Research ArticleNEUROSCIENCE

Pioneer interneurons instruct bilaterality in the Drosophila olfactory sensory map

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Science Advances  23 Oct 2019:
Vol. 5, no. 10, eaaw5537
DOI: 10.1126/sciadv.aaw5537
  • Fig. 1 Neuroglian is required in bilateral sensory map formation.

    (A and B) Adult olfactory system of Drosophila. (A) Sensory neurons from the antenna (white arrowhead) project via the antennal nerves (white arrow) to the bilateral antennal lobes (ALs; dashed circles). (B) Schematic of a single ORN connecting to a synaptic glomerulus at the ipsilateral AL and a homotopic glomerulus in the contralateral hemisphere (neuropil marker N-cadherin in red). Scale bars, 100 (A) and 50 μm (B). (C to E) Unilateral and bilateral olfactory map organizations in Diptera. Unilateral antennal backfill revealed a strict ipsilateral representation of ORN afferents in mosquitoes (C). In contrast, in higher Brachyceran like Drosophila (D), most ORNs project in a bilateral fashion, as indicated by a large commissural tract and labeling of the contralateral AL. In contrast to wild type, Drosophila carrying a mutation in the cell adhesion molecule Nrg displays a strict unilateral afferent innervation (E). (F to I) Labeling of different bilateral ORN populations in Drosophila wild type (F and H) and nrg mutants (G and I) identified not only the complete absence of the antennal commissure (F and G) but also the precise ipsilateral targeting and class-specific ORN axon convergence [asterisks in (H) and (I)]. (J and K) nrg mutants show a specific loss of bilateral ORN connectivity. (J) Wild type projections from a single olfactory sensillum (ac1) containing two bilateral (Ir92a and Ir31, yellow and green, respectively; arrows indicate contralateral projections) and one unilateral (Ir75d, red) ORN classes. Note the higher degree of synaptic arborization within the ipsilateral glomerulus (left insets) compared to the contralateral target side (right insets). (K) In nrg mutant, bilateral ORN axons show a normal level of ipsilateral arborization but fail to extend any contralateral process (yellow/green arrows, contralateral AL not shown). No changes in the connectivity of the unilateral ORN class can be detected. The table summarizes a systematic analysis of 19 ORN classes in nrg mutants, showing a complete switch of all bilateral into unilateral ORNs but no effect on unilateral ORN classes (100%; n ≥ 8 for wild type and nrg mutant). (L and M) The targeted Nrg RNAi in projecting ORNs (n = 16) uncovers a cell-autonomous function in sensory neurons visualized by the unilateral connectivity (Or47b, green). (N and O) Compared to wild type (N and N′), loss of Nrg (O, O′) has no effect on the presynaptic differentiation at the ipsilateral target side as indicated by the localization of Bruchpilot (Brp) protein. Green, Brp::GFP; red, neuropil marker N-cadherin. (P and Q) Targeted RNAi of Nrg in different cell types of the developing olfactory system. Removal of Nrg from PNs (n = 10) does not influence bilateral ORN (green) connectivity (P and P′). In contrast, loss of Nrg in a cluster of ventro-lateral interneurons (vl-LNs) (n = 8) leads to a complete switch into unilateral ORN circuitry (Q and Q′). (R and S) In the adult olfactory system, a vl cluster (white arrows) of LNs displays, in addition to a broad ipsilateral arborization within the AL, a distinct commissural projection (inset R′ and R″). In nrg mutant, ipsilateral dendritic arborizations seem unaffected, whereas the contralateral LN tract is missing (inset S′ and S″). Green, LNs; red, all neurons labeled by anti-Nrg; blue, neuropil marker N-cadherin. (T) Schematics illustrating sensory map connectivity in the Drosophila olfactory system. Within each pair of homotopic glomeruli, bilateral sensory input (red and orange ORNs) onto unilateral PNs is modified by different classes of bilateral LNs. Loss of Nrg in bilateral ORNs and LNs (but not PNs or midline glial cells) leads to a switch of the bilateral into a unilateral sensory representation. Dashed vertical white lines indicate the midline, commissure position is highlighted by white rectangles, and dotted circles show glomerulus boundaries. Scale bars, 20 μm for all images of adult ALs.

  • Fig. 2 Domain-specific organization of cPINs.

    (A and A″) At about 20 hours after pupa formation (APF), Nrg-positive [arrowhead in (A″)] ORN pioneer axons enter the AL and segregate into a lateral and a medial fascicle [arrows in (A) and arrowheads in (A′)], which extend toward the dorsal midline of Nrg-positive fibers [bracket in (A″)]. (B and B″) At the time of ORN axon arrival, a vl cluster (cell bodies indicated by arrowheads) of cPINs has developed localized ipsilateral dendritic arborizations [dashed circles in (B′)] and broad contralateral projection at the dorsal AL [arrowheads in (B′) and (B″)]. ORN axons [arrow in (B″)] and the commissural tract of cPINs [bracket in (B″)] can be identified by their strong expression of the cell adhesion molecule Flamingo [Fmi in (B″)]. (C and C‴) On the basis of the spatial segregation of their dendritic fields in the adult AL, two main classes of cPINs, the lateral and the medial, can be recognized [lateral/medial domain (LD/MD)]. The cell bodies of both cPIN classes are in close proximity [inset, (C″) and (C‴)], but their commissural tracts in the dorsal AL remain separated [dashed rectangles in (C) and brackets in (C′)]. (D and E) In nrg mutants, the dendritic field of each cPIN class in the ipsilateral AL (lateral/medial domain) remains correctly positioned, but the contralateral projection is missing [dashed rectangles in (D) and (E)]. (F and G) Wild type organization of cPIN classes. Before ORN axon arrival, dendritic fields of the cPIN classes segregate in the ipsilateral and contralateral AL and both lateral and medial cPINs have distinct projection patterns. (F and F′) Lateral cPINs have a distinct ipsilateral dendritic field [arrow in (F′)] and a strong commissural tract, which terminate at the dorsal edge of the contralateral AL [arrowhead in (F)]. In contrast, medial cPINs have a thin commissural tract, which extend to ventral region of the contralateral AL (G and G′). CBs, cell bodies. Development of cPIN in wild type (H to J) and nrg mutants (K to M). With the beginning of metamorphosis, cPINs start to extend to the dorsal AL midline, and ventral extensions of the ipsilateral dendritic arborizations become visible. Following the initiation of the ipsilateral dendritic field [green arrowhead in (H)], cPINs project a pioneer commissural track across the dorsal midline [red arrowhead, showing contralateral axon, in (H)], which grows along the medial surface of the contralateral AL (I) to merge with the ipsilateral dendritic arborization at the time of ORN axon arrival (J). In nrg mutants, no changes can be observed for initiation of the dendritic field [green arrowhead in (K)] and the dorsal extension of cPINs within the ipsilateral AL [white arrowheads in (K)]. However, the dorsal process loops back, extends ventrally, and “self-merges” with the ventral ipsilateral processes (L), subsequently forming the appropriate dendritic field in the medial AL region (M). (N) Dendritic fields of cPINs (green) and PNs (red) are spatially segregated within the early AL, with only cPIN localized at the ORN axon entry side in the posterior AL. (O and P) Anterior and lateral view of the AL, respectively. (Q) Model of lateralized ORN axon projection and targeting: ORN axons enter the ipsilateral AL at the posterior domain and are guided by cPINs toward the dorsal ML. With the contralateral hemisphere, ORN axons switch to the anterior domain to recognize their corresponding PN target neurons. Dashed vertical white lines indicate midline, developing AL is indicated by white dashed circles, and lateral and medial domains are indicated by red dashed lines. Scale bars, 10 μm for all images of pupal and 20 μm for adult ALs (C to E).

  • Fig. 3 Sensory neurons bypass their ipsilateral target.

    (A) Two alternative developmental pathways to switch from unilateral to bilateral circuit assemblies: (1) Following the ipsilateral ORN targeting to glomerulus-specific PNs, contralateral innervation is induced via a commissural branch (blue) across the midline (ML). (2) Direct contralateral projection via suppression of ipsilateral targeting followed by the induction of an ipsilateral synaptic collateral (blue). (B to I) Axon growth analysis of a single pioneer ORN class (Ir92a), which targets a ventral medial glomerulus (VM1; see Fig. 1). In wild type, pioneer Ir92a ORN axon enters the AL around 20 hours APF (B) and extends along the medial pathway to the dorsal AL with no signs of accumulation at the putative target region in the ventro-medial AL region (C) (white dashed lines). Following midline crossing and extension to the contralateral target region [red line in (D)], ORN axons converge within the next 20 hours into spatially restricted synaptic glomeruli (E). (F to I) In nrg mutants, ORN axons reach the AL within the temporal period of wild type axons. In contrast to the smooth ipsilateral extension of pioneer axons in wild type, loss of nrg leads to an instant accumulation of pioneer axons at the prospective ventral target region [arrowhead in (F)]. During the period of wild type dorsal extension and contralateral projection (25 to 30 hours APF), nrg mutant pioneer axons converge prematurely at the target region [arrowheads in (G) and (H)], with no differences during the following period of glomerulus maturation (I). (J to Q) Single-cell analysis of pioneer axon branch dynamics. During the period of ipsilateral growth, individual axons of bilateral ORNs induce a large number of lateral processes all along the medial AL neuropil [ventral, central, and dorsal area in (L)] with no enrichment at the prospective target region [TR; red dashed lines in (J), high magnification in (J′), and quantification in (P)]. Following the contralateral projection, the number of ipsilateral filopodia reduces at the dorsal AL and restricts to the prospective ventro-medial target region [(K) and (M); quantification in (Q)]. In contrast to bilateral ORNs, axons of ingrowing unilateral ORNs aggregate at the prospective ventral target region, with filopodia extending into multiple directions [(N) and (O); quantifications in (P) and (Q)]. (R to U) Similarly to the sequence of axon projection, the presynaptic differentiation following contralateral projections, as indicated by the localization of Bruchpilot-GFP, is more restricted in nrg mutants compared to wild type (R and S), but similar pattern of synaptic maturation is observed during glomerulus assembly (T and U). Scale bars, 10 μm for all images of pupal ALs.

  • Fig. 4 Neuroglian-dependent hierarchical interactions coordinate sensory map development.

    (A) Analysis of cell type–specific Nrg RNA interference (RNAi) to define autonomous and non-autonomous functions of sequentially ingrowing cPINs (blue), pioneer (red), and follower (green) ORNs, as well as serotonergic CSD neurons (magenta). The table summarizes the resulting connectivity phenotypes. (E to G) Removal of Nrg in developing cPINs does not only switch bilateral into unilateral interneurons (E) but also disrupt the bilateral projection of pioneer and follower ORNs [(F) and (G), respectively, compared to wild type (B to D)]. (H to J) Down-regulation of Nrg in pioneer neurons interferes with their bilateral projection (I) and the projections of follower neurons (J) but has no effect on bilateral cPIN development (H). (K to M) Restricted removal of Nrg in late-projecting bilateral ORN classes (“followers”) transforms them into a unilateral projection type (M) but leaves the bilateral organization of cPINs (K) and pioneer ORNs (L) unaffected, demonstrating a strict temporal hierarchy of Nrg-mediated interneuronal interactions in bilateral circuit formation. (N to R) Analysis of Nrg domain requirement for two classes of cPINs (N and O) and two classes of bilateral ORN types, pioneer ORNs (P), and follower ORNs (Q). Removal of the consensus sequence for Ankyrin signaling (∆FIGQY) strongly affects the bilateral projection of follower ORNs (Q′) but not pioneer ORNs (P′) and cPINs (N′ and O′). The combined deletion of Ankyrin and PDZ binding domains (∆C) switches all bilateral ORNs into a unilateral connectivity pattern (P″ and Q") but does not interfere with bilateral cPIN development (N″ and O″). The deletion of the Moesin-binding domain (∆FERM) leads to a complete unilateral connectivity pattern for all four classes of bilateral olfactory neurons (N‴ to Q‴). (R) Schematics illustrate domain organization of Nrg and the connectivity phenotype of the deletion mutants (top) and their quantification (bottom). Scale bars, 20 μm for all images of adult ALs. For the list of genotypes used in this study, see table S1.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaaw5537/DC1

    Fig. S1. Unilateral versus bilateral olfactory circuit organization within Diptera.

    Fig. S2. Comprehensive analysis of unilateral and bilateral projecting antennal ORN axon in Neuroglian mutant (see also table in Fig. 1).

    Fig. S3. Neuroglian expression during development.

    Fig. S4. Neuroglian expression in midline glia cells is dispensable for bilateral ORN connectivity.

    Fig. S5. Cell-specific loss of Neuroglian in cPINs affects bilateral olfactory map formation.

    Fig. S6. Ablation of cPINs affects bilateral connectivity of ORNs.

    Fig. S7. cPINs segregate from PNs in early AL development.

    Fig. S8. Cell autonomous and non-autonomous function of Neuroglian in cPINs and ORNs.

    Fig. S9. Neuroglian affects olfactory commissure development of CSD neurons.

    Table S1. Genotype of experiments.

    Reference (49)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Unilateral versus bilateral olfactory circuit organization within Diptera.
    • Fig. S2. Comprehensive analysis of unilateral and bilateral projecting antennal ORN axon in Neuroglian mutant (see also table in Fig. 1).
    • Fig. S3. Neuroglian expression during development.
    • Fig. S4. Neuroglian expression in midline glia cells is dispensable for bilateral ORN connectivity.
    • Fig. S5. Cell-specific loss of Neuroglian in cPINs affects bilateral olfactory map formation.
    • Fig. S6. Ablation of cPINs affects bilateral connectivity of ORNs.
    • Fig. S7. cPINs segregate from PNs in early AL development.
    • Fig. S8. Cell autonomous and non-autonomous function of Neuroglian in cPINs and ORNs.
    • Fig. S9. Neuroglian affects olfactory commissure development of CSD neurons.
    • Table S1. Genotype of experiments.
    • Reference (49)

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