Pioneer interneurons instruct bilaterality in the Drosophila olfactory sensory map

Abstract

Interhemispheric synaptic connections, a prominent feature in animal nervous systems for the rapid exchange and integration of neuronal information, can appear quite suddenly during brain evolution, raising the question about the underlying developmental mechanism. Here, we show in the Drosophila olfactory system that the induction of a bilateral sensory map, an evolutionary novelty in dipteran flies, is mediated by a unique type of commissural pioneer interneurons (cPINs) via the localized activity of the cell adhesion molecule Neuroglian. Differential Neuroglian signaling in cPINs not only prepatterns the olfactory contralateral tracts but also prevents the targeting of ingrowing sensory axons to their ipsilateral synaptic partners. These results identified a sensitive cellular interaction to switch the sequential assembly of diverse neuron types from a unilateral to a bilateral brain circuit organization.

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Kaur et al., Sci. Adv. 2019; 5 : eaaw5537 23 October 2019
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NEUROSCIENCE
Pioneer interneurons instruct bilaterality
in the Drosophila olfactory sensory map
Rashmit Kaur1, Michael Surala1, Sebastian Hoger1, Nicole Grössmann2,3, Alexandra Grimm1,
Lorin Timaeus1, Wolfgang Kallina1, Thomas Hummel1*
Interhemispheric synaptic connections, a prominent feature in animal nervous systems for the rapid exchange
and integration of neuronal information, can appear quite suddenly during brain evolution, raising the question
about the underlying developmental mechanism. Here, we show in the Drosophila olfactory system that the in-
duction of a bilateral sensory map, an evolutionary novelty in dipteran flies, is mediated by a unique type of com-
missural pioneer interneurons (cPINs) via the localized activity of the cell adhesion molecule Neuroglian. Differential
Neuroglian signaling in cPINs not only prepatterns the olfactory contralateral tracts but also prevents the targeting
of ingrowing sensory axons to their ipsilateral synaptic partners. These results identified a sensitive cellular interaction
to switch the sequential assembly of diverse neuron types from a unilateral to a bilateral brain circuit organization.
INTRODUCTION
Coherent integration of perception, cognition, and behavior between
the two brain hemispheres is mediated by commissural axons form-
ing precise connections between homotopic bilateral synaptic areas
(1,2). The formation of bilateral circuits requires not only a variety
of guidance cues and cellular interactions but also the precise regu-
lation of ipsilateral versus contralateral synaptic target recognition
(3,4). Although a diverse set of conserved signaling pathways that
control the guidance of commissural neurons at the CNS midline is
well characterized (5,6), we know very little about the coordinated
regulation of multiple neural components into a bilateral brain circuit.
During brain evolution, novel commissural tracts seem to appear
rather rapidly (2). A prominent example for a structural novelty in
the nervous system is the emergence of the corpus callosum in placental
mammals, according to T. H. Huxley, “the greatest leap anywhere
made by Nature in her brainwork” (7). Comparative studies suggest
an initial change in axonal interactions for establishing novel inter-
hemispheric connections, but the underlying modification in the
developmental program, which causes global brain circuit remodel-
ing, remains obscure (1,8). In this study, we describe how the activity
of a single class of commissural interneurons in the Drosophila
olfactory system induces a switch from unilateral to bilateral neural
circuit organization.
Olfactory systems are characterized by the precise segregation of
sensory neuron projections into distinct synaptic glomerular units,
in which odor information is relayed to matching classes of projection
neurons (PNs) (9,10). With a multitude of sensory class-specific
synaptic glomeruli, interconnected by various types of modulatory
interneurons, vertebrates and insects share main structural and func-
tional features in olfactory map organization (9–11). Although primary
sensory representation in most olfactory systems is strictly unilateral,
flies have evolved a unique bilateral olfactory map in which olfactory
sensory neurons (ORNs) target, in addition to the ipsilateral glomerulus,
a homotopic synaptic region on the contralateral hemisphere via a
commissural extension (Fig.1,AandB) (12). As sensory neurons in
mosquitoes only connect to the unilateral olfactory brain hemisphere
(13), direct bilateral connections may support increased sensitivity
and navigation accuracy in fast-flying Diptera with rather short
antennae (14,15).
Here, we show that the bilateral sensory map in the Drosophila
olfactory system is completely reverted into a unilateral circuit in
mutants of the cell adhesion molecule Neuroglian. We could localize
Neuroglian activity in a small cluster of contralaterally projecting
interneurons, which not only pioneer the commissural sensory tract
but also interfere with synaptic partner recognition of these sensory
neurons on the ipsilateral target region. As olfactory circuit assembly
relies on defined hierarchy of cell type interactions, these findings
offer a rather simple mechanism to switch a complete developmental
program from the ipsilateral to the contralateral hemisphere.
RESULTS
Loss of Neuroglian switches bilateral to unilateral sensory
neuron innervation
To visualize sensory map organization in the olfactory system within
Diptera, we performed unilateral labeling of the antennal nerve and
determined unilateral versus bilateral projection patterns in the
antennal lobes (ALs) of multiple Nematocera and Brachycera spe-
cies (fig. S1). The bilateral sensory representation is absent in most
Nematoceran species like Culicidae and Simuliidae and becomes prom-
inent in basal Brachycera flies like Bombyliidae and Dolichopodidae.
In all Schizophora species analyzed, including Drosophilidae and
Calliphoridae, most sensory neurons display interhemispheric
connection (Fig.1,CandD, and fig. S1), therefore providing an
excellent experimental system to determine the genetic regulation
of bilateral neural circuit formation. In a candidate gene approach in
Drosophila using unilateral antennal labeling of mutants combined
with targeted RNA interference (RNAi) in projecting ORNs, loss of
the immunoglobulin (Ig) family member protein Neuroglian (Nrg)
leads to a striking and highly penetrant connectivity phenotype: Axons
of bilateral ORNs project only to their ipsilateral target glomerulus with
a complete absence of a contralateral connection, thereby switching
1Department of Neurobiology, University of Vienna, Althanstrasse 14A, 1090 Vienna,
Austria. 2Ludwig Boltzmann Institute, Health Technology Assessment (LBI-HTA),
Garnisongasse7/20, 1090 Vienna, Austria. 3Department of Health Economics, Center
for Public Health, Medical University of Vienna, Vienna, Austria.
*Corresponding author. Email: thomas.hummel@univie.ac.at
Copyright © 2019
The Authors, some
rights reserved;
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for the Advancement
of Science. No claim to
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License 4.0 (CC BY-NC).
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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 m (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 m for all images of adult ALs.
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the olfactory sensory map back to the unilateral organization (Fig.1,
E,FtoI,L,andM). A systematic analysis of multiple ORN classes
in the nrg mutant background showed that most bilateral neuron
classes display a precise ipsilateral connectivity pattern similar to
unilateral ORN classes in wild type (Fig.1, table, and fig. S2). Terminal
arborization remains confined within glomerular boundaries (Fig.1,
JandK), and no changes in presynaptic density (Fig.1,NandO) of
ORN axons within the ipsilateral hemisphere can be observed in nrg
mutants. These data indicate a crucial developmental step in olfactory
system formation to determine a unilateral versus bilateral state of
circuit organization.
Non-autonomous function of Neuroglian in bilateral
ORN connectivity
Besides ORNs, Nrg is expressed in various central neurons and glial
cells (16) in the developing and adult olfactory system (fig. S3), rais-
ing the question about the underlying cellular interactions for bilateral
circuit formation. We therefore extended the targeted Nrg RNAi
approach using a collection of developmentally expressed Gal4 lines
for different olfactory cell types. In contrast to a previous study (16),
interfering with Nrg function in various olfactory glia types (via
repo-Gal4 and 442-Gal4) did not affect the wild type connectivity
pattern of bilateral ORNs (fig. S4). Similarly, developmental knock-
down of Nrg in PNs (via GH146-Gal4), the main synaptic partner
neurons of ORNs, has no effect on sensory axon targeting (Fig.1P).
However, loss of Nrg in a specific cluster of ventro-lateral (vl) inter-
neurons (via OK107-Gal4 and OK371-Gal4) results in a unilateral
ORN connectivity phenotype (compare Fig.1, QandM). In the
adult olfactory system, the vl cluster contains different types of uni-
lateral and bilateral interneurons with specific neural arborization
patterns (Fig.1R) (17,18). nrg mutant vl-cluster neurons fail to de-
velop a contralateral projection but show no change of arborization
within the ipsilateral hemisphere (Fig.1,SandT).
Commissural pioneer interneurons prepattern bilateral
ORN projections
Pioneer sensory axons in the Drosophila olfactory system show
high Nrg expression and segregate into a lateral and medial path-
way while extending dorsally toward the midline of the developing
AL (Fig.2A and fig. S3). By the time of sensory neuron ingrowth,
vl-cluster interneurons have established restricted neural arboriza-
tions within the AL and a prominent midline commissure (Fig.2B).
To determine the neuronal diversity of commissural interneurons
critical for bilateral circuit organization, we analyzed a collection of
expression lines (Fig.2,CtoG) (19) and identified a small popula-
tion of commissural pioneer interneurons (cPINs), which, upon
Nrg knockdown, results in a unilateral ORN projection phenotype
(fig. S5). Similarly, targeted ablation of cPINs before ORN innervation
leads to a unilateral sensory map (fig. S6). Further developmental
and clonal characterization revealed two main morphological cPIN
classes, which prefigure the early sensory projections: Most cPINs
(8 to 10 neurons) extend along the dorso-lateral pathway and form
a restricted commissural tract at the dorsal AL (“lateral cPINs”;
Fig.2,CandF). A smaller subset of cPINs (two to four neurons)
projects via the medial AL on the ipsilateral and contralateral hemi-
sphere (“medial cPINs”; Fig.2,CandG). The neural arborizations
of lateral and medial cPINs remain separated in nrg mutants, but
both classes of interneuron fail to extend a commissural tract
(Fig.2,DandE).
During wild type development, outgrowing cPINs start to project
at late third instar larval stage to establish ipsilateral arbors and a
commissural extension, which converge on the contralateral side (Fig.2,
HtoJ, and fig. S7). In nrg mutants, the formation of ipsilateral pro-
cesses is not affected, but the contralateral extensions become rerouted
and merge with the ipsilateral arborizations (Fig.2,KtoM). Co-labeling
of cPINs and unilateral PNs revealed a spatial segregation of their
neuronal fields by the time of pioneer ORN arrival. Here, the entry
side of pioneer ORN axons at the posterior AL is covered by cPIN
arborizations and devoid of PN dendrites, which are enriched at the
anterior AL region. These distinct AL domains for extending ORN
axons, a posterior projection domain and an anterior targeting domain,
support an instructive role of cPINs in bilateral sensory neuron inner-
vation by preventing ipsilateral axon-target interaction via spatial
segregation from the synaptic area (Fig.2, N to Q).
Neuroglian suppresses ipsilateral ORN axon targeting
To induce interhemispheric circuit organization, a likely cellular
scenario would be the formation of a novel contralateral branch fol-
lowing the default developmental program of unilateral axon target-
ing (Fig.3A,1). However, the comparison of initial axon targeting
of unilateral and bilateral pioneer neurons in wild type and nrg mu-
tants points toward an alternative developmental strategy to coordinate
bilateral innervation (Fig.3A,2). In wild type, axons of atonal (ato)–positive
pioneer ORNs form a solid fiber track (Fig.3B), which extends beyond
the ipsilateral target region (Fig.3C). After sending a commissural
process across the dorsal midline (Fig.3D), glomeruli are induced
via bilateral axon convergence (Fig.3E). In contrast, loss of Nrg results
in a strong accumulation of pioneer axons at the ipsilateral prospec-
tive target side (Fig.3,FandG). During the period of contralateral
axon projection in wild type, nrg mutants show an accelerated glo-
merular convergence (Fig.3H) but no obvious differences in glomer-
ulus maturation (Fig.3I). Single-cell analysis revealed a dynamic growth
cone morphology with a dense array of filopodia all along the AL
surface (Fig.3,JtoM). Here again, no signs of filopodia enrichment
at the putative target region can be detected. During ipsilateral ex-
tension, single axons form the same amounts of filopodia in the central
and dorsal AL domains (Fig.3,LandP). By the time of contralateral
axon projection, the number of ipsilateral filopodia reduces in the
dorsal area and processes at the central synaptic target region become
stabilized (Fig.3,MandQ). In contrast, axons of unilateral ORN
display a restricted field of filopodia during initial targeting and axon
convergence (Fig.3,NtoQ). In the subsequent period of glomerulus
assembly, nrg mutants show an increased axonal restriction at the
unilateral presynaptic region (Fig.3,SandU) compared to the bi-
lateral innervation in wild type (Fig.3,RandT). These results show
that the transient suppression of ipsilateral target site recognition
defines a key event in bilateral map formation. Here, ipsilateral
ORN-cPIN interaction shifts the primary ORN-PN recognition
program to the contralateral hemisphere followed by ipsilateral axon
targeting. The functional coupling of delayed axon targeting and
contralateral growth by cPINs ensures an organized assembly of bi-
lateral sensory circuits.
Differential Neuroglian signaling mediates hierarchical
neuron interactions in bilateral circuit formation
Neuron type–specific interference with Nrg function revealed not
only a cell-autonomous role in cPINs and ORNs but also a strict
hierarchy in their cellular interactions [Fig.4,BtoM, summarized
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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 m for all images of pupal and 20 m for adult ALs (C to E).
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in (A)]. The removal of Nrg from cPINs triggers unilateral targeting
of all bilateral ORN classes (Fig.4,FandG), whereas the knock-
down of Nrg in ORNs has no effect on the bilateral organization of
cPINs (Fig.4,HandK). Similarly, axons of amos-positive follower
ORNs rely on Nrg expression in cPINs and atonal-positive pioneer
sensory neurons (Fig.4J) but not vice versa (Fig.4,KandL), indi-
cating a defined sequence of interneuronal interactions, which cor-
relates with the specific window of axon growth (Fig.4A and fig.S8).
Furthermore, Contralaterally projecting Serotonin-immunoreactive
Deutocerebral neurons (CSD) (20), which innervate the AL after
sensory neurons, depend on Nrg specifically for the olfactory com-
missure but not for the development of an evolutionary more an-
cient protocerebral commissure (fig. S9) (20). cPINs subsequently
develop into glutamatergic inhibitory interneurons of the adult ol-
factory system (21), indicating that an efficient bilateral odor repre-
sentation requires the combined segregation of different types of
modulatory interneurons along with sensory neurons.
As Nrg-mediated adhesion triggers different types of intracellular
signaling pathways (22), we tested if the sequence of bilateral neuronal
interactions involves distinct downstream effectors (Fig.4,NtoR).
Loss of the PDZ interacting domain has no effect on bilateral map
formation (Fig. 4R), whereas the interference with Ankyrin binding
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 m for all images of pupal ALs.
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Fig. 4. Neuroglian-dependent hierarchical interactions coordinate sensory map development. (A) Analysis of cell type–specific Nrg RNA interference (RNAi) to de-
fine 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 inter-
neurons (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 organiza-
tion 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 bi-
lateral olfactory neurons (N‴ to Q‴). (R) Schematics illustrate domain organization of Nrg and the connectivity phenotype of the deletion mutants (top) and their quanti-
fication (bottom). Scale bars, 20 m for all images of adult ALs. For the list of genotypes used in this study, see table S1.
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disrupts bilateral organization of amos-positive follower ORNs
(Fig.4Q′) but not cPIN and pioneer ORN connectivity (Fig.4,N′toP′).
The combined deletion of Ankyrin/PDZ interacting domains affects
all bilateral ORN classes, suggesting partially redundant function in
pioneer ORNs (Fig.4,P″andQ″) but not cPINs (Fig.4,N″andO″).
Bilateral cPIN development is mediated via Moesin interaction, with
the complete absence of a contralateral extension following the dele-
tion of the corresponding intracellular domain (Fig.4, N‴andO‴).
These results revealed distinct effector pathways in the Nrg-mediated
hierarchical interactions to coordinate the bilateral assembly of key
circuit components.
DISCUSSION
Olfactory coding relies on the precise synaptic matching of distinct
classes of sensory to the corresponding set of central PNs. During
Drosophila olfactory system development, PN dendrites localize within
the olfactory target field according to their final glomerular position
before sensory neuron arrival and induce the ORN class-specific
axonal convergence by a mostly unknown recognition process (23).
In contrast to a unilateral sensory map, in which sensory neurons
have to connect to a single array of PN classes at the ipsilateral brain
hemisphere, the development of bilateral ORN connectivity has to
coordinate the interaction of projecting sensory axons with two
mirror-symmetric populations of unilateral PNs.
On the basis of the adult morphology, bilateral sensory neuron
innervation in Drosophila has been considered as a sequential pro-
cess where ipsilateral axon targeting is followed by the extension of
a contralateral branch toward the homotopic synaptic PN class (24).
Here, we show that bilaterality is established through direct con-
tralateral targeting of sensory axons, followed by the induction of
synaptic connections on the ipsilateral hemisphere (Fig.3A). We
identified a distinct class of commissural interneurons, which not
only provide a contralateral tract for ingrowing sensory neurons but
also prevent their interaction with ipsilateral PNs. On the basis of
the spatial segregation of PN and cPIN processes in the early target
region, we are proposing a mechanism in which ipsilateral target
site recognition of ORNs is suppressed by a cPIN-based projection
domain distinct from the PN-defined targeting region (Fig.2Q).
ORN axons exit the antennal nerve encounters cPIN processes at
the posterior target region and extend toward and across the dorsal
midline to interact with the contralateral PN field. Nrg function
allows ORN axons to stay within the projection domain, most likely
via a direct interaction with cPIN processes, in which loss of Nrg
in cPIN or the removal of cPIN itself results in ipsilateral circuit
assembly. A small subset of unilateral ORN classes in Drosophila
escapes the cPIN-mediated contralateral guidance mechanisms
(fig. S2D). Here, target regions of unilateral ORNs are clustered in
distinct vl AL domain, indicating an additional level of domain or-
ganization in the target region.
Mutations in the human Nrg homolog, L1CAM, lead to a severe
reduction in corpus callosum formation (25). During cortex devel-
opment, callosal projection neurons build homotopic connections
in a sequential manner, in which axons of early-generated neurons
pioneer the commissural tract, providing a conceptual framework
for corpus callosum evolution (1,8). With a conserved midline
pattern between vertebrates and insects (5,6), the identification
of distinct unilateral and bilateral sensory maps within dipteran
species offers a unique opportunity to determine how evolutionary-
dynamic Nrg expression could support novel cellular interactions.
For example, persisting Nrg-positive larval commissures in close
proximity to the developing adult olfactory system (26,27) could
provide a permissive substrate for unilateral cPIN precursors to
extend across the midline. A similar mechanism has been proposed
for corpus callosum development with novel adhesive interactions
of pioneer fibers with more ancient commissures of the cingulate
cortex and hippocampus (1,8). As sequential afferent interaction
is a common theme not only among sensory neurons in insect
and vertebrate olfactory system development (28–31) but also in
corpus callosum formation (1,8); cPINs could have “hijacked” pre-
existing Nrg-mediated ORN interactions and thereby shifting uni-
lateral olfactory circuit assembly to the contralateral hemisphere
followed by ipsilateral collateral formation. Callosal projection neu-
rons extending to the contralateral hemisphere leave filopodia
behind, from which subsequently interstitial collateral branches
emerge (32).
In an alternative scenario, bilateral cPIN may have appeared
denovo and triggered interhemispheric connectivity of sensory
neurons. The ventral AL neuroblast lineages generate a highly di-
verse population of bilateral neurons (18,26), and a recent study
showed how changes in Hox gene expression within these progeni-
tors result in major remodeling of brain circuit organization (33).
As the larval olfactory system in higher dipterans displays a uni-
lateral connectivity pattern (34), an adult life style with highly agile
flight behaviors seems to be a major determinant for the evolution
of bilateral sensory representation. Even unilateral ORN classes
have established interglomerular connectivity via a unique type of
bilateral PNs (35), indicating a strong requirement for fast inter-
hemispheric communication. The enhanced sensitivity due to a
higher degree of sensory convergence in bilateral versus unilateral
olfactory circuits is accompanied by a substantial reduction in spa-
tial information. It is tempting to speculate that the bilateral olfac-
tory map became stabilized in the course of dipteran evolution by
strengthening directional sensitivity via lateralized synaptic differ-
entiation (36).
How do cPINs cross the midline in the first place? Although a
balanced activity of chemoattraction and chemorepulsion has been
a broadly accepted mechanism in bilateral circuit development for
decades (5,6), recent studies on Netrin signaling have challenged
the concept of long-range guidance also at the midline, and a critical
role for cell adhesion has been proposed in both vertebrates (37,38)
and insects (39). In Drosophila, a midline glial structure [transient
interhemispheric fibrous ring (TIFR)] has been shown to support
ORN axon crossing (16), but interference with Netrin signaling in
this region does not prevent bilateral map formation. In addition,
the different Robo genes show differential expression in the devel-
oping AL, but commissure defects have only been described in
gain-of-function studies (40), further supporting an adhesion-based
mechanism of midline guidance. Although similarities in brain cir-
cuit organization and developmental cell-cell interactions suggest
a Nrg-related mechanisms in the formation of bilateral circuits in
other animals, a direct experimental proof requires the identifica-
tion of cPIN neurons and the analysis of Nrg expression patterns.
With the rapidly improving technologies of targeted gene knock-
out and transgene expression outside Drosophila, our results will
stimulate future comparative studies, especially within more basal
Diptera, to determine how modulation of cell adhesion can trigger
brain circuit evolution.
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MATERIALS AND METHODS
Methods summary
For developmental analysis of individual ORN axon targeting, wild
type single-cell clones were generated using the Flybow (FB) con-
struct (41) in combination with a heat-induced FLPm5 on second
chromosome. Flies expressing FB1.1B transgene under the control
of R86G11-Gal4 (19) were exposed to single heat shock for 90min
at 37°C to induce transient mFLP5 (41) activity between 0and
5hours after pupa formation (APF). Confocal images were pro-
cessed and analyzed using ImageJ and Imaris 9.2 (Bitplane).
To characterize the neuronal morphology of cPIN, wild type
single-cell clones were generated using the hs-FLP and FRT/FLP
system (42). Wild type cPINs were labeled with R19H07-Gal4 (19),
driving the expression of UAS-mCD8::GFP (42). Second instar larvae
were heat-shocked for 20 to 30min at 37°C. Pupae at the desired
stage were dissected. The developmental pattern of pioneer ORNs,
their synaptogenesis and LNs, in wild type and Nrg mutant back-
ground was analyzed in confocal image stacks of stained pupal and
adult brains. R86G11-Gal4 (19) and R20F11-Gal4 (19) driver lines
for ORNs and LNs were used, respectively. Nrg mutant hemizygous
males and heterozygous females (control) were preselected at third
instar and separated in different food vials for both clonal and ex-
pression analysis. White pupae (defined as 0 hours APF) were col-
lected for staging at 25°C. Pupae at desired age and adult flies were
dissected after eclosion. For developmental analysis, 5 to 10 brains
were analyzed at each time point.
Methods
Fly stocks and genetics
Fly stocks and crosses were maintained in standard medium at 25°C,
whereas RNAi experiments were performed at 29°C. To discover
molecules involved in bilateral sensory map formation, an RNAi
screen was conducted against several cell adhesion molecules using
pebbeled-Gal4 [a gift from L. Luo (28)]. To perform Nrg RNAi
knockdown in ORNs, glia, and AL-associated neurons, the follow-
ing stocks were obtained from Bloomington Drosophila Stock Center
(BDSC) or received as gift: ORN drivers, Sg18.1-Gal4 (43) (BDSC
6405), atonal-Gal4 (a gift from B.A. Hassan), and amos-Gal4 [a gift
from T. Chihara (29)]; ORN-specific drivers and markers were pro-
vided by B. Dickson and L. Vosshall (44,45); LN drivers, OK107-Gal4
[BDSC 854 (18)] and OK371-Gal4 [a gift from H. Aberle (17)]; Gal4
and LexA driver lines having expression in subset of LNs were se-
lected on the basis of expression pattern (19) and were obtained
from BDSC; glia-specific drivers, repo-Gal4 (BDSC 7415), 442-Gal4
[a gift from T. Préat (16)], PN-specific GH146-Gal4 (46), UAS-
NrgRNAi (BDSC 38215), GH146-QF, and QUAS-mtdTomato-3×HA
(BDSC 30037). Stocks used for Nrg mutant and intracellular do-
main deletion constructs (P[acman] constructs) were the following:
nrg849 (BDSC 35827), nrg14, and P[acman] constructs were provided
by J. Pielage (22). For visualizing axons and synaptic terminals and
co- labeling of two distinct cell types, reporters of different binary
systems were used: UAS-mCD8::GFP (42), UAS-Brp::GFP (BDSC
36291), UAS-mCherry (BDSC 27392), 10XUAS-mCD8::RFP,13X
LexAop2- mCD8::GFP (BDSC 32229), GH146-QF, QUAS-mtdTomato-
3xHA (BDSC 30037). For generating single-cell clones, hs-FLPm5
(41) (BDSC 56799 and BDSC 35534) and UAS-Flybow1.1B (41)
(BDSC 56803) constructs were used. For cell ablation, the following
stocks were used: UAS-DTI (BDSC 25039) and UAS-hid (a gift from
J. R. Nambu).
Immunohistology
Dissection of the larval, pupal, or adult brains was carried out in 1×
phosphate-buffered saline (PBS) and fixed in 2% freshly prepared
paraformaldehyde (PFA) (prepared in 1× PBS) for 60min for larval
and pupal brains and 90min for adult brains at room temperature.
The fixative was removed, and the brains were washed four times
with 0.3% PBTx (Triton X-100in 1× PBS) for 15min each at room
temperature. Blocking of the samples was performed for 60min
at room temperature in 10% goat serum, prepared in 0.3% PBTx,
and incubated with primary antibody overnight at 4°C. Following
four times washing, 15min each, samples were incubated with sec-
ondary antibody overnight at 4°C. After four times washing, 15min
each, samples were mounted in VECTASHIELD (Vector Laboratories),
an antifade mounting medium for confocal microscopy. Both pri-
mary and secondary antibodies were diluted in 10% goat serum.
Fluorescent samples were analyzed using a Leica TCS SP5II con-
focal microscope. To process and analyze images and quantify pheno-
types, the open source tool ImageJ, Adobe Photoshop, and Imaris
(Bitplane) were used.
Primary antibodies used for this study were rat anti–N-cadherin
extracellular domain [DN-Ex no.8, 1:10; Developmental Studies
Hybridoma Bank (DSHB)], mouse anti-Flamingo (1:5; DSHB), mouse
anti–Neuroglian-180 (BP 104, 1:10; DSHB), rabbit anti-GFP (green
fluorescent protein) (1:1000; Invitrogen), and mouse anti-Fasciclin2
(1:5; DSHB). Secondary antibodies used were as follows: goat anti-
rabbit Alexa 488 (1:500), goat anti-rat Alexa 568 (1:300), goat anti-rat
Alexa 647 (1:500), goat anti-mouse Alexa 568 (1:300), and avidin
Alexa 488 (1:40). For general nuclear staining, TOTO-3 (1:5000) was
used. All secondary antibodies were obtained from Invitrogen.
Unilateral antennal backfills
All experiments were performed on adult flies. Mosquitoes (Anopheles
arabiensis) were provided by International Atomic Energy Agency
Laboratories, Seibersdorf Laboratories, Austria. Almost all diptera
species were collected within the state of Vienna area, with the ex-
ception of Hermetia illucens, which was caught in South Tyrol. Species
identity of fly samples was performed by a diptera determination
key to reach family level and further specific literature to refine the
taxon. In cases where a genus level could not be reached or there
were remaining uncertainties, the help of experts on the diptera forum
(www.diptera.info) was claimed. Living flies were first anesthetized
via CO2 and then inserted into 200-or 1000-l plastic pipette tips,
where parts of the tips have been cut off according to the body size
so that the head could stick out of the tip. The flies were immobi-
lized with plasticine. In a petri dish, the loaded pipette tip was placed
on a plasticine cube with a small indentation for the fly head. To
apply the tracer, a wall was built around the antenna with vaseline,
creating a small cavity and leaving only one antenna exposed. In all
flies, the right antenna was cut, at the base of the third segment, and
completely submerged with a drop of neuronal backfill tracer—2%
neurobiotin (Vector Laboratories) diluted in Millipore water. The
cavity was then completely covered with vaseline to prevent desic-
cation, and the petri dish was kept in 4°C for 90min. Afterward, fly
heads were removed and processed with the abovementioned im-
munohistology process for visualization on the same day (47). DN-
cadherin was used as a general neuropil marker, and anti-Nrg was
used to label the olfactory commissure.
Flyclear
Three- to 5-day-old adult flies were fixed in 4% PFA at 4°C for 90 min,
followed by three times washing with 1× PBS at 4°C for 20min.
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Furthermore, the flies were dipped in Solution-1 (48) for 5 days at
37°C. Flies with the complete depigmentation of the compound eyes
were further processed and washed with 1× PBS for three times at
25°C. Last, the samples were immersed in Solution-2 (48) for a minimum
of 1 day at 25°C and mounted in VECTASHIELD (Vector Laboratories)
for imaging using a Leica TCS SP5II confocal microscope.
Cell ablation
For the genetic ablation of cPINs, two different reagents were used.
The expression of temperature-sensitive diphtheria toxin (UAS-DTI)
was induced directly from Gal4 driver line, and its expression was
developmentally controlled by temperature shifts. In the case of
UAS-hid, Gal4 expression was controlled via Gal80ts. In both cases,
the crosses were raised at 18°C. Late third instar larvae were picked
and kept at 29°C to allow transgene expression. The adult flies were
dissected 3 days after eclosion.
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)
View/request a protocol for this paper from Bio-protocol.
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Acknowledgments: We are grateful to B. A. Hassan, T. Chihara, J. Pielage, H. Aberle,
and T. Préat for providing critical reagents for this study. We would like to thank the BDSC for
Drosophila transgenic lines and DSHB for antibodies. We thank H. Yamada for providing
Anopheles arabiensis. We thank B. Bergkirchner and A. Kasture for their help with filament tracing.
We further thank L. Luo, Y. Chou, and M. Pende for stimulating discussions and members of the
Hummel laboratory for critical comments on the manuscript. Funding: DFG (HU 992/21), Schram
Foundation (T287/22478/2012), and ROL (254) Research Platform Vienna University supported
this work. Author contributions: R.K.: conceptualization and investigation, acquisition of data,
analysis and interpretation of data, and writing the article; M.S., S.H., N.G., and A.G.: acquisition
and analysis of data; L.T.: identification of different fly species; L.T. and W.K.: antennal backfilling;
T.H.: conceptualization, design, supervision, analysis and interpretation of data, writing (original
article), and funding acquisition. Competing interests: The authors declare that they have no
competing interests. Data and materials availability: All data needed to evaluate the
conclusions in the paper are present in the paper and/or the Supplementary Materials.
Additional data related to this paper may be requested from the authors.
Submitted 4 January 2019
Accepted 28 September 2019
Published 23 October 2019
10.1126/sciadv.aaw5537
Citation: R. Kaur, M. Surala, S. Hoger, N. Grössmann, A. Grimm, L. Timaeus, W. Kallina, T. Hummel, Pioneer
interneurons instruct bilaterality in the Drosophila olfactory sensory map. Sci. Adv. 5, eaaw5537 (2019).
on October 23, 2019http://advances.sciencemag.org/Downloaded from

Pioneer interneurons instruct bilaterality in the Drosophila olfactory sensory map
Thomas Hummel
Rashmit Kaur, Michael Surala, Sebastian Hoger, Nicole Grössmann, Alexandra Grimm, Lorin Timaeus, Wolfgang Kallina and
DOI: 10.1126/sciadv.aaw5537
(10), eaaw5537.5Sci Adv
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