateral (il), lateral anterior (la), lateral central (lc), and lateral posterior (lp; G, H). For other abbreviations: see List of Abbreviations. Bar: 25μm.

Figure 5

Formation of the ellipsoid body from an anterior and posterior primordium. Panels of the left column (A, B, E, F, J, K) show Z-projections of frontal confocal sections of pupal brains in which a subset of columnar neurons of the central complex are labeled by R19G02-Gal4 (green); neuropil is labeled by anti-DN-cadherin (white). Middle column (C, C′, G, H, L, M) presents digitally-tilted horizontal sections of brains labeled with R19G02-Gal4 (C, G, L) or Poxn-Gal4 (H, M). Upper two rows (A–C′) show 12h pupa (P12), middle two rows (E–H) 24h pupa (P24), and bottom two rows 48h pupa (P48). Frontal sections of left column alternatingly show a more posterior plane, containing the primordium of the fan-shaped body (prFB; A, E, J) and an anterior plane with primordium of the ellipsoid body (prEB) and lateral accessory lobe (prLALg; B, F, K). Panels (D, I) are schematic representations of pupal brain hemisphere viewed from dorsally, illustrating architecture of central complex primordia at an early pupal stage (P12, shown in D) and early-mid pupal stage (P24-48, shown in I). In these schematics, neurons of the DALv2 lineage, forming R-neurons of the ellipsoid body, are rendered in red; DALcl1/2d neurons, connecting anterior optic tubercle with bulb, are in blue; columnar neurons of the posterior type II lineages (PB-EB-gall neurons) are in green. (D′, I′) are enlarged views of boxed areas in (D, I). Light blue arrows in (C, C′, D, D′, I, I′) point at prFB; dark blue arrows in (C, C′, D, D′) indicate prEBp, which at this early stage forms part of the prFB; red arrows in (C, C′, D, D′) indicate prEBa; purple arrow in (I, I′, L, M) indicates prEB after the anterior and posterior domains (prEBa, prEBp) have merged. Panels (N–S) are Z-projections of frontal confocal sections of pupal brains (N, O: P12; P, Q: P24; R, S: P48) labeled with anti-Neuroglian (secondary axon tracts; magenta) and anti-DN-cadherin (neuropil; white). Left column (N, P, R) represent posterior plane, containing left half of fan-shaped body primordium (prFB); Right panels (O, Q, S) show anterior planes with right halves of ellipsoid body primordium (prEB). Arrows in (N, P, R) point at longitudinal bundles of PB-EB-LALg axons penetrating the prFB. For other abbreviations see List of Abbreviations. Bar: 25μm.

Figure 6

Development of the glial layer of the central complex. Panels of the first and second column show Z-projections of frontal confocal sections of late larval (A, B) and pupal brains at stages indicated in upper left corner. The third and fourth columns present sagittal sections at the corresponding stages. Panels of the first column (A, E, I, M, Q, U) represent a posterior plane, showing the primordium of the fan-shaped body (prFB); second column panels (B, F, J, N, R, V) are taken at a more anterior plane representing the primordium of the ellipsoid body. Panels of the third column (C, G, K, O, S, W) show sagittal plane right at midline [indicated by arrow in panel (A)], panels of the fourth column (D, H, L, P, T, X) represent a parasagittal plane at approximately 5–10μm lateral of midline [indicated by arrowhead in (A)]. Neuropil glia is labeled by NP6520-Gal4 (A–D), 442-Gal4 (E–L), or Nrv2-Gal4 (M–X; magenta). In all panels except (N, R, V), secondary axon tracts (green) are labeled by anti-Neurotactin (A–D), anti-Neuroglian (E–L) or anti-Fasciclin II (M–X). Processes of neuropil glia forming stable sheaths around individual commissural tracts, or combinations of neighboring tracts, can be recognized in sagittal sections (panels of third column) from larval stages to the late pupa and adult. In the larva and early pupa (C, D, G, H), a common sheath surrounds a system of tracts that includes the primordia of the fan-shaped body (prFB, including posterior primordium of ellipsoid body), anterior ellipsoid body (prEBa), several commissures (supraesophageal commissure, SEC; part of superior arch commissure, SAC) and the tip of the medial lobe of the mushroom body (ML). After P24, a layer of glial processes starts to separate the medial lobe from the ellipsoid body primordium (orange arrow in T, X; orange arrowhead in P, representing 24h pupa, points at position where glial septum will appear at later stages). Concomitantly, glial processes grow in between the primordia of the ellipsoid body and fan-shaped body (white arrows S, T, W, X). Panels (N, R, V) represent the frontal plane that contains the emerging glial sheath (arrowhead in N: no glia present yet; arrows in R, V: glial septum has formed) between ellipsoid body and fan-shaped body primordium; note increasing intensity of the Nrv2 signal, attesting to the growth in thickness of the glial layer. Other abbreviations: see List of Abbreviations. Bar: 25μm.

Figure 7

Elements of the AVP in the early larva. (A–B″) Z-projections of frontal confocal section of larval brain hemisphere labeled with Insc-Gal4>UAS-mcd8GFP (magenta; secondary neurons) and anti-Neuroglian (green; primary neurons) at the L2 stage (48h post hatching; A, A′) and early L3 stage (64h post hatching; B–B″). Note close association of primary and secondary fiber bundles of DALcl1 and DALcl2 (DALcl1/2dp and DALcl1/2ds; DALcl1/2vp and DALcl1/2vs) in (A′) and (B′), respectively. (B″) shows enlarged view of early secondary tracts of DALcl1/2 and DALv2, labeled by Insc-Gal4>UAS-mcd8GFP (white; all other labeled lineages were digitally erased; mushroom body (MB) shown as reference). (C–C‴) Frontal confocal sections of late third instar larval brain hemisphere labeled with anti-Neurotactin (secondary neurons) and Per-Gal4>UAS-mcd8GFP (labels secondary and primary neurons of the DALv2 lineage). In (C), only the Neurotactin labeling is shown; secondary tracts of lineages DALcl1/2 and DALv2 were digitally rendered green and magenta, respectively. (C′–C‴) show Per-Gal4 labeling of both primary neurons (DALv2p) and secondary neurons (DALv2s) of the DALv2 lineage. In (C′), Neurotactin labeling of secondary DALcl1/2 tract and DALv2 tract (digitally rendered green and magenta, respectively) is left; Neurotactin signal of all other secondary neurons was digitally erased. (C″) and (C‴) show Per-Gal4> mcd8GFP in DALv2 in brain of two different L3 specimens. Small arrowhead in (C′–C‴) indicates secondary DALv2 tract (DALv2s); large arrowhead in (C′) and (B″) points at convergence of secondary tracts of DALv2 and DALcl2v. Arrow in (C′–C‴) indicates primary DALv2 tract (DALv2p). (D–E) Z-projections of frontal confocal section of first instar larval brain hemisphere, showing labeling of primary neurons of DALcl1/2d [R48B06-Gal4>UAS-mcd8GFP in (D)] and DALv2 [Poxn-Gal4>UAS-mcd8GFP in (E)]. Antibody against DN-cadherin labels neuropil (magenta). (F) Z-projection of frontal confocal section of late third instar larval brain hemisphere. Constructs labeling postsynaptic sites (DenMark; green) and presynaptic sites (Syntaxin; Syt; magenta) were driven by DALv2 driver Poxn-Gal4. Bars: 20μm (A, B, D–E); 50μm (C–C‴)

Figure 8

Schematic representation of projection patterns of lineages building the central complex at the larval stage (A) and adult stage (B).