Developmentally Arrested Precursors of Pontine Neurons Establish an Embryonic Blueprint of the Drosophila Central Complex

Abstract

Serial electron microscopic analysis shows that the Drosophila brain at hatching possesses a large fraction of developmentally arrested neurons with a small soma, heterochromatin-rich nucleus, and unbranched axon lacking synapses. We digitally reconstructed all 802 "small undifferentiated" (SU) neurons and assigned them to the known brain lineages. By establishing the coordinates and reconstructing trajectories of the SU neuron tracts, we provide a framework of landmarks for the ongoing analyses of the L1 brain circuitry. To address the later fate of SU neurons, we focused on the 54 SU neurons belonging to the DM1-DM4 lineages, which generate all columnar neurons of the central complex. Regarding their topologically ordered projection pattern, these neurons form an embryonic nucleus of the fan-shaped body ("FB pioneers"). Fan-shaped body pioneers survive into the adult stage, where they develop into a specific class of bi-columnar elements, the pontine neurons. Later born, unicolumnar DM1-DM4 neurons fasciculate with the fan-shaped body pioneers. Selective ablation of the fan-shaped body pioneers altered the architecture of the larval fan-shaped body primordium but did not result in gross abnormalities of the trajectories of unicolumnar neurons, indicating that axonal pathfinding of the two systems may be controlled independently. Our comprehensive spatial and developmental analysis of the SU neurons adds to our understanding of the establishment of neuronal circuitry.

Keywords: Drosophila; brain; central complex; circuitry; development; lineage; serial electron microscopy.

Figure 1

Lineages DM1-DM4 form two types of columnar neurons. The compartments of the central and lateral accessory complex are schematically shown in a dorsal view (PB: protocerebral bridge; FB: fan-shaped body; NO: noduli; EB: ellipsoid body; LAL: lateral accessory lobe). Four bilateral pairs of lineages, DM1-DM4, located in the posterior brain cortex (top), generate the columnar neurons whose axons (grey bars and lines oriented along the vertical axis; filled circles symbolize terminal arborizations) interconnect the compartments of the central complex in a strict topographical order. The location of DM1–4 cell bodies within the cortex is reflected in the position at which their corresponding tracts enter and terminate within the central complex (20–23), as indicated by the color code. DM1 axons (blue) enter through the medial segments of the PB (1–3, after [34]), followed by DM2 (green, PB segments 4–5), DM3 (red, segments 6–7), and DM4 (yellow, segments 8–9). Each DM lineages generates multiple sublineages. These fall into two main types, unicolumnar neurons (light colors) and pontine neurons (saturated colors). Unicolumnar neurons (representing the large majority of DM neurons) interconnect compartments of the central complex along the anterior-posterior axis. They subdivide the PB, FB, and EB into four quadrants, as indicated by the coloring. For example, DM4 interconnects the lateral quarter of the PB (segments 8–9) with the lateral half of the ipsilateral FB, and the ventral half of the ipsilateral EB. This is followed by DM3, that projects from PB segments 6–7 to the medial half of the ipsilateral FB and dorsal half of the ipsilateral EB. DM2 and DM1 neurons interconnect the medial PB with the contralateral PB and FB, respectively, as indicated. The projection pattern of one class of unicolumnar neurons, E-PG (nomenclature according to [46]; “1” at top of figure), is shown representatively for lineage DM3; this neuron type is illustrated as a line drawing at the upper right of the figure (after [11], with permission). The second main type of neurons generated from DM1–4 are the pontine neurons (“2”, solid line). Axons of pontine neurons enter the central complex alongside the unicolumnar neurons, but pass through the PB without branching (see line drawing at lower right of figure, after [11], with permission). Projections of pontine neurons are confined to the FB, where they interconnect two columns along the transverse axis. The topology of this connectivity, as elaborated by Hanesch et al. [11], is indicated by horizontal bars rendered in saturated colors.

Figure 2

Structural features of small undifferentiated (SU) neurons in the early larval brain. (A) Electron micrograph showing differentiated neuron (top), SU neuron (middle) and quiescent neuroblast (bottom). (B) Histogram showing size distribution of differentiated neurons (grey; number counted =50) and SU neurons (orange: all SU neurons of right brain hemisphere; n =405; yellow: SU neurons forming fan-shaped body primordium (prFB); n =54). Units on horizontal axis are in μm2 and represent area of cut surface of cell body. (C) Electron micrograph of cortex-neuropil boundary, showing bundle of axons (surrounded by hatched line) formed by the axons of the two DALcl lineages as they enter the neuropil. Within this bundle, axons of small undifferentiated neurons (“SU axon”) from a coherent contingent of thin, electron dense fibers; they are surrounded by thicker and typically more electron-lucent axons of differentiated neurons (“regular axon”). (D, D′) 3D digital model of part of one primary lineage (BAlc), illustrating structural aspects of SU neurons. BAlc includes local interneurons and projection neurons connecting the antennal lobe with higher brain centers [47]. Shown are one differentiated projection neuron (mPN iACT A1, white) and one differentiated local interneuron (Broad D1: grey) whose dendritic arborizations outline the antennal lobe; the axon of the projection neuron follows the antennal lobe tract. Shown in yellow are the SU neurons which were identified for BAlc. They each form an unbranched axon that follows the axons of the differentiated neurons as they enter the antennal lobe neuropil (black arrow in D′) and extends into the antennal lobe tract (grey arrow in D′) where they end approximately midway (white arrow in D). The model is presented in lateral view; anterior to the left, dorsal up. The mushroom body (MB) is outlined in grey for spatial reference. (E, F) Electron micrographs show club-shaped endings of SU axons, featuring membrane lamellae (E) and short filopodia (F). Bars: 1μm (A, E), 0.25μm (B, D, E), 5μm (F)

Figure 3

Pattern of SU neurons and primary axon tracts (PATs) in the early larval brain. (A-C) 3D digital model of first instar larval brain in lateral view (A; anterior to the left, dorsal up) and anterior view (B, C; dorsal up). Cell bodies and axons of all SU neurons of one hemisphere are shown as colored spheres and lines. Differential coloring indicates association of SU neurons with spatially contiguous groups of lineages; labels of lineages are shown in corresponding colors (A, B; for nomenclature of lineages and fascicles see [30]). In (A, B), outline of brain and ventral nerve cord is rendered in grey; in (C), outlines of mushroom body lobes, located in center of neuropil, are shaded, with tips of left and right medial lobe pointed out by arrowheads. Note that lineage-associated bundles of SU axons follow discrete fascicles identified for the brain throughout development. Among the examples made explicit are the BAmv axons that form the longitudinal ventromedial fascicle (loVM); axons of BAlp constitute the longitudinal ventro-lateral fascicle (loVL); DALd axons form the central protocerebral descending fascicle (deCP); CM lineages the medial equatorial fascicle (MEF); DPMpm the longitudinal superior-medial fascicle (loSM) and primordium of the fan-shaped body (prFB). (D-I) Primary lineage tracts imaged light microscopically can be identified with electron microscopically reconstructed SU axon tracts. (D) and (E) show representative frontal confocal section (D: anterior section at level of MB lobes; E: posterior section at level of MB calyx); primary neurons and their axon tracts are labeled by anti-Neuroglian (white). Primary axons converge at the cortex-neuropil boundary at invariant positions to form tracts with characteristic trajectories. A subset of tracts, reconstructed from a confocal stack of an anti-Neuroglian labeled brain hemisphere, is shown in panels (F) and (G). In these models, the mushroom body lobes (ML medial lobe; VL vertical lobe) and antennal lobe (AL) are shown for reference. Representative anterior lineages (BAla1/2, BAla3/4, BAlc, BAmd1, BAmv1/2, DALcl1/2, DALcm1/2) are shown in (F)(anterior view of one hemisphere, medial to the right); representative posterior lineages (CP1, CP2/3, BLVp2, DM1–4) appear in (G)(posterior view, medial to the right). Lineage-associated tracts are shown as colored pipes; spheres indicate location of cell bodies belonging to the tracts illustrated. Panels (H, I) show digital models extracted from the serial EM stack. As in (F, G), the mushroom body and antennal lobe is shown as reference. SU neurons of the same lineages presented in (F, G) are shown as colored lines; individual cell bodies appear as spheres. Tracts formed by SU neurons can be identified with light microscopically-defined lineage-associated tracts based on specific location of entry and trajectory, as pointed out by numbered arrows that indicate the following features: (1) Entry of DALcm lineage pair dorso-laterally of anterior peduncle; upper tract passes over peduncle and turns ventrally; lower tract projects anteriorly and then medially, passing in front of MB vertical lobe; (2) Entry of DALcl lineage pair laterally of anterior peduncle; upper tract passes over peduncle and turns ventrally; lower tract turns ventrally and medially, passing underneath peduncle; (3) Entry of BAlc at lateral surface of antennal lobe (AL); (4) Entry of lineage pairs BAla1/2 and BAla3/4 at posterior-ventral surface of antennal lobe; (5) Tracts of BAlc and BAla1 converge at posterior-medial surface of antennal lobe and project dorsally, forming antennal lobe tract; (6) Entry of BAmv1/2 lineage pair medially of antennal lobe; tract turns posteriorly, forming longitudinal ventromedial fascicle (loVM); (7) Entry of BAmd1 anteriorly of MB medial lobe; tracts diverge, and upper tract passes over medial, joining upper DALcm tract on its medial turn; lower tract turns ventrally and then medially, forming antennal lobe commissure; (8) Entry of CP2/3 lineage pair laterally of MB calyx-peduncle junction; upper tract projects dorso-anteriorly, passing peduncle dorsally and forming oblique posterior fascicle; lower tract turns ventrally and then anteriorly, forming dorsal component of postero-lateral fascicle; (9) Entry of CP1 medially adjacent to CP2/3; upper tract follows CP2/3 axons in oblique posterior fascicle; lower tract projects ventrally and anteriorly, forming lateral equatorial fascicle; (10) Entry of BLVp2 ventrally of CP2/3; tract continues anteriorly, forming ventral component of postero-lateral fascicle; (11) convergence of DM1–4 forming the primordium of the fan-shaped body. See also Figures S1–S5, Table S1 for more detailed information of entry points. See also Data S1. Bars: 20μm

Figure 4

SU neurons of lineages DM1–4 form the primordium of the fan-shaped body (prFB) and give rise to pontine neurons. (A) Electron micrograph of dorso-medial brain cortex showing clusters of contiguous SU neurons (shaded in blue) associated with lineages DM1–4. (B, C) Digital 3D models of L1 brains (B: dorsal view; C: lateral view) showing SU neurons associated with DM1–4 rendered in different colors (DM1 blue; DM2 green; DM3 magenta; DM4 yellow). Outline of mushroom body (representing center of brain neuropil) is rendered in light gray. Only subsets of SU neurons contributing to the prFB are shown. Note that in addition to DM1–4, lineages DPMm2 and CP2 contribute three fibers (white) to the prFB. (D) Electron micrograph of brain midline, showing supraesophageal commissure. The prFB appears as a bundle of electron-dense axons embedded in fascicles of lighter axons belonging to differentiated neurons (shaded blue). Glial lamella (shaded green) flanks the prFB. Note conspicuous, medially located nucleus of glial cell (white arrow) right adjacent to the prFB. (E, F) Z-projections of confocal sections of first instar brain (E) and late third instar brain (F). Glial nuclei are labeled by anti-Repo (blue); glia surrounding supraesophageal commissure (=interhemispheric ring glia) is labeled by Drl-lacZ (white); neurons of prFB are marked by driver line R45F08-Gal4>UAS-mcd8GFP (green). Interhemispheric ring glia forms several channels containing individual commissural fascicles. One channel contains the R45F08-Gal4-positive axon bundle constituting the prFB [orange arrowheads in (E, F)]. This channel widens as prFB gains in volume towards late larval stages (F; arrowheads). Note pair of large glial nuclei (arrows) located near the midline, posteriorly adjacent to the prFB. These cells correspond in shape, size and position to the prFB-associated glial cells that appear in electron micrographs (see panels D, G). (G-I) Topographical order of DM1–4 SU axons in the fan-shaped body primordium. Panels (G, H) show electron micrographs of prFB sectioned at the two planes shown by hatched lines in panel (B). Sectioned profiles of FB pioneers were assigned to their cell bodies of origin and are shaded in colors following the same scheme used in the 3D digital models in (B, C). Axons belonging to a lineage form a coherent bundle within the prFB. Note that bundles of corresponding lineages of the left and right hemisphere project together (e.g., light blue profiles of left DM1 are closest to saturated blue profiles of right DM1). Panel (I) shows digital 3D model of FB pioneers rendered of right hemisphere in different colors; antero-medial view. Note relationship between lineage identity and axonal projection (axon position within prFB, length of axons) among FB pioneers. DM1 fibers are located dorsally (blue arrowhead) and project furthest in the contralateral hemisphere (blue arrow); DM4 is located ventrally (yellow arrowhead) and barely reaches the midline (yellow arrow). DM2 and DM3 fall in between these extremes. (J-M) Frontal confocal sections of central complex of adult brain, showing single cell clones of neurons descended from FB pioneers. (J, L) are sections at the level of the protocerebral bridge (PB), (K, M) at the level of the fan-shaped body (FB). Labeled neurons are color coded following the same scheme used in panels (G-I). (J, K) show a pontine neuron of lineage DM1 (blue), with a medially located cell body and axon entering at the medial PB (J), and terminal axons branching in an ipsilateral medial column (arrowhead in K) and a contralateral lateral column (arrow in K). (L, M) show a DM4 neuron (yellow) and DM2 neuron (green). (N) Histogram depicting average length of FB pioneer axons belonging to lineages DM1–4. Upper horizontal bar of each pair belongs to left hemispheric lineage, lower bar to corresponding right hemispheric lineage. The sharp medial turn of axon as it enters the prFB (white arrowhead in panel B) was taken as origin on x-axis; arrow at top demarcates brain midline. (O) Schematic of Hanesch et al. [11; with permission], showing fan-shaped body (FB) divided into eight columns (A-H; arrow at top indicates midline). Based on Golgi-preparations, Hanesch et al. [11] distinguished between four classes of bi-columnar pontine neurons, indicated by solid lines on left side, and hatched lines on right side. Our data indicate that the four classes described by Hanesch et al. [11] correspond to the four lineages of origin of the pontine neurons. Other abbreviations: sg secondary glia labeled by Drl-lacZ. Bars: 2μm (A, D); 10μm (E, F); 2μm (G, H); 25μm (J-M)

Figure 5

Growth and development of the prFB during the larval period. Panels show Z projections of frontal or horizontal confocal sections of larval brains at different stages (48h, 72h, 96h after hatching). Global labeling of secondary (larvally born) neurons with anti-Neurotactin (magenta in A-C, E-G, H, N, Q; white in B′, C′, F′, G′, T) or neuropil with anti-DNcadherin (magenta in I, J, M, O, P, R, S; white in K, L). FB pioneers are labeled by R45F08-Gal4>mcd8UAS-GFP (green in A-C, E-J). (A-C) At 48h (second larval instar) FB pioneers form a single bundle, as in freshly hatched first instar (see Figure 4E). The outgrowing secondary axons tracts (SATs) of DM1–4 (magenta in B) fasciculate with FB pioneers (green in B). At this stage, R45F08-Gal4 expression is still confined to Neurotactin-negative primary FB pioneers (C, C′). (D) Average numbers and standard deviations of R45F08-Gal4-positive DM1–4 neurons between 24 and 96h after hatching (n=5). Horizontal hatched lines give average for all four DM lineages. (E-G′′) At 72h after hatching (early third instar) the FB pioneer axons have split into several commissural bundles (arrow in E, G). Gaps between these bundles are filled with Neurotactin-positive secondary fibers formed by lineages DM1–4 (G-G′′). Numerous Neurotactin-positive secondary neurons have joined the set of R45F08-Gal4-positive FB pioneers (arrow in F, F′). (H-L) At 96h after hatching (late third instar) secondary DM1–4 axons have increased in number and form a system of crossing bundles, the posterior plexus of the fan-shaped body (FBppl in H, J, K). Both R45F08-Gal4-positive FB pioneers and secondary axons display tufts of filopodia which appear as DN-cadherin-positive domains (prFB, prEBp, prEBa in panels I-L). (M-P) Labeling of secondary E-PG neuron precursors (nomenclature after [46]) by R19G02-Gal4 driver. Terminal filopodia of these neurons are found in ventral prFB, prEBp and prLALgall (see also [35]). (Q-S) R83H12-Gal4 is expressed in a different subset of secondary neuronal precursors, mainly PB-FN and P-EN (nomenclature after [46]). Note in (Q) chiasmatic architecture of projection of labeled neurons towards the fan-shaped body, with all four bundles turning medially, but only DM1 and DM2 crossing towards contralaterally, and DM3/4 remaining ipsilaterally. This chiasmatic projection is typical for all unicolumnar neurons, as demonstrated in panel (T) (12h after puparium formation), where anti-Neurotactin (white) globally labels massive bundles of secondary axons of DM1–4. Bar: 25μm (for all panels except B-C′, F-G′′); 10μm (for B-C′, F-G′′). See also Figure S6.

Figure 6

Structure of the prFB after ablation of FB pioneers. (A-C) Z-projections of horizontal confocal sections of wild-type late third instar brains, illustrating entry of lineages DM1–3, posterior plexus of fan-shaped body (FBppl), and filopodial tufts forming primordia of fan-shaped body neuropil (prFB) and ellipsoid body neuropil (prEBa). For a clear depiction of the FB pioneers, males lacking UAS-hid;rpr and tub-Gal80ts are shown. (D-F) Same views of late larval brain as in (A-C) after ablation of FB pioneers by activating UAS-hid;rpr from 24–48h with R45F08-Gal4. (E and E′) show two different representative specimens. Note that FBppl is reduced in diameter (arrow in D, F) compared to control (arrow in A, C). Also primordium of fan-shaped body neuropil (prFB in B, E, E′) is separated by wider gap in ablated specimens (arrowheads in B, E, E′). (G, H) Z-projection of horizontal confocal sections of one brain hemisphere of control (G) and ablated (H) specimen, showing normal branching pattern of secondary axon bundles of DM4 lineage (yellow arrowheads). The neurotactin and DNcadherin pattern of non-temperature shifted female controls (which contain UAS-hid;rpr and tub-Gal80ts), in which no ablation occured, were indistinguishable from A-C, G (not shown). (I, J) Schematic fan-shaped body primordium at first larval instar (L1) and late third instar (L3) in control (I) and ablated specimen (J). Bars: 25μm (A, B, D-E′); 10μm (C, F, G, H)